For many neurological diseases, the efficacy and outcome of treatment depend on early detection. Diagnosis is currently based on the detection of symptoms and neuroimaging abnormalities, which appear at relatively late stages in the pathogenesis. However, the underlying molecular responses to genetic and environmental insults begin much earlier and non-coding RNA networks are critically involved in these cellular regulatory mechanisms. Profiling RNA expression patterns could thus facilitate presymptomatic disease detection. Obtaining indirect readouts of pathological processes is particularly important for brain disorders because of the lack of direct access to tissue for molecular analyses. Living neurons and other CNS cells secrete microRNA and other small non-coding RNA into the extracellular space packaged in exosomes, microvesicles, or lipoprotein complexes. This discovery, together with the rapidly evolving massive sequencing technologies that allow detection of virtually all RNA species from small amounts of biological material, has allowed significant progress in the use of extracellular RNA as a biomarker for CNS malignancies, neurological, and psychiatric diseases. There is also recent evidence that the interactions between external stimuli and brain pathological processes may be reflected in peripheral tissues, facilitating their use as potential diagnostic markers. In this review, we explore the possibilities and challenges of using microRNA and other small RNAs as a signature for neurodegenerative and other neuropsychatric conditions. Central nervous system disorders encompass a broad spectrum of neurodegenerative, oncological, inflammatory, and developmental conditions. Several mechanisms exist that evolved in order to isolate and protect the CNS from insult; interestingly, these effectively also act as barriers to diagnosis. Surrogate markers of disease are thus critical to facilitate disease detection, stratification of patients into subpopulations, prediction of prognosis, evaluation of response to treatment, and eventually allow better understanding of etiopathology.To be of maximum diagnostic benefit, biomarkers would predict disease early, before the onset of clinical symptoms. Finding and testing such biomarkers would be best achieved by a longitudinal study in a large patient population at risk of developing the disease, a resource-intensive process that requires a long commitment and careful planning. However, the more common cross-sectional association studies are equally valuable in biomarker discovery. Brain imaging techniques and their modifications, as well as genotype studies to identify susceptibility alleles—the latter frequently employed in predicting tumor prognosis—are being used successfully to understand complex neurological conditions. In parallel, as techniques evolve rapidly and new hypotheses emerge, we see novel methods being applied to biomarker discovery. Thus, with the recent rapid acceleration in the field of non-coding RNA research, the potential predictive and diagnostic uses of these molecules have also attracted significant attention. Among non-coding RNA, microRNAs have been most intensely studied and their biology has repeatedly been proven critical for diverse cellular functions. More importantly, recent evidence indicates that miRNAs can be detected in peripheral tissues and can be used to “capture” changes in the cell of origin, including neurons. This has generated substantial interest in the use of small non-coding RNAs, in particular miRNAs, as biomarkers for CNS pathology. One advantage of molecular markers such as small RNAs over imaging technology is that samples can be frozen down for retrospective analysis, which enables larger studies. This manuscript aims to provide an overview of recent advances in the field of miRNA-based biomarker discovery for CNS disease.As RNA is continually transcribed, translated, and turned over in response to physiological and pathological stimuli, the RNA profile of a cell, interpreted appropriately, could serve as a reflection of its current functional state. Current technologies enable transcriptome analysis on an unprecedented scale. In the human CNS we often need to rely on extracranial or peripheral sources of RNA to obtain a live readout of the disease state. The choice of potential sources for representative RNA is wide and includes body fluids such as blood, plasma, or cerebrospinal fluid as well as non-neuronal tissue or cells such as lymphocytes