Motivation: Recent technological advances revealed that an unexpected large number of proteins interact with transcripts even if the RNA-binding domains are not annotated. We introduce catRAPID signature to identify ribonucleoproteins based on physico-chemical features instead of sequence similarity searches. The algorithm, trained on human proteins and tested on model organisms, calculates the overall RNA-binding propensity followed by the prediction of RNA-binding regions. catRAPID signature outperforms other algorithms in the identification of RNA-binding proteins and detection of non-classical RNA-binding regions. Results are visualized on a webpage and can be downloaded or forwarded to catRAPID omics for predictions of RNA targets.Availability and implementation:catRAPID signature can be accessed at http://s.tartaglialab.com/new_submission/signature.Contact:gian.tartaglia@crg.es or gian@tartaglialab.comSupplementary information:Bioinformatics online. The algorithm is an extension of the catRAPID approach  use RNA-binding domains (RDs) to recognize target RNAs and to regulate co-/post-transcriptional processes. Examples of classical RDs include RNA-recognition motif (RRM), double-stranded RNA-binding domain (dsRRM), K-homology (KH), RGG box and the Pumilio/FBF (PUM) domain , we built a method for the (i) identification of ribonucleoproteins and (ii) prediction of RNA-binding regions:For each protein, we calculated the fraction of residues with correlation coefficients above the cutoffs that are associated with physico-chemical properties and RDs , which we then used to train catRAPID signature shows an AUC = 0.76 for discrimination of 950 RBPs from 950 negative cases , which suggests that the N-terminus contributes to the RNA-binding ability of the full-length FMRP.The input of the server is a FASTA sequence. To illustrate the output with an example, we studied the RNA-binding ability of Fragile X Mental Retardation Protein FMRP. catRAPID signature addresses this limitation by detecting binding regions through physico-chemical features. Our algorithm will be helpful to investigate components of ribonucleoprotein complexes and to identify RNA-binding regions.As newly discovered RDs are not annotated, traditional domain-detection tools fail their identification. Supplementary DataClick here for additional data file.


Bunyaviridae represents the largest family of segmented RNA viruses, which infect a staggering diversity of plants, animals, and insects. Within the family Bunyaviridae, the Phlebovirus genus includes several important human and animal pathogens, including Rift Valley fever virus (RVFV), severe fever with thrombocytopenia syndrome virus (SFTSV), Uukuniemi virus (UUKV), and the sandfly fever viruses. The phleboviruses have small tripartite RNA genomes that encode a repertoire of 5–7 proteins. These few proteins accomplish the daunting task of recognizing and specifically packaging a tri-segment complement of viral genomic RNA in the midst of an abundance of host components. The critical nucleation events that eventually lead to virion production begin early on in the host cytoplasm as the first strands of nascent viral RNA (vRNA) are synthesized. The interaction between the vRNA and the viral nucleocapsid (N) protein effectively protects and masks the RNA from the host, and also forms the ribonucleoprotein (RNP) architecture that mediates downstream interactions and drives virion formation. Although the mechanism by which all three genomic counterparts are selectively co-packaged is not completely understood, we are beginning to understand the hierarchy of interactions that begins with N-RNA packaging and culminates in RNP packaging into new virus particles. In this review we focus on recent progress that highlights the molecular basis of RNA genome packaging in the phleboviruses.The  Phlebovirus genus represents one of five Bunyavirus genera . The International Committee on Taxonomy of Viruses cites nine species within the Phlebovirus genus, to which at least 70 distinct serotypes belong [Phlebovirus genus members awaiting a species designation [The s belong . The genignation . ExcludiPhleboviruses have become medically and economically significant around the world within the past century. The Uukuniemi-like viruses are transmitted by ticks, while the sandfly fever viruses are spread by mosquitoes or phlebotomine flies. Outbreaks caused by the sandfly fever viruses, including sandfly fever Sicilian virus (SFSV), sandfly fever Naples virus (SFNV), were documented during the World War II era, during which epidemics were documented amongst the Allied troops in Italy . SFSV anAedes aegypti mosquitos, which can also carry a number of other human pathogens including Zika, Yellow fever, and Dengue viruses. RVFV infection is endemic to parts of sub-Saharan Africa and the Arabian Peninsula, and has caused notable localized epidemics with significant human mortality and morbidity that coincide with heavy rainfall and the prevalence of mosquito vectors. RVFV causes a wide range of clinical manifestations in humans, from general malaise and flu-like illness to blindness, meningoencephalitis, hemorrhagic fever and death [The most studied phlebovirus, Rift Valley fever virus (RVFV), was first isolated in Kenya in the 1930’s and has since spread throughout the African continent and more recently into the Arabian Peninsula ,7. The vnd death ,9,10,11.nd death . In contnd death ,15,16,17Phlebovirus genus is the use of an ambisense coding strategy in the smallest of the three genome segments, the S segment, whereby the nucleocapsid (N) protein mRNA is transcribed from the viral sense, and the mRNA for the virulence factor, termed non-structural protein on S segment, (NSs), is transcribed from the antisense RNA intermediate [The molecular virology of the bunyaviruses has been actively studied for years, and the major attributes of their replication cycles and interactions with host cells have been described as the result of myriad scientific approaches by many laboratories around the globe ,19. The rmediate ,21. The rmediate ,23. The rmediate .Phlebovirus virions exhibit icosahedral symmetry and measure approximately 100 nm in diameter . VirionsTranscription of vRNA to mRNA requires genomic RNA coated in N protein as a template. Each vRNA segment is accompanied in the virion by the RdRP ,30. PrimViral assembly and budding occur at the Golgi apparatus, which is a distinguishing characteristic of bunyaviruses . HoweverIn the following sections we summarize recent research on genome packaging in the phleboviruses, highlighting the molecular mechanisms that drive the processes. We focus first on the critical binding between a single N protein and its cognate vRNA, then discuss the subsequent RNA packaging that builds upon the nucleocapsid-RNA (N-RNA) interface, including subsequent N protein oligomerization and RNP formation. Finally, the higher order protein and RNA based contributions that mediate RNP packaging into infectious particles are outlined . UnderstThe genomes of negative sense RNA viruses are covered and protected by the viral nucleoprotein or N protein. N proteins fulfill many essential jobs, including protection of vRNA from detection by host defenses and compaction of the genomic RNPs. N protein is required for transcription and replication . N proteArenaviridae, Orthomyxoviridae, and Bunyaviridae families. This is in stark contrast with the capsid proteins of non-segmented viruses belonging to the Mononegavirales, which exhibit much greater homology amongst order members. Even within the Bunyaviridae, the structural properties of the N proteins vary widely. For example, the N protein of Crimean Congo hemorrhagic fever virus (CCHFV), of the Nairovirus genus, shows greater similarity with the N protein from Lassa virus (LASV), from the Arenaviridae family, than it does with RVFV N protein [Currently available structural comparisons show very limited similarity amongst nucleocapsid proteins of segmented negative sense RNA viruses classified in the Multinegavirales order, which includes the  protein . Althoug protein . Interes protein . RVFV, T protein ,44,45,46The ~25–30 kDa phleboviral N proteins carry out all of their known essential tasks in the host cytoplasm. Therefore, it has been assumed that N protein must have a mechanism for distinguishing between viral and host RNA binding partners. The two crystal structures available for RVFV may provide clues about how phleboviral N proteins mediate RNA binding. The first published RVFV N protein structure showed a monomeric N protein, devoid of RNA, that did not address the nature of N-RNA binding and N protein oligomerization along the RNA molecule . A seconPhleboviruses do not encode a phosphoprotein (P), a multifunctional protein in nonsegmented RNA viruses that prevents newly synthesized nucleoprotein from prematurely binding to cellular or vRNA. For example, nascent N proteins of Sendai virus (SeV) and vesicular stomatitis virus (VSV) can form non-specific aggregates with RNA in the absence of bound P. The phosphoprotein helps to maintain N proteins in an unbound state until the nucleoprotein is at the site of virus assembly ,51,52,53Based on the structural data from N-RNA complexes, the energetics of RNA binding to N are dominated by electrostatic and hydrophobic interactions built into the RNA binding groove of N protein. A patch of basic residues that are highly conserved in phleboviruses  flank the central region of the globular N protein body and confirm the location of the RNA binding domain . MutatioThe RNP is the vehicle that protects, transports, and packages the viral genomic RNA. RNPs serve as the templates for the synthesis of mRNA, cRNA, and new copies of the vRNA. Phleboviral RNPs are built from individual N-RNA interactions and form circularized pseudo-helical structures with the 5′ and 3′ complementary ends of the S, M, or L segments interacting via base pairing and bound by the L protein. It has yet to be determined how small planar crystalized RNPs  are expanded to generate full-length native RNP complexes that coat each genome segment. Recent work on TOSV indicates that RNA binding stimulates conformational changes at the protein interface between adjacent N proteins that causes rotation between N subunits . This roThe direct mechanism by which the RNPs are guided to the site of viral budding at the Golgi compartment remains unclear. Bunyaviral RNPs have been shown to accumulate in cytoplasmic aggregates for hantaviruses, tospoviruses and nairoviruses, implying that the RNPs alone do not localize to the Golgi ,61,62. RIn many viruses, viral matrix proteins serve as the master mediators of virion assembly and provide structural integrity for budding virus particles. Matrix proteins function by bridging the interaction between the genomic RNPs with the glycoproteins in the lipid envelope and may intrinsically induce membrane curvature or even hijack host protein budding scaffolds. Bunyaviruses are unique amongst other negative-sense RNA viruses in that they do not encode a matrix protein, suggesting an essential role for the viral glycoproteins in concert with the nucleocapsid proteins in anchoring the RNPs into the budding virion framework .Bunyaviridae possess large Gn C-terminal tails that extend outward on the cytoplasmic face of the Golgi membrane. Enzymatic digestion of membrane bound Karimabad virus (KARV) glycoproteins during infection indicated that around 12% of the glycoproteins were readily digested, suggesting that a large portion of the Gn-Gc complex is not membrane bound and is thus accessible for engaging in protein-protein interactions with the N proteins [The viral envelope glycoproteins Gn and Gc are cotranslationally cleaved from a polyprotein precursor to form the mature proteins encoded within the M segment ,65,66. Wproteins .Given the lack of a matrix protein within this virus family, it is not surprising that many lines of evidence point to the association of N protein with Gn and Gc. For example, in UUKV infected cells, Gn/Gc can be co-immunoprecipitated with the N protein . TunicamEfficient RNP packaging in RVFV and UUKV minigenome and virus like particle (VLP) systems requires a direct interaction between the Gn cytoplasmic tail and N protein ,72. MutaEvidence indicates that Gn is also responsible for recruiting the viral polymerase to the site of assembly at the Golgi. The bunyaviral RdRP is synthesized from the L segment on cytosolic ribosomes and maintains a diffuse cytoplasmic localization in the absence of other viral proteins. In RVFV, when Gn is expressed with the RdRP, the polymerase colocalizes with Gn . ConversBunyaviridae whose Gn proteins lack zinc-finger (ZF) domains in their cytoplasmic tails. CCHFV and Andes virus (ANDV) Gn proteins each have dual ZF with the traditional ββα fold, but they are positioned close together so that they form a globular domain, which may be important for RNA recognition and/or for RNP binding [Bunyaviridae, recruit host factors that play a role in the viral budding process.Interestingly, phleboviruses are the only genus within the  binding ,75,76. I binding ,77. ThisViral genome assembly strategies are strikingly diverse but can be inferred based on the characteristics of the nucleic acid to be packaged. For example, the steric and charge constraints conferred by viral dsDNA  genomes results in their packaging into preformed protein capsid shells using viral molecular motors driven by ATP hydrolysis. In contrast, more flexible single stranded RNA genomes can be manipulated into diverse conformations driven by protein-RNA interactions. The non-enveloped positive sense single stranded tobacco mosaic virus (TMV) RNA genome can assemble into rigid rod-like helical capsids in vitro via spontaneous electrostatic interactions between the capsid protein and vRNA . LikewisBunyaviridae, Orthomyxoviridae and Arenaviridae families, which are also ssRNA viruses, but encode multiple genome segments. In order for a segmented RNA virus to be infectious, a nascent virus particle must contain at least one copy of each genome segment. This implies that RNP packaging is not only contingent upon differentiation between viral and host nucleic acids, but also between diverse viral nucleic acids, including sense and antisense genome. While genome segmentation offers evolutionary advantages, it clearly creates the need for increased specificity in genomic packaging to ensure production of viable virions. The possibility of acquiring beneficial traits via reassortment must therefore outweigh this cost of increasing complexity conferred by genome segmentation.Genome packaging increases in complexity for members of the Orthobunyavirus genus, exhibits a particle:PFU ratio of 2.6–7.2, further implying that viruses have built in mechanisms to ensure their genomes are packaged into nascent particles [Historically, arguments have been posed in support of both segment-specific and random packaging mechanisms for multi-segmented RNA viruses. Eight negative sense single stranded RNA genome segments must be incorporated into infectious progeny of the prototypical orthomyxovirus, influenza A (IAV). When compared with VSV mentioned above, IAV also forms helical structures, but there is a non-uniform association of RNPs with the matrix proteins, resulting in a diverse pool of viral particles. However, observed IAV particle:PFU ratios are significantly lower than statistical estimates based on random chance genome incorporation models, indicating that there must be some degree of specificity inherent in the gathering and parceling of all eight segments. Likewise, the tri‑segmented Bunyamwera virus, of the articles .Within the phleboviruses, our understanding of RNP co-packaging is only beginning to take shape. In addition to the overall packaging mechanism (random versus segment specific), an active area of research is focused on whether the characteristics of one segment make that segment more likely to be incorporated into progeny virus particles over another. There are abundant examples illustrating the existence of RNA based packaging signals across many diverse families of viruses that impact genome packaging. Even in the model bacteriophage MS2 system, positive sense RNA genome assembly is driven by coat protein recognition of degenerate RNA packaging signals . RNA sigRecent studies in IAV indicate that for each segment, RNA packaging signals interact with other vRNA elements in coding and noncoding regions to facilitate segment specific co-incorporation ,87,88,89cis-acting signal within the coding region that determines whether sense or antisense genome is packaged. In this study the 5’ and 3’ non-coding regions (NCRs) were left intact, but the NSs and N protein open reading frames were swapped. In the RVFV MP12 virus more genomic S RNA was packaged, however in the MP12 clone with the coding regions reversed, more antigenomic S was packaged [It has also been shown that S antigenomic RNAs are packaged in UUKV virions and all three antigenomic segments may be packaged in RVFV particles ,94. Workpackaged .Experimental results also show that there is genome packaging variation across species and cell lines. For example, multiple viable four-segmented RVFV variants have proven capable of spreading, albeit with reduced growth and virulence, in mammalian cell lines, but are unable to propagate in mosquito cells . In the Bunyaviral genome NCRs have genus specific complementary sequences that can base pair to form the panhandle shaped RNPs and contain essential replication promoter elements, transcription and encapsidation signals. Given these already established essential roles, the NCRs are a logical place to search for packaging signals. In fact, Murakami and coworkers demonstrated a strong overlap between the packaging and replication signals within the terminal 15–25 nts comprising the panhandles of the S, M, and L segments in RVFV . MinirepL segment based minigenomes exhibit the strongest packaging efficiencies compared to the S and M segments for both UUKV and BUNV viruses. Reporter activity following serial passage of virus minireplicon genomic RNAs packaged into virions shows that L is maintained for several passages and M is retained for fewer passages, while the S signal rapidly decreases ,101. IntStudies using RVFV suggest that in infected cells, vRNA accumulates S > M > L . It has The work summarized above indicates that there is both innate flexibility and variability in the RNA packaging mechanisms employed by phleboviruses. The initial and most fundamental event in RNA packaging is the interaction between the viral nucleocapsid protein and its cognate RNA, since subsequent interactions build upon this base. Within the past few years there has been a wealth of structural and biochemical data that show the N-RNA interaction can be both specific and promiscuous. Future work should focus on identifying the molecular triggers that control the RNA binding modes of N protein. We know surprisingly little about the nature of phleboviral RNPs. We do not know if co-packaging of RNPs is driven by protein-protein interactions or via nucleic acid interfaces. The NCRs are critical for packaging, but RNA genomic elements that mediate segment co‑packaging have yet to be identified. In light of recent research demonstrating the importance of inter-segment signaling in segmented RNA viruses, we can be certain that there are phleboviral co‑packaging signals awaiting discovery. Segmented RNA viruses must be rapidly adaptable to new environmental challenges and stimuli, and also not so rigid in their packaging strategies that they ablate the possibility of creating genetic reassortants. Thus, it is hardly surprising that there is a fundamental plasticity in the RNA packaging mechanisms exploited by these viruses.


Human adenoviruses (HAds) encode for one or two highly abundant virus-associated RNAs, designated VA RNAI and VA RNAII, which fold into stable hairpin structures resembling miRNA precursors. Here we show that the terminal stem of the VA RNAs originating from Ad4, Ad5, Ad11 and Ad37, all undergo Dicer dependent processing into virus-specific miRNAs . We further show that the mivaRNA duplex is subjected to a highly asymmetric RISC loading with the 3′-strand from all VA RNAs being the favored strand, except for the Ad37 VA RNAII, where the 5′-mivaRNAII strand was preferentially assembled into RISC. Although the mivaRNA seed sequences are not fully conserved between the HAds a bioinformatics prediction approach suggests that a large fraction of the VA RNAII-, but not the VA RNAI-derived mivaRNAs still are able to target the same cellular genes. Using small RNA deep sequencing we demonstrate that the Dicer processing event in the terminal stem of the VA RNAs is not unique and generates 3′-mivaRNAs with a slight variation of the position of the 5′ terminal nucleotide in the RISC loaded guide strand. Also, we show that all analyzed VA RNAs, except Ad37 VA RNAI and Ad5 VA RNAII, utilize an alternative upstream A start site in addition to the classical +1 G start site. Further, the 5′-mivaRNAs with an A start appears to be preferentially incorporated into RISC. Although the majority of mivaRNA research has been done using Ad5 as the model system our analysis demonstrates that the mivaRNAs expressed in Ad11- and Ad37-infected cells are the most abundant mivaRNAs associated with Ago2-containing RISC. Collectively, our results show an unexpected variability in Dicer processing of the VA RNAs and a serotype-specific loading of mivaRNAs into Ago2-based RISC. Human adenoviruses (HAd) are non-enveloped DNA viruses with a linear double-stranded DNA genome of about 30 000–40 000 base pairs. More than 60 types of HAds have been described so far. They are classified into seven distinct subgroups A to G based on immunological, biological and biochemical characteristics All HAds encode at least one virus-associated RNA (VA RNA), which is homologous to the well-characterized VA RNAI from Ad2/5  During a lytic adenovirus infection only a small fraction (2–5%) of the Ad5 VA RNAs are processed into mivaRNAs All HAd VA RNAs are similar in length that varies between 149–177 nucleotides. The primary nucleotide sequence of the VA RNAI and VA RNAII genes from a specific HAd differs considerably in sequence although the matching VA RNA genes between members of the same subgroup are highly conserved 2. For infection virus stocks were diluted to 5 FFU per cell in DMEM without serum. The medium was removed from the plate, and the diluted virus inoculum was added, followed by incubation for 1 h at 37°C, in 7% CO2). After the 1 h incubation the medium was removed and DMEM, supplemented with 10% FCS, 1% PEST was added and plates were returned back to the CO2 incubator.Viruses used in this study were Ad4, Ad5, Ad11 and Ad37. Virus titers were measured as fluorescence forming units (FFU) HeLa cells were transfected with 50 nmol of siRNA using Lipofectamine 2000 (Invitrogen) according to the manufacture’s instructions. To target Dicer, a pool of two siRNAs (5′-UGCUUGAAGCAGCUCUGGAtt-3′ and 5′-UUUGUUGCGAGGGCUGAUtt-3′) was used. To detect specific effects of the Dicer siRNA a scrambled ON-TARGETplus Non-targeting siRNA Pool (Thermo Scientific) was used as a control.2, 1% NP-40), followed by two rounds of Phenol:Chloroform:Isoamylalcohol extraction and one extraction with Chloroform:Isoamylalcohol. The RNA was precipitated with Ethanol and dissolved in H2O. Total RNA was extracted using the TRI Reagent (Sigma) according to the manufacture’s instructions.Cytoplasmic RNA was prepared by lysis with IsoB-NP-40 buffer . Membranes were hybridized with the probe mixtures containing an equal molar concentration of serotype-specific γ-32P-ATP labeled oligonucleotides (for probe nucleotide sequences see Immunoprecipitation of the Flag/HA tagged Ago2 protein was done as described previously www.scilifelab.se). Enrichment of small RNAs in the size range of 10–40 nucleotides was done by using the PureLink miRNA Isolation Kit (Invitrogen) according to the manufacturer’s protocol. RNA samples were treated with tobacco acid pyrophosphatase (TAP), which removes the γ and β phosphates from the 5′-ends of RNA and thereby generates 5′-monophosphorylated RNAs. Small RNA libraries were constructed using the SOLiD Total RNA-Seq Kit . Strand specific adapters were hybridized to the RNA before reverse transcription. The cDNA was then size-selected on a 10% TBE-Urea gel (Life Technologies) and the libraries were constructed after amplification (15 cycles). Emulsion PCR was performed using the EZ Bead System (Life Technologies) and the small RNA libraries were then sequenced on the SOLiD 5500xl system . Sequencing reads were mapped to the corresponding adenovirus genome (www.ensembl.org).The cDNA library construction and RNA-seq data was generated by the service provided by the Scilifelab Uppsala  supplemented with 1 U/ml of Benzonase (EMD Millipore), was added and cells suspended by vortexing and incubation for 1 h at 4°C. Cells were further disrupted by addition of 10% SDS and 1M DTT to final concentrations of 1% and 100 mM followed by a boiling step of 5 min Protein samples were separated on a 4–20% gradient precast gel (Bio-Rad). Proteins were electro-transferred (Bio-Rad blotting chamber) onto a Immobilon-FL Western blot nitrocellulose membrane (Millipore) at 200 mA for 2–6 h, using Towbin transfer buffer . The membrane was blocked with Odyssey blocking buffer (LI-COR) for 1 h at 4°C and thereafter incubated with the primary antibody, diluted in the blocking buffer, overnight at 4°C. The membrane was washed 4 times for 5 min in 1xPBS followed by incubation with the fluorescent secondary antibodies (LI-COR) diluted 1∶10 000 in the blocking buffer, for 1 h at room temperature. The membrane was washed as described above and finally rinsed in 1xPBS and scanned with the Odyssey scanner (LI-COR). The following primary antibodies were used: anti-laminB (1∶4000) (Abcam), anti-Ad5 (1∶5000) (Abcam), anti-TRBP (1∶1000) (Abcam), anti-Dicer (1∶1000) (Abcam), anti-Ago2 (1∶1000) (Abnova).35S Protein Labeling Mix (50 µCi per 2 ml) was added and the cells were further incubated for 2h in a CO2 incubator . The cells were harvested and total protein extracts were prepared as above.Prior to pulse labeling of cells, the growth medium was removed and the cells were washed once in 1xPBS and incubated in Methionine/Cysteine free DMEM (Invitrogen)  for 1 h. After this starvation period the medium was removed and fresh Methionine/Cysteine free DMEM supplemented with 35S-methionine pulse labeling. The main read-out in this experiment is the detection of de novo translation of viral late proteins, which indicate the growth rate of the viruses. As shown in de novo viral protein synthesis had essentially ceased in Ad4, Ad5 and Ad37-infected cells  indicating that the viral life cycle has reached completion for these serotypes. In conclusion, all studied serotypes actively synthesize viral late proteins at 24 hpi although Ad11 showed a slightly slower growth kinetic compared to the other three serotypes in HeLa cells.Different human adenovirus serotypes show significant genome diversity, which can affect multiple aspect of the virus life cycle like cell entry, replication efficiency, interaction with the host cell pathways and the innate immune response. To monitor the lytic growth properties of HAds belonging to different subgroups we infected HeLa cells with Ad4, Ad5, Ad11 and Ad37 and followed the progress of the infection by To investigate the impact of the different HAds on the RNAi/miRNA pathway, we investigated the impact of the HAd infections on the steady state accumulation of some of the main RNAi/miRNA components see , lane 6 The secondary structures of the VA RNAs share a great deal of similarity and fold into hairpin-like structures with a terminal and apical stem separated by a more complex central domain  and RISC bound (referred to as RISC-IP) small RNA pools isolated at 24 hpi from Ad4, Ad5, Ad11, and Ad37-infected 293-Flag-Ago2 cells. Since the VA RNAs are primary RNA polymerase III transcription products the 5′-mivaRNAs from both VA RNAs would be expected to have a mixture of mono-, di- and tri-phosphorylated 5′-ends The small RNA libraries (15–35 nucleotides) prepared from cytoplasmic RNA and RISC-IP were subjected to deep sequencing using the SOLiD technology. Sequencing reads were mapped against the corresponding adenovirus genomes . The size distribution of reads in the cytoplasmic small RNA pool showed a broad distribution of RNAs ranging from 20–35 nucleotides in length with a peak around 22 nucleotides . As expeThe Ad2/5 VA RNAI gene has previously been shown to initiate transcription from two sites, generating the major G start and the minor A start located three nucleotides upstream Previous studies have shown that the Ad5 3′-mivaRNAI and 3′-mivaRNAII are heterogeneous at their 5′-terminus suggesting that Dicer cleavage in the terminal stem varies slightly (see Discussion). Also, since the length of the VA RNAs varies slightly  the numbThe VA RNAII genes differ significantly in length between the three serotypes tested . TherefoCollectively our results suggest that the asymmetry in RISC loading observed in Ad5-infected cells Here we have characterized mivaRNA expression from four HAds belonging to different subgroups  in orderIn mammalian cells Dicer recognizes stem-loop structured pre-miRNAs and cleaves both strands to generate 21–25 nucleotide dsRNA duplexes with a two nucleotide 3′-overhang and a phosphate group at the recessed 5′-end  strand. From this point it is interesting to note that in three of the analyzed HAds the 5′-mivaRNAI generated from the VA RNAI A start is the predominant 5′-mivaRNAI in RISC , 3′-mivaRNAI (B), 5′-mivaRNAII (C) and 3′-mivaRNAII (D) were detected both in the input and RISC fractions by Northern blot using mixtures of 32P-labelled oligonucleotide probes complementary to the corresponding mivaRNA sequences (equences . The det(TIF)Click here for additional data file.Figure S2Size distribution of the viral small RNA reads in the cytoplasmic (Cyto) and RISC-bound (RISC-IP) fraction in 293-Ago2-infected cells.(TIF)Click here for additional data file.Figure S3Mapping of Ad4 and Ad5 small RNA reads to the 5′- and 3′-end of the VA RNA genes. The data shown is similar to (TIF)Click here for additional data file.Figure S4Mapping of Ad11 and Ad37 small RNA reads to the 5′- and 3′-end of the VA RNA genes. The data shown is similar to (TIF)Click here for additional data file.Figure S5Computational prediction of mivaRNAI target genes. The number of targeted human genes by the most abundant small RNA generated from the Ad4, Ad5, Ad11 and Ad37 5′- (A) or 3′- (B) mivaRNAI were predicted using the miRanda software and presented as a Venn diagram. Numbers in the intersecting circles indicate the number of genes predicted to be a common target by the mivaRNAIs from the different HAds.(TIF)Click here for additional data file.Table S1List of the human adenovirus (HAd) serotypes used in this study.(PDF)Click here for additional data file.Table S2Nucleotide sequences of DNA oligonucleotides used.(PDF)Click here for additional data file.Table S3List of total reads and length variations of the mivaRNAs expressed in the HAd infections.(PDF)Click here for additional data file.


RNA-binding proteins play a key role in the regulation of all aspects of RNA metabolism, from the synthesis of RNA to its decay. Protein-RNA interactions have been thought to be mostly mediated by canonical RNA-binding domains that form stable secondary and tertiary structures. However, a number of pioneering studies over the past decades, together with recent proteome-wide data, have challenged this view, revealing surprising roles for intrinsically disordered protein regions in RNA binding. Here, we discuss how disordered protein regions can mediate protein-RNA interactions, conceptually grouping these regions into RS-rich, RG-rich, and other basic sequences, that can mediate both specific and non-specific interactions with RNA. Disordered regions can also influence RNA metabolism through protein aggregation and hydrogel formation. Importantly, protein-RNA interactions mediated by disordered regions can influence nearly all aspects of co- and post-transcriptional RNA processes and, consequently, their disruption can cause disease. Despite growing interest in disordered protein regions and their roles in RNA biology, their mechanisms of binding, regulation, and physiological consequences remain poorly understood. In the coming years, the study of these unorthodox interactions will yield important insights into RNA regulation in cellular homeostasis and disease.The online version of this article (doi:10.1186/s12964-016-0132-3) contains supplementary material, which is available to authorized users. DNA is well known as the molecule that stores genetic information. RNA, a close chemical cousin of DNA, acts as a molecular messenger to execute a set of genetic instructions (genes) encoded in the DNA, which come to life when genes are activated. First, the genetic information stored in DNA has to be copied, or transcribed, into RNA in the cell nucleus and then the information contained in RNA must be interpreted in the cytoplasm to build proteins through a process known as translation. Rather than being a simple process, the path from transcription to translation entails many steps of regulation that make crucial contributions to accurate gene control. This regulation is in large part orchestrated by proteins that bind to RNA and alter its localisation, structure, stability, and translational efficiency. The current paradigm of RNA-binding protein function is that they contain regions, or domains, that fold tightly into an ordered interaction platform that specifies how and where the interaction with RNA will occur. In this review, we describe how this paradigm has been challenged by studies showing that other, hitherto neglected regions in RNA-binding proteins, which in spite of being intrinsically disordered, can play key functional roles in protein-RNA interactions. Proteins harbouring such disordered regions are involved in virtually every step of RNA regulation and, in some instances, have been implicated in disease. Based on exciting recent discoveries that indicate their unexpectedly pervasive role in RNA binding, we propose that the systematic study of disordered regions within RNA-binding proteins will shed light on poorly understood aspects of RNA biology and their implications in health and disease.RNA-binding proteins (RBPs) assemble with RNA into dynamic ribonucleoprotein (RNP) complexes that mediate all aspects of RNA metabolism , 2. Due Early on, it was recognised that not all RNA-binding activities could be attributed to classical RBDs. Computational predictions based on transcriptome complexity suggested that 3-11 % of a given proteome should be dedicated to RNA binding, whereas only a fraction of this number could be identified by homology-based searches for classical RBDs , 15. MorIn vitro studies in yeast identified dozens of proteins lacking classical RBDs as putative RBPs, including metabolic enzymes and DNA-binding proteins  -mediated RNA binding also plays a role in nuclear RNA export, as illustrated by the nuclear RNA export factor 1 (NXF1). While NXF1 harbours an RRM capable of binding RNA , most ofFragile X mental retardation protein (FMRP) is another RBP with a well-characterised, RNA-binding RGG-box Fig. . InvolveA number of other RBPs use an RGG-repeat region to target G-rich and structured RNA targets and are implicated in neurological disease as well as cancer Table . These RBasic residues often cluster in RBPs to form basic patches that can contribute to RNA-binding. Analysis of mammalian RNA-binding proteomes showed that such motifs are abundant among unorthodox RBPs , 27. BasArginine rich motifs (ARMs) Table  are probin vitro selected RNA aptamers [in vitro selected R-rich peptides that include additional serine, glycine, and glutamic acid residues [Similar to Tat proteins, lentiviral Rev auxiliary protein binds a structured RNA element  present in partially-spliced and unspliced viral RNAs to facilitate nuclear export of viral RNA , 95. Theaptamers , highligresidues –107 — thresidues . These fThe basic amino acid lysine can form disordered poly-lysine peptides that interact with RNA. 47 proteins identified in the human RNA-binding proteome have a long poly-K patch but lack known RBDs, suggesting these motifs are good candidates for RNA binding . The K-rBasic tails in RBPs share physicochemical similarities with analogous sequences in DNA-binding proteins (DBPs) . In DNA-One open question is whether basic tails can distinguish between DNA and RNA. The AT-hook, defined as G-R-P core flanked by basic arginine and/or lysine residues, binds DNA and is found in many nuclear, DNA-binding proteins , 122. HoRNA processing and storage is often undertaken in the context of dynamic, membraneless organelles that vary in size, composition, and function. These organelles include the nucleolus, PML bodies, nuclear speckles and cajal bodies in the nucleus as well as P–bodies, stress and germ granules in the cytoplasm –130. RNAC. elegans [in vitro [in vitro and in vivo [Several recent studies have shown that disordered, low complexity regions in a number RBP have a capacity to form such granules , 139–141 elegans . Similarin vitro . Further in vivo , 143–146 in vivo . In yeas in vivo . Notably in vivo . A recen in vivo . Given tInterestingly, different types of low complexity sequences may help to form different types of aggregates and ways to embed RNA. A recent study showed that while low complexity sequences promote formation of both P-bodies and stress granules in yeast, these granules differ in their dynamic properties, P-bodies displaying more dynamic/fluid phase transition than more solid-like stress granules . GranulePost-translational modifications can modulate protein’s interaction properties . A numbeIn other contexts, it is known that alternative splicing can alter the sequence and function of proteins. Several global analyses have reported that short, regulatory sequences such as sites for post-translational modifications and protein-protein interactions are often subjected to alternative splicing –159. CouRNA-binding activity can also be modulated by competitive or cooperative interactions Table , Fig. 3.We have discussed the contribution of RS-, RG-, and K/R-rich, disordered regions to RNA interactions, and given examples of how they participate in co- and post-transcriptional regulation of RNA metabolism; how defects in these interactions can lead to disease; and how disorder in RBPs can be utilised by viruses during their infection cycle. Disordered regions are emerging as malleable, often multifunctional RNA-binding modules whose interactions with RNA range from non-specific to highly selective with defined target sequence or structural requirements Fig. . How speMuch remains to be learnt about disorder-mediated protein-RNA interactions. How do disordered regions interact with RNA? How many functionally relevant disorder-RNA interactions exist? Can more refined motifs be identified among the different classes of RNA-binding disordered regions? Are there further subclasses of motifs within RS-, RG-, basic, and other RNA-binding disordered regions with distinct binding characteristics? How is RNA binding regulated post-translationally, by alternative splicing, or by competitive interactions with other biomolecules? How do mutations in disordered regions involved in RNA binding cause disease? Fundamental principles of disorder-RNA interactions are likely to have close parallels to what has been elucidated for protein-protein and protein-DNA interactions, where disorder-mediated regulation has received much more attention over the past decade , 165–170Structure-to-function paradigm  has pers


Agarose hydrogels are widely used for three-dimensional cell scaffolding in tissue engineering and cell biology. Recently, molecular profiles have been obtained with extraction of a minimal volume of RNA using fluorescent-tagged quantitative polymerase chain reaction (qPCR), which requires high integrity RNA. However, the agarose interferes considerably with the quantity and quality of the extracted RNA. Moreover, little is known about RNA integrity when the RNA is extracted from cell/agarose construct. Thus, in order to obtain RNA of sufficient integrity, we examined various extraction methods and addressed reproducible methodologies for RNA extraction from cell/agarose constructs using spectrophotometry and microfluidic capillary electrophoresis.With various extraction methods using a mono-phasic solution of phenol and guanidine isothiocyanate, we evaluated quantity and quality of total RNA from cell/agarose construct. Extraction with solution of phenol and guanidine isothiocyanate followed by a silica based membrane filter column gave sufficient RNA integrity number, which allowed us to proceed to fluorescent-tagged qPCR for evaluating various cellular activities.The RNA extraction methods using phenol and guanidine isothiocyanate solution and a silica membrane column can be useful for obtaining high integrity RNA from cell/agarose constructs rich in polysaccharide and extracellular matrix. Our study contributes to further investigation using agarose hydrogels and other materials rich in polysaccharide in the field of cellular and tissue engineering. Agarose hydrogels have been conveniently used for three-dimensional 3-D) cell scaffolding. This is largely because the gels promote the maintenance of chondrogenic phenotypes, e.g., the synthesis of cartilaginous extracellular matrix (ECM), for neocartilage engineering and cell biology applications –3.-D cell s® Reagent , has recently been used for RNA extraction from multiple organisms. After dissolving agarose gels with this solution and adding chloroform to separate the phases, subsequent RNA precipitation with isopropanol results in contamination of RNA with agarose. This contamination frequently hinders downstream applications, including gene expression analysis using fluorescent-tagged quantitative polymerase chain reaction (qPCR). Although the extraction methods were modified to reduce the agarose contamination [Cells embedded in the agarose are supported by the 3-D environment and homogeneously distributed, so that the gel is suitable for investigating cellular functions in response to biophysical stresses –6. Recenmination , 10, infg for 5 min.Bovine forelimbs  were purchased from a local abattoir. Full-depth articular cartilage pieces were harvested from the humeral condyle using a surgical blade . The harvested cartilage pieces were washed with Dulbecco’s phosphate-buffered saline , minced, and digested with 0.2 % type I collagenase  dissolved in Ham’s F-12 medium (Life Technology) with gentle rotating for 18 h at 37 °C. Bovine articular chondrocytes (bACs) were isolated from the digestion with a cell strainer  by removing non-digested debris, followed by rinsing twice with D-PBS and centrifugation at 220×For making cell/agarose constructs, 1.5 % culture-grade agarose  dissolved in D-PBS was autoclaved at 120 °C for 15 min followed by maintaining at 37 °C. Five hundred thousands cells were suspended in 100 µl of the warm agarose at 37 °C. The cell/agarose suspension was poured on a non-adherent 100-mm culture dish (Fisher) and cooled to room temperature for agarose solidification . For the agarose control, the same amount of agarose without cells was poured on the non-adherent culture dish. The bAC/agarose constructs were incubated in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 (1:1) medium (Life Technology) including 10 % fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin at 37 °C and 5 % CO2 in air for 4 days.For the cell-only control, the same number of the cells were seeded to 12-well plates . To reveal the presence of sulfated glycosaminoglycan, a 10-µm section was stained with 0.5 % Toluidin blue-O at pH 4.0 (Fisher).Cell distribution and chondrocytic phenotypes within agarose hydrogels were evaluated histologically. A cell/agarose construct was harvested at day 4, fixed with 2 % paraformaldehyde (Fischer) in 0.1 M cacodylic acid , and embedded in methacrylate resin  with a pellet pestle . We examined those homogenized samples with various RNA extraction methods as follows .After phase separation with chloroform, the aqueous phase was transferred to a separate tube. A half volume of high-salt solution composed of 1.2 M sodium chloride (NaCl) and 0.8 M sodium citrate and a half volume of isopropanol were then added, followed by centrifugation  , 11.® Gel and PCR Clean-Up, Macherey–Nagel). The column binding RNA was washed with ethanol-based buffer  and eluted with low ionic strength conditions using alkaline buffer .After TZ or TZ-Salt preparation and centrifugation, the precipitant was dissolved in 200 µl of potassium thiocyanate buffer  per 100 mg of agarose and incubated at 50 °C for 10 min. The sample in NTC buffer was mixed with an equal volume of 70 % ethanol, followed by centrifugation with a silica spin column .® Reagent was mixed with a half volume of 100 % ethanol, followed by centrifugation with a silica based membrane filter . The RNA bound to the membrane filter was washed with guanidine salt and ethanol based buffer (RW1) and eluted with RPE buffer (not defined by manufacturer) following the manufacturer’s protocol (TZ-Plant) [One volume of sample homogenate in TRIzol® Reagent was mixed with one volume of 100 % ethanol followed by centrifugation with a silica based membrane filter  (TZ-Direct). The RNA bound to the membrane filter was washed with Direct-zol™ RNA PreWash and RNA Wash Buffer followed by elution with RNase-free water following the manufacturer’s protocol.One volume of sample homogenate in TRIzolThe cell/agarose constructs were harvested and immediately homogenized in 1-ml of phenol and guanidine isocyanate reagent . RNA was separated in the channels of the microfabricated chip (Agilent) due to molecular weight and detected with a fluorescence detector. RNA integrity was expressed as the RNA integrity number . RIN values ranged from 10 (intact) to 1 , with an RIN higher than 8.0 indicative of high integrity [We evaluated the concentration, purity, and integrity of RNA extracted with various extraction methods from the bAC/agarose hydrogel construct. We measured the optical density (OD) of 1.5-µl of extracted RNA at 230, 260 and 280 nm with a spectrophotometer . The RNA concentration was converted from OD at 260 nm, and the purity was evaluated by the OD ratio at 260/280 nm for protein contamination and the OD ratio at 260/230 nm for polysaccharide, phenol and other contaminations. We also measured the concentration and the integrity of RNA with a fluorescent-tag and microfluidic capillary electrophoresis  were calculated for all measurements.The final precipitations of RNA with isopropanol in both the TZ and TZ-Salt groups were gel-like stiff, large and glossy compared to the ones in the cell-only control group Fig. . This prbACs were distributed homogeneously throughout an agarose hydrogel Fig. .Fig. 4BoThe bACs produced a metachromatic matrix around the cells, representing cartilage ECM. Picnotic cells were not seen, indicating that the bACs maintained their biosynthetic capabilities within the agarose hydrogel constructs.® Reagent could interfere with OD 260 nm. A higher level of extracted RNA was seen by spectrophotometry compared to microfluidic capillary electrophoresis, which was also seen in TZ and TZ-Salt. Moreover, in the TZ and TZ-Salt groups, the concentrations by spectrophotometry were higher than cell-only control, whereas the concentrations by microfluidic capillary electrophoresis were lower than the cell-only control. In TZ-NTC and TZ-2NTC, the concentrations were similar by both spectrophotometry and microfluidic capillary electrophoresis. By adding salt to TZ-NTC and TZ-2NTC, the concentrations in TZ-Salt-NTC and TZ-Salt-2NTC showed lower than without salt by spectrophotometry and microfluidic capillary electrophoresis. Using the Plant kit, the concentrations by spectrophotometry showed a higher trend compare to by microfluidic capillary electrophoresis. Using the Direct-zol kit, the concentrations showed a decreased trend by both spectrophotometry and microfluidic capillary electrophoresis  by spectrophotometry and microfluidic capillary electrophoresis, respectively. The RNA concentration was determined by OD at 260 nm, which can be compromised by the presence of DNA and phenol . One of is Figs. , 6.Fig. The ratio of OD 260/280 nm and OD 260/230 nm were 1.93 ± 0.06 and 0.56 ± 0.12 in the cell-only control, respectively. The mean OD 260/280 showed a higher trend (>1.8) in the TZ-Salt, TZ-NTC, TZ-2NTC, TZ-Salt-NTC and TZ-Plant methods. The mean OD 260/230 showed a lower trend in all methods, though the TZ-NTC and TZ-Plant methods produced the best values , so we were motivated to use the same kit. The commercially available NTC buffer, which includes potassium thiocyanate, has been used for purification of DNA and RNA fragments from agarose gels. We used it for isolation of RNA from the agarose-contaminated precipitation with isopropanol. In TZ-NTC, the concentration and RIN were successfully obtained by microfluidic capillary electrophoresis. Our results showed that TZ-NTC was a reliable method for RNA extraction from cell/agarose constructs. For additional improvement, we attempted to use 2× volume of NTC to completely dissolve the precipitation instead of 1× volume of NTC buffer for TZ-NTC and TZ-Salt-NTC. However, there was no visible effect produced by 2× volume of NTC.Secondly, RNA recovery from agarose gel after electrophoresis has been performed with NTC buffer and a silica membrane kit (NucleoSpin® Mini Plant kit was designed for extracting RNA from plant tissue. The similarities between our agarose gel and polysaccharide rich plants motivated us to incorporate this Plant kit as well. Recently, Wang and his colleagues extracted RNA from human mesenchymal stem cells and murine chondrocytes embedded in agarose gel using this kit, but a RIN was not reported [The RNeasyreported . In addiThe TZ-NTC and TZ-Plant methods can be used to extract high integrity RNA from cell/agarose constructs rich in polysaccharide and ECM for fluorescent-tagged qPCR. Our findings contribute to a further investigation using agarose hydrogels and other materials rich in polysaccharide in the field of tissue engineering.