Figure . The queThe use of genetic material from blood cells to screen for biomarkers of neurological conditions has been used as early as 1975  could be as low as 46% . Techniques previously used to probe the cellular small RNAome are diverse and each of them has unique advantages and disadvantages to it, mainly associated with (1) whether detection is done in solid state or in solution and (2) whether or not previous knowledge of the target molecules is required [reviewed in Cissell and Deo , and statistical comparison of conditions of interests , typically used to detect a feature pattern without prior knowledge about sample grouping. In situations where the RNA profiles of the groups under comparison exhibit a high level of dissimilarity, they cluster into distinct groups by an unsupervised clustering algorithm. Alternatively, a “modified unsupervised clustering” where clustering is performed after feature selection may also be used  to a single individual. Therefore, predictions of sensitivity, specificity, and accuracy are often more useful than estimates of significant differences between patient and control groups.Blood cells, plasma, and CSF have all been used as starting material to develop miRNA biomarkers for CNS malignancies as well as neurodegenerative and other neurological diseases. One of the first studies to compare miRNA profiles from blood mononuclear cells between patient and control populations showed mir-34a and mir 181b to be upregulated in mononuclear cells from the blood of patients with Alzheimer's disease. In addition, gender and APOE4 status were also found to influence the PBMC miRNA profiles within the group of AD patients , and data from proteomics and other high throughput approaches. Proteomics-based biomarkers for neurodegenerative and other neurological diseases have been studied and new avenues for biomarker discovery such as metabolomics continue to emerge; an LC/MS based approach , as well as those specifically associated with sequencing-based detection methods and extraction strategies.As is often the case when working with human tissue, samples from different sources show wide variability in profile as a result of handling, sample preparation and preservation. These are especially pronounced when a highly sensitive technique like sequencing is used. In addition, because the source of tissue are primarily human patients that may be on medication, proper consideration of these  cofactors is essential, as medication pursues restoration of the biological balance and this may include alterations in the molecule of interest. When RNA profiles are altered after drug treatment, it can be a challenge to dissect the direct effects of treatment on RNA expression from those connected with disease remission , often studies strive to uncover molecules that can serve as a biomarker and be used as therapeutic targets. Evidence from PBMCs indicates that there is indeed a considerable coherence between the central neuronal response and the peripheral response in blood and that there is a cross-talk between these two tissues. It remains to be experimentally established whether this correlation can also serve to better understand neuronal physiology in the healthy and the disease situation. In this respect, the development of novel, unbiased technologies to detect even the smallest amounts of miRNAs peripherally in combination with studies in model systems has proven critical.Finally, as already mentioned, there is the issue of how faithful the peripheral profile is to the original biological situation in the CNS. Although this is not most critical for biomarker discovery All in all, despite current limitations, miRNA-based biomarkers constitute an exciting field in biomedical research. For neuroscience, where the search for remotely accessible markers to understand the brain is essential for human studies, the field has elicited considerable interest and as the costs of NGS continue to decrease, it is likely to become a routine approach to generate individual patient profiles and allow targeted therapeutic intervention.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Streptomyces coelicolor A3(2). Peptide nucleic acid and expressed antisense RNA silencers successfully inhibited actinorhodin production. Synthetic RNA silencing was target-specific and is a new tool for gene regulation and metabolic engineering studies in Streptomyces.We demonstrate the first application of synthetic RNA gene silencers in  These RNAs include riboswitches  resulted in decreased growth, reduced protein production and synthesis of the red-pigmented antibiotic, undecylprodigiosin. A second example of a cis-encoded sRNA α-abeA was identified in S. coelicolor as part of a four-gene cluster involved in the enhanced production of the blue-pigmented antibiotic, actinorhodin, although the exact role of α-abeA has yet to be elucidated Trans-encoded sRNAs are analogous to eukaryotic miRNAs, and, in many cases, require an RNA chaperone to mediate regulation In vitro and in silico evidence of a trans-encoded antisense sRNA micX, a putative activator of actinorhodin biosynthesis, has been reported in vivo evidence for a S. coelicolor trans-encoded sRNA (scr5239) acting as a repressor of extracellular agarase expression cis- and trans-encoded sRNAs are thought to regulate gene expression as antisense RNA silencing molecules (asRNAs) that bind to their mRNA target, inhibit ribosome access and/or trigger mRNA degradation The genus Streptomyces species use sRNAs to regulate gene expression; as such it is attractive to consider ways to exploit these molecules in practical applications. The use of conditional antisense RNA silencing may be of use not only in the elucidation of secondary metabolite regulation, but also in studies where gene knockouts are unsuitable e.g. when monitoring the affect of transcript abundance on gene expression; determining the minimally required levels of expression of essential genes trans-acting RNAs that are not naturally expressed by the host, has yet to be demonstrated in the genus Streptomyces. Oligo-nucleobase antisense RNA silencers, such as peptide nucleic acid (PNA) and phosphorodiamidate morpholino oligomers (PMO), are DNA mimics that maintain naturally occurring DNA bases but have non-natural linkages between bases. PNAs and PMOs, conjugated to peptide carrier molecules, have been used to silence gene expression in a number of Gram-positive and Gram-negative species E. coli, most likely through the stabilization, and thereby increased abundance, of the asRNA The studies cited above demonstrate that Streptomyces. Both peptide-PNA conjugants and paired-termini antisense RNA were used to silence the expression of a gene required for actinorhodin biosynthesis. The development of antisense RNA gene silencers for Streptomyces species provides complementary tools to conventional genetics for the elucidation of regulatory pathways and gene function and will be a valuable tool in metabolic engineering.In this study, we demonstrate, for the first time, the successful application of synthetic RNA silencing in actI-ORF1). Two peptide-PNAs (Sc001 and Sc002) were designed and applied as antisense agents targeting the translation initiation region of actI-ORF1  S. coelicolor MT1110 and actinorhodin production was monitored in a number of media. For liquid and agar R5 media, both S. coelicolor MT1110/pAS01 and pAS02 showed visible reduction of actinorhodin production when induced with thiostrepton and ε–caprolactam, respectively (DNA sequences (120–160 bp) covering the 5′UTR, RBS and >50 bp of the coding region of ectively . In liquactI-ORF1, growth and antibiotic production were monitored for MT1110/pAS01 and control cultures in R5 liquid medium . This genus is responsible for the production of a large number of high value compounds and the development of techniques for improving titre through metabolic engineering are industrially significant micX asRNA with that of the synthetic asRNA designed for this study revealed possible areas for the future development of asRNAs for use in Streptomyces: The micX asRNA has a predicted paired-end structure that contains regions of non-complementary nucleotides  and thus the design of synthetic asRNAs based on naturally occurring trans-encoded asRNAs may yield more effective gene silencers. It is also likely that a better understanding of the mechanism of antisense RNA gene silencing in Streptomyces will lead to improvements in asRNA design. For example, in many species, limited complementarity between natural trans-encoded asRNAs and the corresponding mRNA requires the RNA chaperone Hfq to facilitate RNA-RNA interactions S. coelicolorStreptomyces. Long stretches of base pairing, or high concentrations of the asRNA, as in the case of the synthetic asRNAs used in this study, may obviate a chaperone requirement trans-encoded sRNA repressor of extracellular agarase expression, has five stem-loop structures that presumably require facilitated