The power consumption is 220 μW with 3.3 V supply. The input parasitic capacitances within the range of −250 fF to 250 fF can be cancelled out automatically, and the required calibration time is lower than 10 ms.Capacitive sensing schemes are widely used for various microsensors; however, such microsensors suffer from severe parasitic capacitance problems. This paper presents a fully integrated low-noise readout circuit with automatic offset cancellation loop (AOCL) for capacitive microsensors. The output offsets of the capacitive sensing chain due to the parasitic capacitances and process variations are automatically removed using AOCL. The AOCL generates electrically equivalent offset capacitance and enables charge-domain fine calibration using a 10-bit R-2R digital-to-analog converter, charge-transfer switches, and a charge-storing capacitor. The AOCL cancels the unwanted offset by binary-search algorithm based on 10-bit successive approximation register (SAR) logic. The chip is implemented using 0.18 μm complementary metal-oxide-semiconductor (CMOS) process with an active area of 1.76 mm Capacitive microsensors based on microelectromechanical system (MEMS) technologies are used in various application areas, including accelerometers, gyroscopes, pressure sensors, touch-screen sensors, proximity sensors, and so on because of their small form factor, low-power characteristics, good temperature dependency, and low cost ,2,3,4,5.This paper presents a fully integrated low-noise capacitive sensing circuit with automatic offset calibration loop (AOCL). The output offsets of the capacitive sensing chain due to the parasitic capacitances and process variations are automatically removed using the automatic offset cancellation loop (AOCL). The AOCL generates an electrically equivalent offset capacitance, and it enables charge-domain fine calibration using a 10-bit R-2R digital-to-analog converter, charge-transfer switches and a charge-storing capacitor. The presented circuit enables the automatic offset calibration for capacitive sensors without using external test equipment.2) for a general metal-insulator-metal (MIM) capacitor. Assuming a sensing capacitance of several femtofarads, to achieve the high offset accuracy, offset calibrations with sub-fF steps are required. In this design, to implement a calibration capacitance smaller than the physical design rule, charge-domain fine calibration scheme is implemented, as shown in o) and charge-domain equivalent capacitance (Ceq) can be expressed as Equation (1). In this design, the charge storing capacitor, offC, is 250 fF, and the 10-bit R-2R DAC generates voltage from GND to VDD in 1024 steps. Thus, the charge-domain calibration circuit can generate an electrically equivalent offset capacitance in the range of ±250 fF with 0.488 fF step. The digital inputs of R-2R DAC are automatically determined by binary search algorithm of AOCL.in1) stores the input voltage signal and noise in Φ1 phase, and the stored charge is dumped in a feedback capacitor in Φ2 phase. The gain of PGA can be adjusted from 0 dB to 30 dB by programming Cf1, as expressed in Equation (2). SDA works with the opposite phase of PGA, and the differential gain of SDA can be adjusted from 0 dB to 36 dB by programming Cf2, as expressed in Equation (3). The output signal from the SDA is sampled on Φ3 phase.The proposed AOCL circuit is shown in An operation example of AOCL is illustrated in 2. A die-photograph of the fabricated capacitive sensing IC is shown in Z-axis capacitive accelerometer are mounted on the vibration exciter, as shown in To evaluate the performance of the fabricated IC, the IC and MEMS RMS, respectively. The input-output characteristics with MEMS Z-axis accelerometer are presented in The measurement results are presented in 2. The power consumption was 220 μW with 3.3 V supply. The input parasitic capacitances within the range of −250 fF to 250 fF were cancelled out automatically, and the required calibration time was lower than 10 ms. The input referred capacitance noise density and integrated noise with 400 Hz bandwidth were 1.275 aF/√Hz and 25.5 aFRMS, respectively. The scale factor, input range, and non-linearity were 0.1892 V/g, ±7.5 g, and 0.81% FSO, respectively. The presented circuit enabled the automatic offset calibration for capacitive sensors without using external test equipment, and enabled low-cost manufacturing of the capacitive sensors.A fully integrated low-noise capacitive readout circuit with automatic offset cancellation loop (AOCL) for capacitive microsensors has been presented. The output offsets of the capacitive sensing chain due to the parasitic capacitances and process variations were automatically removed using the AOCL. The AOCL cancelled the unwanted offset by binary-search algorithm based on 10-bit SAR logic and charge-domain calibration circuits. The chip was implemented using 0.18 μm 1P6M CMOS process with an active area of 1.76 mm


B cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma (DLBCL) and Burkitt’s lymphoma (BL) (B-UNC/BL/DLBCL) is a new category of tumors that have features resembling both DLBCL and BL. These tumors have large and medium sized cells with greater irregularity of nuclei and more prominent nucleoli than BL. Approximately 35% to 50% have C-MYC rearrangements, although half are non-immunoglobulin variants. We identified six cases of B-UNC/BL/DLBCL with low-level IGH amplification. Four patients died with a median survival of 7 months . In conclusion, to our knowledge low-level IGH amplification has not been previously described and should be evaluated for in this patient population. The 2008 World Health Organization (WHO) Classification of Tumours of Haematopoietic and Lymphoid Tissues introducWe present six cases with this intermediate phenotype due to their atypical features as demonstrated by analysis with immunohistochemistry, conventional cytogenetic analysis and fluorescence in-situ hybridization (FISH). In addition to already well defined cytogenetic abnormalities seen in the intermediate phenotype, such as C-MYC rearrangements and IGH/C-MYC translocations, these cases each demonstrated low level amplification of the immunoglobulin heavy chain (IGH) gene. To the best of our knowledge, IGH amplification has not yet been reported in patients with B-UNC/BL/DLBCL.TM) with the following key words: ‘Burkitt-like lymphoma’, ‘atypical Burkitt’s’, ‘high-grade lymphoma’, ‘grey-zone lymphoma’, and ‘B-cell lymphoma, unclassifiable’. These cases met the criteria for B-UNC/BL/DLBCL as defined by the 2008 edition of the WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues[A retrospective search covering a 10-year period was performed using our database software  tissue sections were stained with routine hematoxylin and eosin (H&E) for morphologic analysis. Additional samples for select cases were submitted for flow cytometric and cytogenetic analysis, including FISH when possible. The morphology was reviewed by a hematopathologist and a diagnosis of B-UNC/BL/DLBCL was made based on morphologic, immunophenotypic, and cytogenetic analysis with clinical correlation.Immunohistochemical stains were performed on paraffin-embedded sections of the lymph node and/or extranodal tumor biopsies using Bond-maX autostainer  according to the manufacturer’s protocol. Mouse anti-human CD3, CD5, CD20, CD79a, BCL2, BCL6, Ki-67, and TdT  were used as the primary antibodies. Horseradish peroxidase-labeled rabbit anti-mouse polyclonal antibodies were employed to convert the chromogen substrate. All stains were performed with appropriate positive and negative controls.TM 2000 four-color flow cytometer according to the standard protocol . Directly conjugated monoclonal antibodies  to the following antigens were used for this analysis: CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD19, CD20, CD23, CD38, CD45, CD79a, BCL2, FMC7, HLA-DR, and terminal deoxynucleotidyl transferase (TdT). Polyclonal and monoclonal rabbit anti-human immunoglobulin light chains were obtained . All antibodies were conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), or PerCP-Cy5.5. The flow cytometric data was analyzed using CellQuest software .Flow cytometric analysis was performed using the FACSCaliburConventional G-banded cytogenetic analysis on 20 metaphase spreads was performed on two cases of tumor tissue and one case of lymph node tissue using methods previously described. The karyotypes were reported according to the 2013 International System for Human Cytogenetic Nomenclature[9/l, ranging from 3.6 to 11.4 × 109/l . None of the patients presented with central nervous system (CNS) involvement. During the course of disease in two patients (cases 5 and 6), intracerebral lesions were found by computerized tomography (CT) scanning and lymphoma cells were detected in cerebrospinal fluid analysis. Bone marrow and peripheral blood involvement were detected in one patient (case 5) during the course of disease. All patients were treated with intensive chemotherapy regimens (IC) supplemented with rituximab. Three patients  underwent autologous hematopoietic stem cell transplant (HSCT) for treatment of relapses. One patient (case 5) underwent external beam radiation therapy of an extranodal tumor. Four patients  died with a median survival of 7 months  after progressing to disseminated disease. Two patients (cases 3 and 4) are alive at 105 and 42 months after diagnosis, respectively, at this time, with the former experiencing multiple relapses.During the 10-year search period, we identified six cases that met the 2008 WHO criteria for B-UNC/BL/DLBCL[Morphologic and immunophenotypic data for all six patients are summarized in Table Cytogenetic data for all six patients are summarized in Table B-UNC/BL/DLBCL represents a heterogeneous group of high grade B-cell lymphomas that cannot be adequately classified as either BL or DLBCL. Cases wThe morphologic characteristics of B-UNC/BL/DLBCL include features of both BL and DLBCL. MonomorTo our knowledge, this study is the first to document the presence of IGH amplification in association with B-UNC/BL/DLBCL. Low-level amplification of other genes such as C-MYC, BCL2, and BCL6, has been described. Foot et al. studied Three cases in this series also showed low-level C-MYC amplification. B-UNC/BL/DLBCL associated with C-MYC amplification but without IGH/C-MYC translocation or any of its variants is associated with a poor prognosis. Low-levAnywhere from 35 to 50% of B-UNC/BL/DLBCL cases have C-MYC translocations and about 15% of these are accompanied by either BCL2 or BCL6 rearrangements. These “double-hit” lymphomas are among the best characterized subset of B-UNC/BL/DLBCL and carry a poor prognosis. Li et aOver half of patients with B-UNC/BL/DLBCL present with widespread, often extra-nodal disease. In contrast with BL, these tumors do not show any preferential localization in particular sites, such as the ileocecal region or jaws. Marrow and CNS involvement are not uncommon. B-UNC/BL/DLBCL follows an aggressive course and no appropriate therapeutic approach has been established. The prognosis in our case series was poor since four of six patients in our series died within six to 20 months of initial diagnosis. In a recent study, clinicaIn conclusion, IGH amplification is a previously undescribed cytogenetic abnormality in patients with B-UNC/BL/DLBCL. The prognosis of B-UNC/BL/DLBCL is known to be poor and this was confirmed in most of our patients. Interestingly, the patient with the longest survival was found to have low-level IGH amplification as its only cytogenetic abnormality. The prognostic significance of low-level amplification of other gene loci such as C-MYC, BCL2, and BCL6 remains uncertain[This study did not meet the definition of research by the Institutional Review Board at Stony Brook University Medical Center.The authors declare that they have no competing interests.MB searched the literature and drafted the manuscript. AZ supplied the cytogenetics data and helped draft the cytogenetics section of manuscript. TA helped draft the manuscript. HL performed the cytogenetic studies. YM supplied the morphologic images and helped draft the manuscript. YH originated the study and helped draft the manuscript. All authors read and approved the final manuscript.