opening in order for annealing to occur, leading the authors to speculate that Streptomyces species may require proteins to carry out the functions of Hfq Streptomyces could aid in the development of synthetic asRNAs since synthetic RNAs could then be designed to mimic naturally occurring trans-acting sRNAs. The use of peptide-PNAs in Streptomyces is of particular interest as genetic transformation is not required for gene silencing, thus they can be used in species in which genetic tools have not been established or to simply accelerate the process of genetic analysis. The current synthesis costs and high concentrations required when using these molecules in agar-based assays makes optimization studies prohibitive. However, improvements in micro-culture of StreptomycesStreptomyces use endogenous transcripts to regulate gene expression, and here we show for the first time that synthetic strategies using expressed RNA or a delivered DNA mimic can provide useful levels of RNA silencing. As a method, RNA silencing can be used to improve our understanding of Streptomyces biology and possibly alter metabolic flux in industrial applications.In this study we have demonstrated that synthetic asRNA can be designed and used to regulate gene expression in leotides ; in E. c430).The strains and plasmids used in this study are listed in 6 colony forming units [CFU]) of the experimental and control strains were inoculated into 20 ml of R5 liquid medium containing the appropriate antibiotics to an OD450 of ≈ 0.05. Cultures were grown for 18 h before 1 ml was transferred to 50 ml of R5 liquid medium containing appropriate antibiotics and inducers (for induction samples). Actinorhodin and undecylprodigiosin samples from three biological replicates were quantified according to standard protocols Bacillus mycoides ATCC 6462 as an indicator strain Spores  via a flexible ethylene glycol linker S. coelicolor MT1110 spore suspension (1.5×108 CFU/mL) were used to prepare a confluent lawn of growth (24 h incubation at 28°C) on MPCA agar plates . Peptide-PNAs were applied directly to the lawn at concentrations of 1, 10 and 50 µM and incubation continued at 28°C for 72 hrs. To determine whether inhibition of actinorhodin production was due to RNA silencing of actI-ORF1 or to non-specific toxicity of the PNAs, subcultures from the zones of application were inoculated onto MPCA agar plates. ISP-4 agar  that supports both undecylprodigiosin and actinorhodin production in S. coelicolor MT1110 was used to evaluated the specificity of gene silencing: agar plates were overlaid with 0.5% ISP-4 agar containing 2.3×106 CFU/mL S. coelicolor MT1110 spores, 10 µL of PNAs (50 µM) were applied to antibiotic assay discs (Whatman) and placed onto the soft agar. After 96 hours of incubation, the disks were removed and the agar plates were photographed.Sc001 and Sc002, antisense PNAs specific for actI-ORF1 gene) was amplified from S. coelicolor MT1110 gDNA using primers modified to include paired-termini sequences SphI and PstI restriction sites for convenient excision and replacement of the antisense RNA expressing fragment of the vectors.A 155 bp fragment  using a modified Kirby mix, phenol/chloroform extraction and DNAse I treatment RNA was extracted from actI-ORF1 mRNA. SCO4742 a hypothetical gene that shows little variation in expression 10 dilution factors against the Ct values. The efficiency was corrected based on multiple samples RT-PCR was performed using the Promega Access RT-PCR system with 400 ng RNA per sample. The RT-PCR conditions were as follows: for cDNA synthesis, 45°C for 45 min, followed by 94°C for 2 min. For PCR amplification, 20 cycles at 94°C for 30 s, 55°C for 1 min, and 68°C for 2 min and 1 cycle at 68°C for 10 min. Two primer pairs (actIORF1258f/actIORF1497r and actIORf1860f/actIORF11148r) were used to amplify distinct regions of the Figure S1Schematic representation of the cloning strategy used to construct antisense RNA expressing vector pAS02.(TIFF)Click here for additional data file.Figure S2Calcium-dependent antibiotic assay. The assay was performed as previously reported B. mycoides as the indicator strain (A) wild-type S. coelicolor MT1110. (B) MT1110/pAS01 induced by adding thiostrepton to the agar. Plates were photographed and half images from each strain were aligned to compare the size of the zone of inhibition.(PNG)Click here for additional data file.Table S1Off-target matches in S. coelicolor for PNAs used in this study.(DOCX)Click here for additional data file.