The intricate network of interactions observed in RNA three-dimensional structures is often described in terms of a multitude of geometrical properties, including helical parameters, base pairing/stacking, hydrogen bonding and backbone conformation. We show that a simple molecular representation consisting in one oriented bead per nucleotide can account for the fundamental structural properties of RNA. In this framework, canonical Watson-Crick, non-Watson-Crick base-pairing and base-stacking interactions can be unambiguously identified within a well-defined interaction shell. We validate this representation by performing two independent, complementary tests. First, we use it to construct a sequence-independent, knowledge-based scoring function for RNA structural prediction, which compares favorably to fully atomistic, state-of-the-art techniques. Second, we define a metric to measure deviation between RNA structures that directly reports on the differences in the base–base interaction network. The effectiveness of this metric is tested with respect to the ability to discriminate between structurally and kinetically distant RNA conformations, performing better compared to standard techniques. Taken together, our results suggest that this minimalist, nucleobase-centric representation captures the main interactions that are relevant for describing RNA structure and dynamics. Ribonucleic acid (RNA) plays a fundamental role in a large variety of biological processes, such as enzymatic catalysis , proteinde novo prediction of RNA three-dimensional structure still represents a challenging task  after optimal superposition  is knowna priori known patterns, as is done for instance in annotation methods  a scoring function for RNA structure prediction   in a non-trivial manner ,33. Suchet al. . This feet al. .j, k indexes should be included in the sum since the vectors H. marismortui large ribosomal subunit. With this definition, a lower weight is assigned to structures with pairs of bases in relative positions not compatible with the training set, including those with steric clashes. The probability distribution http://research.microsoft.com/en-us/um/people/minka/papers/minka-summation.pdf). A non-redundant set of high-resolution structures  in all cases, and it diminishes at large time lag. The CV for the bona fide matches or known motif variants, while the residual discrepancies are due to differences in the employed cutoff value (RNA molecules display a great variety of base–base interaction patterns (RNA motifs) that play a key role in RNA folding and that mediate important biological processes . A strucdatabase  release database , the mosentclass1pt{minimaff value . We refeA fully atomistic description of an RNA molecule requires on average ≈20 non-hydrogen atoms per nucleotide, and thus ≈60 degrees of freedom per nucleotide. Biologically relevant RNA structures are composed of hundreds or even thousands of nucleotides that display an intricate network of interactions. The complexity of the problem is further increased when considering that many RNA molecules are highly dynamic entities, and multiple chain configurations are needed for describing their mechanism and function . In ordeAs shown in Figure de novo predictions of RNA structure.A second aspect intimately connected with the choice of molecular representation is the definition of a proper structural proximity measure. Typically, structural deviations are used either to analyse and compare three-dimensional structures . These MSD, INF ) or to rMSD, INF –31. The leobases  and anisleobases . We note measure , that all motifs . Howevery Figure , ii) theentclass1pt{minimaIn this study we introduce a minimalist representation of RNA three-dimensional structures that solely considers the relative orientation and position of nucleobases. Based on this representation, we construct a scoring function for RNA structure prediction and we define a metric for calculating deviation between RNA three-dimensional structures. In both cases, the results are on par or better compared with state-of-the-art techniques. Taken together, the presented results suggest that this minimalist representation captures the main interactions that are relevant for describing RNA structure and dynamics. As a final remark, we note that the methodology presented here can be readily applied to DNA molecules as well.https://github.com/srnas/barnaba.Python scripts to compute Supplementary Data are available at NAR Online.


The results demonstrate that iron(II) supramolecular helicates inhibit Tat-TAR interaction at nanomolar concentrations by binding to TAR RNA. These studies provide a new insight into the biological potential of metallosupramolecular helicates.The interaction between the HIV-1 transactivator protein Tat and TAR (transactivation responsive region) RNA, plays a critical role in HIV-1 transcription. Iron(II) supramolecular helicates were evaluated for their  The trans activation response region (TAR) of RNA  represenTat binds to TAR RNA at the three-nucleotide pyrimidine bulge and interacts with two base pairs above and below the bulge in the major groove of the TAR RNA molecule2467892L3]Cl4  metallosupramolecular triple helicates [FeM- and P-enantiomers of [Fe2L3]Cl4 with the TAR RNA by using thermal denaturation, electrophoretic mobility shift assay, and RNase A footprinting. In addition, we used the ADP-1 polypeptide  of TAR RNA reflects to some extend the strength of binding. The melting curves of TAR RNA and fully matched RNA duplex (Tm (Tm of the helicate-RNA complex - Tm of the free RNA) values are presented in The ability of A duplex  in the aM- and P-[Fe2L3]Cl4 increased the Tm of the TAR RNA by 12.1 °C and 11.2 °C at 1:1 helicate:RNA ratio, respectively, and by 13.3 °C and 12.9 °C at 2:1 helicate:RNA ratio, respectively. Doubling the helicate:RNA ratio from 1:1 to 2:1 had little effect (<2 °C) on the thermal stability of the TAR RNA, which indicates that there is a single dominant binding site for the helicates on the TAR RNA. The Tm values of the fully matched RNA duplex were affected only negligibly by the presence of the helicates.The melting temperatures of the TAR RNA and fully matched RNA duplex in the absence of the helicates were 67.4 °C and 65.7 °C, respectively. The presence of K) of the helicates to TAR RNA, calculated from ethidium bromide displacement experiments .Binding constants  was used to explore the stability of the helicate:TAR RNA complex. The autoradiogram of the electrophoresis gel run at 5 °C  shows th2L3]4+ appear in the gel.Inspection of the gel in It supports the presence of only one major binding site for the helicates on the TAR RNA. We performed the same experiment under the same conditions using the fully matched RNA duplex but no new bands corresponding to the formation of the helicate:RNA complex were observed in the gel see .M- and P-[Fe2L3]Cl4 on the TAR RNA, we probed the helicate:TAR RNA complexes with RNase A. Since this enzyme prefers cleavage of single-stranded to double-stranded regions of the RNA, it is particularly well suited for investigating drug binding to the single stranded parts, including the bulge and loop regions. The autoradiogram of the RNA cleavage-inhibition patterns for the TAR RNA is shown in 2L3]Cl4. In contrast, the cutting is increased between nucleotides C30 and U31 located in the loop. It is likely that the base protection effects induced by the helicates result from the direct interaction with the helicates, but it cannot be excluded that the protection arises from helicate-induced structural changes. In any case, the results of the RNase A footprinting suggest that the M- and P-[Fe2L3]Cl4 preferentially bind to the TAR RNA bulge or in its close proximity.In order to identify potential binding sites of M- and P-[Fe2L3]Cl4. It can be seen that the ADP-1-TAR RNA interaction is almost completely inhibited in the presence of both enantiomers at the concentration of 16 nM. It might be possible that the helicates disrupt formation of the ADP-1-TAR complex by binding to the peptide rather than to the TAR-RNA. In order to probe this eventuality, we recorded CD spectra of M-[Fe2L3]Cl4 at constant helicate concentration (14 μM) and increasing ADP-1 concentrations . Acrylamide and bis(acrylamide) were from Merck KgaA . RNase A was from Roche .The iron(II) helicates [FeTm) was calculated within the thermal heating program by applying a first derivative calculation. The Tm values could be determined with an accuracy of ±0.5 °C. Each DNA melting experiment was carried out at least three times and the Tm values represent the mean. The concentration of oligoribonucleotides was 3 μM per strand. The buffer conditions were sodium phosphate buffer  and 0.5 mM EDTA.The stability of DNA bulges in the presence of the helicates was monitored by measuring the absorbance at 260 nm  as a function of temperature. The experiment was run simultaneously on six masked 1 cm pathlength microcuvettes of 0.2 mL volume using a Peltier controlled 6-sample cell-changer in a Varian Cary 4000 UV/vis spectrophotometer. Melting temperature (32P]ATP and then it was purified by denaturing polyacrylamide gel electrophoresis (PAGE). The RNA concentration was determined by the UV absorbance at 260 nm. RNA was annealed by heating at 90 °C and slow cooling to room temperature in sterile water. RNA, helicates, and the ADP-1 peptide were mixed in a buffer (5 μL) containing Tris-HCl , KCl (50 mM), DTT (100 mM), and Triton X-100 (0.05%) and incubated for 10 min on ice. The samples were analysed by electrophoresis on 15% polyacrylamide (PAA) gels in 0.5 × TB buffer and electrophoresed at 5 W and 5 °C. Gels were exposed to a phosphor imaging plate and scanned with a FUJIFILM BAS-2500 bio-imaging analyzer.RNA was 5′-end labeled using T4 polynucleotide kinase and [γ-32P]ATP and then purified by denaturing PAGE. TAR RNA was annealed by heating at 90 °C and slow cooling to room temperature in sterile water. 5 μL solutions containing 2 × 10−6 M TAR RNA in 10 mM Tris-HCl (pH 7.4) and various concentrations of the helicates were incubated for 10 min at 25 °C. Cleavage was initiated by the addition of 1 μL of RNase A diluted in the precedent experiment to the concentration (5 × 10−7 unit) that was sufficient to achieve partial cleavage of the TAR RNA. Samples were allowed to react for 5 min at room temperature before quenching with 5 μL of 2× concentrated formamide loading buffer followed by incubation at 90 °C for 3 min. 2 μL of the mixture containing RNA cleavage products were then withdrawn and resolved by PAGE under denaturing conditions (24%/8 M urea PAA gel). Gels were exposed to a phosphor imaging plate and scanned with a FUJIFILM BAS-2500 bio-imaging analyzer.TAR RNA was 5′-end labeled using T4 polynucleotide kinase and [γ-How to cite this article: Malina, J. et al. Iron(II) supramolecular helicates interfere with the HIV-1 Tat-TAR RNA interaction critical for viral replication. Sci. Rep.6, 29674; doi: 10.1038/srep29674 (2016).