Drosophila antiviral RNAi response to four different viruses using high-throughput sequencing. We found that each virus was uniquely targeted by the RNAi pathway; dicing substrates included dsRNA replication intermediates and intramolecular RNA stem loops. For instance, a putative intergenic RNA hairpin encoded by Rift Valley Fever virus generates abundant small RNAs in both Drosophila and mosquito cells, while repetitive sequences within the genomic termini of Vaccinia virus, which give rise to abundant small RNAs in Drosophila, were found to be transcribed in both insect and mammalian cells. Moreover, we provide evidence that the RNA species targeted by Dicer-2 can be modulated by the presence of a viral suppressor of RNAi. This study uncovered several novel, heavily targeted features within viral genomes, offering insight into viral replication, viral immune evasion strategies, and the mechanism of antiviral RNAi.RNA silencing pathways play critical roles in gene regulation, virus infection, and transposon control. RNA interference (RNAi) is mediated by small interfering RNAs (siRNAs), which are liberated from double-stranded (ds)RNA precursors by Dicer and guide the RNA-induced silencing complex (RISC) to targets. Although principles governing small RNA sorting into RISC have been uncovered, the spectrum of RNA species that can be targeted by Dicer proteins, particularly the viral RNAs present during an infection, are poorly understood. Dicer-2 potently restricts viral infection in insects by generating virus-derived siRNAs from viral RNA. To better characterize the substrates of Dicer-2, we examined the virus-derived siRNAs produced during the  Drosophila melanogaster have been extensively characterized and allow for detailed dissection of the mechanisms governing RNAi-mediated gene silencing. The siRNA silencing pathway is responsible for controlling the expression of both endogenous and exogenous targets in somatic cells. siRNA biogenesis is carried out by Dicer-2 (Dcr-2), which generates predominantly 21 nucleotide (nt) products from several types of precursors RNA silencing pathways are critical regulators of gene expression in organisms ranging from yeast to humans. RNA silencing is initiated by the action of RNase III enzymes, which target RNAs with dsRNA character to generate small RNA duplexes Drosophila, siRNAs are preferentially loaded into Argonaute 2 (Ago2), while miRNAs predominantly bind Argonaute 1 (Ago1) Drosophila, including the thermodynamic characteristics of the small RNA duplex, specific terminal nucleotide sequences, and the extent of central mismatches or pairing within the duplex In C. elegans utilize the activity of RNA silencing pathways to degrade viral RNAs and thus limit virus infection arthropod-borne viruses), and studies of natural mosquito vectors have demonstrated that the RNAi pathway is a critical component of the insect antiviral response toward these RNA viruses Drosophila has provided an excellent model for the study of antiviral RNAi, due in part to the fact that the organism is readily infected by a diverse panel of viruses and that its small RNA silencing pathways are well-characterized Many invertebrates such as plants, insects and Drosophila siRNA pathway is both antiviral and exquisitely poised to recognize dsRNA, the prevailing view is that viral dsRNAs generated during replication of RNA viruses are the target of the antiviral silencing system Drosophila cells. We sequenced the small RNAs generated from three RNA viruses with different coding strategies, and one DNA virus. The viruses include the rhabdovirus Vesicular Stomatitis virus (VSV), the human arbovirus Rift Valley Fever virus (RVFV), the natural Drosophila picorna-like virus Drosophila C virus (DCV), and the dsDNA poxvirus Vaccinia virus (VACV). By studying the antiviral targeting of this diverse set of viruses, we found common features of viral RNA that are preferentially targeted by the cellular machinery, along with novel, heavily targeted viral RNA structures that have not previously been characterized in either RNA or DNA viruses.Since the Drosophila, and serves as a model for medically important pathogens such as measles, rabies, and Ebola virus Drosophila cells. We found that the majority of VSV vsiRNAs are 21 nt in length  coding sequence, but the L mRNA is over 6 kb in length , which cla cells , S3. TheBunyaviridae family, one of the main classes of medically relevant arboviruses. RVFV carries a ∼12 kb trisegmented negative sense genome comprised of the large (L) medium (M) and small (S) segments  of total RVFV vsiRNAs mapped to a particular region in the S segment antigenome . Specifila cells . To deteed cells . Althouged cells . Thus, iDrosophila pathogen that has co-evolved with the host, we hypothesized that the antiviral pathway may be uniquely poised to restrict DCV replication. Indeed, RNAi pathway components are potently antiviral against DCV Drosophila cells revealed that vsiRNAs are predominantly 21 nt in length  and thus might be unusually sensitive to cytoplasmic small RNA silencing pathways n length . The 21 n length . We alsoWe reasoned that VACV vsiRNAs may be generated through two mechanisms: bidirectional transcription at a particular locus could yield dsRNA that is processed into vsiRNAs, or structured regions within single transcripts could be targeted by the RNA silencing machinery or other cellular nucleases. Again, we depleted the enzymes involved in RNA silencing and confirmed functional depletion by small northern blotting . We founAs noted above, a significant fraction of the VACV vsiRNAs map to the two genomic termini . Upon knin silico results suggested that transcription of the VACV tandem repeats gives rise to long, structured RNAs that serve as siRNA and miRNA pathway substrates. Indeed, we found that this region is expressed in Drosophila and mammalian cells , genomic RNAs (DCV), and unique, heavily targeted predicted hairpins . Moreover, the distribution pattern of vsiRNAs produced from viral dsRNA can mirror the distribution observed from a synthetic dsRNA bearing the same sequence . Thus, tDrosophila cells and adult flies, supporting the notion that the observations made in cell culture reflect the function of the RNAi pathway in vivo.These findings complement and expand upon previous studies examining arbovirus-derived vsiRNAs. Deep sequencing of vsiRNAs from various mosquito species and mosquito-derived cell lines has revealed that vsiRNAs produced from Sindbis virus, Semliki Forest virus, and Dengue virus type 2 are present in approximately equal ratios (∼1.5∶1 genome:antigenome) and are distributed along the length of the genome To our knowledge, this study is the first to investigate the production of vsiRNAs from a DNA virus. Many mammalian DNA viruses, most notably Herpesvirus family members, co-opt host RNA silencing machinery in order to express virus-encoded miRNAs, which are transcribed as stem loop precursors and require processing by the host miRNA biogenesis pathway Another important facet of the antiviral RNAi response is the ability of the virus to overcome the restrictive pressure placed on it by the pathway. Thus, many pathogenic plant- and insect-specific viruses encode suppressors of silencing AGO2-deficient flies, creating a favorable condition for DI particle formation, a preference for 5′ end targeting was observed Although many insect viruses encode protein suppressors of RNA silencing, such as DCV-1A, our identification of unique, heavily targeted RNAs such as the VSV DI particles  and the Drosophila DL1 cells were grown and maintained as previously described Aedes aegypti Aag2 cells Aedes albopictus C6/36 cells were obtained from ATCC, grown and maintained at 25°C in L-15 medium (Gibco). Mouse embryonic fibroblasts (MEFs) RNAi was performed as previously described Total RNA was extracted and analyzed by traditional northern blotting, small RNA northern blotting and RT-PCR as previously described 40 µg of total RNA was separated on a 15% TBE-urea gel and small RNAs ∼15–29 nt were excised and eluted. Following ethanol precipitation, small RNA-seq libraries were produced using the Small RNA Sample Prep v1.5 kit  as per manufacturer’s instructions.All raw small RNA sequence data was deposited in Gene Expression Omnibus (GEO) under the accession GSE43031. This includes 24 total sequence datasets that include control as well as Dcr-1, Dcr-2, Drosha, Ago1, and Ago2 knockdowns for all four viruses.Detailed descriptions of small RNA mapping procedures and vsiRNA profile generation are found in Supporting Information. The genomic location of individual vsiRNAs or siRNAs relative to viral genomes or synthetic dsRNAs are found in Figure S1Related to,,, and.Distribution of non-21 nt vsiRNAs across viral genomes. (A) Distribution of non-21 nt (between 15 and 29 nt) VSV-derived vsiRNAs (control LacZ-depleted library) across the viral genome. vsiRNAs mapping to genomic strand are depicted in blue, antigenomic strand in red. (B) Distribution of non-21 nt (between 15 and 29 nt) DCV-derived vsiRNAs (control LacZ-depleted library) across the viral genome. vsiRNAs mapping to genomic strand are depicted in blue, antigenomic strand in red. (C) Distribution of non-21 nt (between 15 and 29 nt) VACV-derived vsiRNAs (control GFP-depleted library) across the viral genome. vsiRNAs mapping to the (+) strand are depicted in blue, (−) strand in red. (D) Distribution of non-21 nt (between 15 and 29 nt) RVFV-derived vsiRNAs (control LacZ-depleted library) across the viral genome. vsiRNAs mapping to genomic strand are depicted in blue, antigenomic strand in red.