Rho is a ring-shaped, ATP-dependent RNA helicase/translocase that dissociates transcriptional complexes in bacteria. How RNA recognition is coupled to ATP hydrolysis and translocation in Rho is unclear. Here, we develop and use a new combinatorial approach, called time-resolved Nucleotide Analog Interference Probing (trNAIP), to unmask RNA molecular determinants of catalytic Rho function. We identify a regulatory step in the translocation cycle involving recruitment of the 2′-hydroxyl group of the incoming 3′-RNA nucleotide by a Rho subunit. We propose that this step arises from the intrinsic weakness of one of the subunit interfaces caused by asymmetric, split-ring arrangement of primary RNA tethers around the Rho hexamer. Translocation is at highest stake every seventh nucleotide when the weak interface engages the incoming 3′-RNA nucleotide or breaks, depending on RNA threading constraints in the Rho pore. This substrate-governed, ‘test to run’ iterative mechanism offers a new perspective on how a ring-translocase may function or be regulated. It also illustrates the interest and versatility of the new trNAIP methodology to unveil the molecular mechanisms of complex RNA-based systems. Many key biological processes, such as replication, transcription or virus packaging, involve the translocation of nucleic acids (NAs) by NTP-dependent molecular motors. These motors often adopt a ring-shaped oligomeric structure encircling the NA substrate . This co-dependent 5′→3′] RNA translocase that contributes to the regulation and protection of bacterial genomes by mediating the dissociation of transcription elongation complexes and/or unwinding of RNA:DNA hybrids (R-loops) ,5. In co′→3′ RNA Figure  as that Figure  or in a Figure ,9. WithiFigure  responsiFigure  and ATP-Figure ,14. ImpoFigure ,14. HoweFigure  as well In the first model, ATP-dependent interconversion of protomers in the ‘trimer of dimers’ mediates movement of two triplets of Lys283 ‘levers’ pulling on RNA within the central channel . DownstrIn the second translocation model, each Rho subunit escorts RNA through the central channel by sequentially adopting one of the six distinct subunit conformations found in the asymmetric ring structure . At any 12 ligand used for crystallization and empty PBS clefts in the structure  and steric constraints at periodic intervals. We propose a new, comprehensive model for Rho translocation that accounts for this and most other information. Overall, our work reconciles conflicting data on Rho mechanisms, reveals principles for the functioning and regulation of a model ring-shaped translocase that are exquisitely linked to its molecular architecture, and illustrates the remarkable potential of the new trNAIP methodology to dissect the mechanisms of dynamic RNA-based systems.To clarify these essential features, we first compared NAIM penalty profiles obtained upon Rho-directed unwinding of RNA:DNA substrates having distinct architectures and compositions. Next, we explored the ‘chemo-kinetic’ framework  of the Rho helicase in depth using a new, time-resolved Nucleotide Analog Interference Probing (trNAIP) assay. Our data show that Rho translocates RNA residues non-uniformly irrespective of the substrate identity and support structural coupling between the PBS and SBS during translocation. In addition, we show that the 2′-hydroxyl-dependent stepping behavior of Rho  is due tEscherichia coli (amino acids numbers refer to this protein throughout the paper) and Poly[rC] fragments (>300 nts) were prepared and purified as described previously (in vitro transcription of the resulting DNA templates with wild-type (WT) or Y639F mutant of T7 RNA polymerase. Conditions for in vitro transcriptions  were adjusted as described previously ). The bipartite and tripartite RNA:DNA substrates were prepared from individual components (one of which contained a 32P-end label) by heat denaturation/renaturation and PAGE purification protocols as described previously  were from Promega and Glen Research, respectively. The 2′-OMe-CTPαS and 2′-OMe-GTPαS analogs were obtained from Biolog-LSI (Germany). The Rho protein from eviously . Double-eviously . Both uneviously ,22,23 toeviously . All RNAeviously ,19. Subs57A oligodesoxyribonucleotide released upon duplex unwinding) were added before further incubating the helicase mixture at 30°C. Four volumes of quench buffer  sodium dodecyl sulphate (SDS), 4% (w/v) Ficoll-400) were added to mixtures at times corresponding to ∼20% of reaction extent (32P-labeled products were separated and purified on 7.5% PAGE gels that contained 0.5% (w/v) SDS. The phosphorothioate linkages in the transcripts were cleaved by treatment with 1 mM iodine for 3 min at 37°C. The resulting cleavage products were resolved by denaturing PAGE (we used 9–20% PAGE gels to resolve different RNA sizes) and quantified with a Typhoon Trio imager and ImageQuant TL v8 software  using the full, undistorted dynamic range of the gel scans . Analysis of NAIM experiments was performed as described previously by converting raw NAIM signals obtained from gel band ratios into normalized λ interference factors  modified substrates were performed as described previously ,22. Brien extent . The 32P factors ,22,23.32P labeled RNA molecules present in B1 fractions were detached from the bead-bound biotinylated DNA strands and recovered in the S2 fractions. Buffer compositions of the S1 and S2 fractions were then equalized in order to avoid reactivity biases during subsequent treatment with iodine. Analog-containing transcripts were then cleaved with iodine and analyzed by denaturing PAGE as described in the preceding section. For each position of analog incorporation (i), a reaction progress curve was deduced from the fraction of RNA:DNA hybrids  unwound at time t defined as: Ft,i = I(S1)i/(I(S1)i + I(S2)i), where I(S1)i and I(S2)i are the intensities of the bands corresponding to analog incorporation at position i in the S1 and S2 fractions, respectively.Preliminary trNAIP experiments indicated that selection of reaction species by PAGE, as in NAIM, was a method bottleneck plagued by poor recovery of low-abundance species (e.g. unwound species at the reaction onset) upon elution from gel bands (data not shown). These issues were addressed by opting for affinity bead partitioning as follows. For each trNAIP experiment, about 0.4 mg (40 μl) of streptavidin-coated magnetic beads  were washed thrice with 80 μl of BW buffer  before incubation with 4 pmoles of biotinylated RNA:DNA substrate in 80 μl of helicase buffer II for 1 h at 18°C. Beads were then separated from supernatant, washed twice with 80 μl of buffer II, and incubated for 10 min at 25°C with 17.8 pmoles of Rho hexamer in 220 μl of buffer III . After elimination of the supernatant containing unbound Rho hexamers, the helicase reaction was initiated by addition of 220 μl of pre-warmed buffer III containing Mg-ATP , poly[rC] (3 μM in rC residues) and non-biotinylated DNA competitor . The reaction mixture was incubated at 25°C and shacked at 300 revolutions per minute in a thermomixer (Eppendorf) to homogenize the bead suspension. Suspension aliquots (20 μl) were removed at various times, mixed with 1 volume of quench buffer , and kept on ice before separation of the bead (B1) and supernatant (S1) fractions . Beads from the B1 fractions were mixed with 40 μl of DB buffer  and incubated for 2 min at 72°C. Supernatant (S2) fractions were then quickly collected on a MagRack  that was pre-heated at 72°C. In this way, ∼100% of the Rut (Rho utilization) site to stabilize the Rho:RNA complex during its formation and conversion into a catalytically active isoform  and minimal 3-piece RNA:DNA substrates  components can activate the RNA-dependent ATP hydrolysis and motor activities of Rho , we compared the patterns of interference effects obtained after Rho-directed unwinding of substrates A, B and C (∼20% reaction extent in each case) that were modified randomly with a 2′-deoxy-NαS analog in their tracking RNA arm. For the three substrates, we observed significant 2′-deoxy-NαS interference effects Figure  and B thDespite the differences mentioned above, positions of step-like increases in cumulative profiles Figure  as well To determine the origin of the 2′-hydroxyl-dependent translocation events, we sought to quantitate the kinetic effects of chemical substitutions of the 2′-OH moiety for each position of the RNA track. Since the individual preparation and testing of tens of chemically modified RNA:DNA substrates by classical methods is impractical, we envisioned a new combinatorial approach, trNAIP, wherein reaction progress curves (named ‘kinetic traces’ hereafter) attributable to single substitutions of the 2′-OH moiety are compiled quickly from a succession of NAIM selections performed at different reaction times.10 tail (substrate CBt) on streptavidin-coated magnetic beads . This indicates that the phosphorothioate tag moieties do not play a major role in the 2′-deoxyNαS modification effects described above, which can thus be essentially ascribed to the absence of the 2′-hydroxyl moieties. Taken together, these observations validate the trNAIP approach and confirm that the 2′-OH moieties of the RNA track are not all considered equal by the translocating Rho helicase (A majority of kinetic traces are undistinguishable (∼13%) or only slightly distinct (∼60%) from the control trace , where Dk is the rate of dissociation of the transient Rho:RNA complex and Uk is the rate of the next unwinding step (Dk or decreasing its Uk (Supplementary Table S1). Such a transient, localized destabilization effect would be consistent with SBS:RNA contacts that are continuously remodeled during translocation and could reflect an elaborate regulatory mechanism whereby Rho checks its substrate lattice before undertaking translocation/unwinding steps (see Discussion). An alternative possibility could be that Rho becomes stalled on its RNA track at the position of 2′-deoxyNαS modification. In this case, both Uk and Dk would be significantly decreased. Although we cannot formally exclude this scenario, we suggest that stabilization of the Rho:substrate complex (lower Dk) upon depletion of a key substrate's 2′-OH group  is unlikely. This suggestion is in line with experiments comparing RNA and DNA substrates which show that 2′-OH groups are not only required for enzymatic activity but also for binding of the substrate to Rho's SBS  is governed by competing dissociation and unwinding pathways and is given by the ratio of ing step . The obsho's SBS ,12.Using NAIM, we observed that the sequence of the RNA:DNA duplex affects the 2′-deoxy penalty landscape of Rho-directed unwinding . ConsistBt substrates containing 2′-OMe-NαS analogs. The 2′-O-methyl group is about twice bulkier than the 2′-hydroxyl moiety (Supplementary Table S2) and can only act as a H-bond acceptor . The 2′-F atom is slightly smaller than the 2′-hydroxyl moiety (Supplementary Table S2) but is a strong H-bond acceptor . This step is a key event initiating the rate-limiting, multistep formation of a catalytically active Rho:RNA complex  bulk . Sequenk Figure  and, to k Figure  structur3 analogs bind per Rho hexamer at saturating nucleotide concentrations  . We propose that, without RNA bridging its PBS subsites, the ‘weak’ interface is even more susceptible to breakage when it goes through the E state. This premise defines a regulatory state in the translocation cycle of Rho every time the ‘weak’ interface goes through the E state  contact with its 2′-OH group . Although other interfaces entering the nucleotide-exchange state E may be susceptible to some extent to the same factors, RNA bridging their PBS clefts should restrict the large structural changes required to inactivate Rho irreversibly  should be strongly sensitive to RNA bulk, thereby explaining sequence   and 2′O-p Figure  interfery Figure . In thisy Figure ,42, thery Figure . All they Figure .Our model is reminiscent of the burst (translocation) and dwell (regulation) operating mechanism proposed recently for the ring-shaped DNA translocase ϕ29 . In bothIn sum, our data provide key insights into the translocation and regulatory mechanisms of Rho, an important specimen of ring-shaped molecular motor. Moreover, the versatility and depth of the trNAIP methodology should make it particularly useful for the mechanistic analysis of other RNA-based systems. The key step in trNAIP is time-dependent affinity bead partitioning of reactive species Figure . This prSupplementary Data are available at NAR Online.