(TIF)Click here for additional data file.Figure S2Related to , , , and . miRNA and siRNA pathway components are depleted by RNAi. (A) Robust loss-of-function phenotypes were verified by small RNA northern blotting of RNA samples used to create small RNA libraries. Loss of miRNA pathway components (left panel) affected steady-state levels of bantam miRNA (mature and pre-miRNA forms), while loss of siRNA pathway components resulted in a depletion of the endogenous siRNA esi-2.1. Equal loading was verified by probing 2S rRNA. (B) OligodT-primed cDNA from cells depleted of the indicated RNA silencing factor was subjected to PCR and quantified relative to Rp49 levels. Data is expressed as the normalized mRNA expression in the depleted cells compared to the control cells bathed in a non-targeting dsRNA (LacZ). A representative experiment is shown. Error bars indicate standard deviation.(TIF)Click here for additional data file.Figure S3Related to. Detection of VSV mRNA and putative DI hairpin RNAs. Northern blot from (TIF)Click here for additional data file.Figure S4Related to .VACV terminal repeat-derived siRNA biogenesis and Argonaute stabilization. (A) vsiRNAs produced from a “hot spot” region may be generated due to bidirectional transcription, which generates complementary RNAs with the potential to base pair and form a dsRNA target of the RNAi machinery. A VACV vsiRNA “hot spot” region is highlighted in orange, and the two VACV genes that could potentially produce overlapping, bidirectional transcripts are diagrammed in green. (B) The most abundant repeat-derived vsiRNAs of each size class are diagrammed in reference to the terminal repeat sequence, shown in green. Their relative abundance is calculated as a percentage of all repeat-derived vsiRNAs with >10 reads cloned in the control library. (C) Quantification of the abundant vsiRNAs described in (A) upon Dcr-1 and Dcr-2 depletion. (D) Quantification of the abundant vsiRNAs described in (A) upon Ago1 and Ago2 depletion.(TIF)Click here for additional data file.Table S1Related to . Start location, end location, size, copy number, mismatches and strand for VSV vsiRNA libraries.(XLSB)Click here for additional data file.Table S2Related to . Start location, end location, size, copy number, mismatches and strand for RVFV vsiRNA libraries.(XLSB)Click here for additional data file.Table S3Related to . Start location, end location, size, copy number, mismatches and strand for Control, Dcr-2, and Ago2 DCV vsiRNA libraries.(XLSB)Click here for additional data file.Table S4Related to . Start location, end location, size, copy number, mismatches and strand for Drosha, Dcr-1 and Ago1 DCV vsiRNA libraries.(XLSB)Click here for additional data file.Table S5Related to . Start location, end location, size, copy number, mismatches and strand for VACV vsiRNA libraries.(XLSB)Click here for additional data file.Text S1Supporting experimental procedures.(DOCX)Click here for additional data file.


PVX CP is capable offorming viral ribonucleoprotein complexes (vRNP) not only with homologous, butalso with foreign RNAs. This paper presents the structure and properties of vRNPassembledin vitroupon incubation of PVX CP and RNAs ofvarious plant and animal viruses belonging to different taxonomic groups. Wehave shown that the morphology and translational properties of vRNPs containingforeign (heterologous) RNA are identical to those of homological vRNP (PVX RNA– PVX CP). Our data suggest that the assembly of the“mixed” vRNPin vitrocould be started at the5’-proximal region of the RNA, producing a helical structure of vRNPswith foreign nucleic acids. The formation of heterologous vRNPinvitrowith PVX CP appears not to require a specific 5’ endRNA nucleotide sequence, and the PVX CP seems to be able to pack foreign geneticmaterial of various sizes and compositions into artificial virus-like particles.Potato