C. elegans has had a major impact on scientific research, led to the rapid development of RNAi tools and has inspired RNA-based therapeutics. Astonishingly, nematodes, planaria and many insects take up double-stranded RNA (dsRNA) from their environment to elicit RNAi; the biological function of this mechanism is unclear. Recently, the E. coli OxyS non-coding RNA was shown to regulate gene expression in C. elegans when E. coli is offered as food. This was surprising given that C. elegans is unlikely to encounter E. coli in nature. To directly test the hypothesis that the E. coli OxyS non-coding RNA triggers the C. elegans RNAi pathway, we sequenced small RNAs from C. elegans after feeding with bacteria. We clearly demonstrate that the OxyS non-coding RNA does not trigger an RNAi response in C. elegans. We conclude that the biology of environmental RNAi remains to be discovered.The discovery of RNA interference (RNAi) in  C. elegans genes can induce RNAi when fed to the animalsC. elegans in particular, should respond to environmental dsRNA, is an exciting question that remains unanswered. We recently discussed some potential roles of environmental RNAi in animal to animal communication The biology underlying RNAi is still only partially understood. Bacteria engineered to express dsRNA homologous to C. elegans is a multi-step process  pathway geneche-2 mRNA regulationsid-1sid-2C. elegans that results in gene expression changes and behavioral responses.E. coli strains to C. elegans either for one or three generations: an OxyS over-expressing strain (AZ121), an OxyS deletion strain (GS035) and the common C. elegans food strain (HB101). We isolated total RNA from young adult stage animals and prepared 5′ independent small RNA libraries which enables the sequencing of both 1° and 2° siRNAsE. coli strain (data not shown). In animals fed with the AZ121 strain, small RNAs mapping to the OxyS RNA show a peak at 21–22 nt both in one or three generation fed animals  or chloramphenicol (25 µg/ml) respectively (OD595 = 0.6–0.8). Cultures were centrifuged and the re-suspended pellets were seeded onto NGM plates. For one generation feeding experiments, animals were placed on the seeded NGM plates and allowed to grow until young adult stage. For three generation feeding experiments, L1 larval stage animals were placed on the seeded NGM plates and allowed to grow for three generations. Total RNA from harvested animals was isolated using the TriSure reagent (Bioline). Small RNA sequencing libraries of C. elegans were prepared using the NEBNext Multiplex Small RNA Library Prep set (NEB) and the small RNA sequencing libraries of E. coli are prepared using the TruSeq Small RNA set (Illumina). Libraries were either sequenced on a MiSeq or a HiSeq 1500 machine (Illumina). Data analysis was performed as previously described


Here we demonstrate that 3-way junctions can also form in trans as a result of the binding of microRNAs in an unconventional manner with mRNA by splinting two non-contiguous regions together. This may be used to reinforce the base of a stem-loop motif being targeted by an RNA-binding protein. Trans interactions between non-coding RNA and mRNA may be used to control the post-transcriptional regulatory code and suggests a possible role for some of the recently described transcripts of unknown function expressed from the human genome.The wide array of vital functions that RNA performs is dependent on its ability to dynamically fold into different structures in response to intracellular and extracellular changes. RNA-binding proteins regulate much of this activity by targeting specific RNA structures or motifs. One of these structures, the 3-way RNA junction, is characteristically found in ribosomal RNA and results from the RNA folding in  RNA performs a broad variety of fundamental cellular functions, many of which depend on its ability to actively fold into numerous conformational structures. These RNA structures can form motifs, which are frequently bound by an assortment of RNA-binding proteins (RBPs). In eukaryotic organisms, this provides a means to couple transcription with various levels of post-transcriptional gene regulation such as splicing, nuclear export, RNA localization, turnover, and translation . Conventional models of miRNA regulation are based on perfect or imperfect linear hybridization to mRNAs, which results in the targeted mRNA being degraded and/or inhibited at the translation level. The former's mechanism is well explained by direct cleavage of the mRNA, however the latter's is not yet perfectly elucidated. It is possible that in searching for a mechanism similar to the originally identified destructive pathway, scope was artificially limited to cases with reduced translation of targeted mRNAs. However, as an mRNA cannot be “slightly cleaved,” it is logical to assume that repressive effects of imperfect miRNA hybridization result from a wholly separate, second process rather than simply an attenuated version of the first. If the second process is more generic, it could conceivably manage multiple downstream responses, perhaps via affecting third party elements such as RBPs. In fact, examples of increased translation of a miRNA targeted mRNA have been reported  as part of a RIP. This has become increasingly true as more comprehensive genomic tools, such as tiling-arrays and next generation sequencing technologies have been used. Although experimentally challenging to definitively prove, at least a portion of this ncRNA appears to be cis within the message  are common subdomains critical to the function of numerous structured RNAs  and form when the RNA folds to produce three separate helices meeting around a central unpaired region  and for general hybridization characteristics .To find potential examples of our model, we used the well-characterized HSL motif and determined the potential for miRNAs to target this structure and form a 3WJ. We first identified the set of HSL containing human histone mRNAs present in the Refseq hg19 annotation by using the PatSearch program , it is yet to be shown that these HSL-sxRNAs naturally form or if they play a functional role in histone metabolism. However, histone metabolism is a very elegant and tightly regulated essential cellular process that has many layers, any of which could use the type of post-transcriptional regulation we have predicted Figure . For exam Figure .We have identified similarly predicted sxRNAs for the iron-response element (IRE), which is the target of the IRE-binding proteins that regulate the post-transcriptional expression of genes involved in iron metabolism. A dramatic example of this is found in the erythroid-specific ALA synthase gene (ALAS2), a critical protein whose misregulated activity is associated with both sideroblastic anaemia  and this may be the single most biologically important attribute of RNA. This property permits RNA to serve both as an intermediate for conveying the genetic code while also creating unique surfaces for performing protein-like enzymatic functions in the cell. This also allows mRNA to simultaneously encode for a protein as well as regulate where, when and how much of the mRNA will be translated into this protein. Accordingly, eukaryotic genes are not exclusively transcriptionally regulated, but are also highly regulated at the post-transcriptional level, typically mediated by RBPs, which have repeatedly been shown to facilitate the expression of different mRNA isoforms, localize specific mRNAs and influence variable mRNA translation, and it is reasonable to assume that at least some will be found to play a regulatory role  manner, by eliminating the RBP binding motif, or a positive (inducer) mode by assisting an RBP in binding. Additionally, the miRNA could even help form the RBP motif by actually comprising a portion of it  An RBP whose binding site is hidden by an alternate structure. In the presence of the associated trans-acting ncRNA, the site is revealed. (B) An RBP that recognizes a double stranded binding site that only forms via interaction with a separate ncRNA. (C) An RBP whose binding site must be presented in a particular orientation, with respect to its flanking sequence, that is facilitated by a 3WJ. (D) An RBP whose binding site is hidden by its sequence hybridizing with an ncRNA. (E) An RBP whose binding site structure is weak and may sample alternate structures over time and an sxRNA stabilizes the active conformation, allowing the protein to bind. This model allows a single mRNA transcript variant to exhibit multiple behaviors amongst cell types via the combinatorial absence and presence (in varying concentrations) of both the relevant RBPs and, more specifically, ncRNAs. It is these latter that may facilitate a distinct regulation for sub-populations of expressed mRNAs, where a much broader group may hold the potential for control by the same RBP.Figure An additional level of sxRNA regulation could occur by indirectly influencing tertiary structure to reveal (or hide) complex regulatory motifs that must be properly oriented in space. Figure A) interact with nascent pre-mRNA to affect alternative splicing, perhaps by affecting splicing factor recognition sites or (as depicted here) by binding to and “joining” exon regions on both sides of a particular splice junction, (B) affect the structural conformation of an miRNA primary transcript, determining its availability for trimming to pre-miRNA and nuclear export, (C) reinforce a stem loop binding motif for a localization RBP, thereby determining mRNA shuttling destination, (D) further lessen translation via iron regulatory protein by stabilizing a 5′ IRE in particular mRNAs, (E) alternatively, enhance mRNA stabilization via iron regulatory protein by stabilizing a 3′ IRE in particular mRNAs (F) activate structure dependent ribozymes, (G) act in a combinatorial fashion with multiple signals in a single mRNA.We have envisioned a number of hypothetical biological effects of sxRNA regulation, which are depicted in Figure trans as opposed to a small molecule or metabolite regulating cis structural changes (Serganov and Nudler, This additional role for miRNA we have suggested could explain a variety of observed downstream effects and underscores the importance of ncRNA as a major post-transcriptional gene regulator. This model could also allow for the regulated masking or revealing of many conformational dependent mRNA regulatory elements beyond RBP binding motifs. By stabilizing or disrupting these regulatory elements, miRNAs, and possibly many other ncRNAs, could perform diverse functions with respect to individual or multiple mRNAs. In some aspects, this model is analogous to the behavior observed for riboswitches but here a miRNA is influencing shape in trans-acting post-transcriptional regulatory network in which various sxRNA arrangements could sequentially influence multiple RBP regulatory elements or vice versa. Analogous to the classical attenuator regulatory sequence found in some prokaryotic operons, structural changes to one or more sxRNA could then result in the conformational alterations of the structure of the mRNA such that a second RBP regulatory element can now be revealed or masked, and this could continue in a manner that would enable the post-transcriptional regulation of multi-functional genes via combinations of regulatory elements embedded in the UTRs or even the coding region of the mRNA. The sxRNA model proposed here resonates well with an earlier prosed idea of ncRNAs acting as intelligent scaffolds for the dynamic regulation of the information landscapes (Laurent et al., Additionally, and perhaps most biologically compelling, is the potential for nature to use sxRNA interactions to form an intricate combinatorial Despite compelling informatic support, the sxRNA model we have proposed will need substantial supporting experimental data before it can be recognized as credible and we encourage interested researchers to participate in this process. We have successfully RIPed informatically predicted sxRNA complexes using a bacteriophage MS2-tagged-RNA construct (Yoon et al., Francis Doyle and Scott A. Tenenbaum contributed equally to this manuscript.Scott A. Tenenbaum and Francis Doyle are inventors on sxRNA intellectual property that is owned by SUNY-Research Foundation and may stand to profit in accord with the SUNY-Research Foundation Patents and Inventions Policy. The SUNY-Research Foundation has entered into a licensing agreement with HocusLocus, LLC to develop and market the sxRNA technology.