virus X (PVX) and some other potexviruses can be reconstituted As a result, the structureand biological activity of the virus can be reconstituted. The self-assembly(repolymerization) of a low-molecular-weight coat protein can occur in the absenceof RNA, yielding particles with a structure identical to that of viruses, butpossessing an unlimited length [The protein capsid of a number of phytoviruses consists of identical coat proteinsubunits folded on the basis of helical symmetry. Genomic viral RNA is helicallyarranged between the turns of coat protein subunits and follows their folding. Thepossibility of reversible dissociation of virions into coat proteins and RNAfollowed by the d length .The self-assembly procedure enables one to obtain a “mixed” vRNPconsisting of the viral coat protein and a heterologous RNA , 5. The Another advantage of vRNP is the possibility of controllable activation of thetranslation of the RNA encapsulated in the coat protein. Viruses and a“mixed” vRNP can change their structure under the effect of anumber of factors .The spiral viruses of plants, which are rather highly stable at high temperatures,non-physiological pH values of the environment and in the presence of hydrolyticenzymes, are preferable for the construction of vRNP. Moreover, the length of aspiral virus depends on the size of the nucleic acid; as opposed to isometricviruses, hence, vRNP formed in vitro can include RNA of unlimited length. Lookingahead, it is reasonable to assume that the spiral phytoviruses that are modified andreconstructed in a “mixed” manner can serve as containers tostore and deliver “therapeutic” genes and pharmaceutical agentsinto cells .Potexvirus genus of the family Flexiviridae . PVX virions are flexible thread-like bodies 515 nm longand 13.5 nm in diameter. A viral particle contains approximately 1350 helicallyfolded identical coat protein (CP) subunits and a viral RNA enclosed between thehelix turns [Potato virus X (PVX) is one of the phytoviruses with a spiral structure, a typicalrepresentative of ix turns . A turn ix turns . The genix turns . The RNAix turns . in vitro  not only with the homologous RNA, but with certainheterologous RNAs as well [It has been shown that the coat protein of potexviruses is capable of formingvRNP as well , 10.in vitro  by the incubation of PVX CP with the RNA of anumber of plant and animal viruses belonging to various taxonometric groups. TheRNAs of potexviruses ,tobamovirus (TMV – tobacco mosaic virus), bromovirus (BMV –brome mosaic virus), and Mengo picornavirus  were used as heterologousRNAs.The present study was aimed at investigating the structure and the properties of vRNPobtained Purification of PVX specimens, coat protein, MP1 and PVX RNA Daturastramonium L.  according to the procedure described by Atabekovet al . [The PVX specimen (Russian strain) was extracted from infected plants et al . . PVX CP et al . . RNAwaset al . with modet al . .In vitro obtainment of vRNPIn order to obtain vRNP, the RNA and the coat protein were mixed at a RNA : CP weightratio of 1 : 10. The incubation was carried out under the standard conditions : in 20 µIn vitro translation 35S-methionine for 60 min at 25°С. The amount of RNA in the sample was40 µg/µl . Recombinant MP1 for the translationalactivation of RNA within vRNP was added at a PVX: MP1 molar ratio = 1 : 100; i.e.,1.4 µg of MP1 per 1 µg of RNA (20 µg of the virus).RNA translation was carried out in a cell-free protein-synthesis system consisting ofa wheat germ extract according to the procedure described earlier , in the Transmission electron microscopyThe specimens (15 µl) were sorbed onto copper grids for electron microscopy, coatedwith formvar film (a 0.5% formvar solution in dichloroethane was used for filmcoating) for 15–20 s. Then, the specimens on the grids were contrastedwith a 2% uranyl acetate solution and viewed on a JEOL JEM-1011 electron microscope at 80 kV. The images were obtained using a Gatan ErlangshenES500W digital camera and Gatan Digital Micrograph™software.Atomic force microscopy (AFM) The scanning was performed on the Nanoscope 3a  and SmartSPM  microscopes in air in the resonancemode. The typical scan rate was 1 Hz. Cantilevers fpN01S with a resonance frequencyof 118–190 kHz, rigidity of 5.3 N/m, and guaranteed needle bending radiusof 10 nm  wereused. FemtoScan Online software  was usedto process and visualize AFM images. For sample preparation, 5–10 µl ofthe specimen at the required concentration was applied to freshly cleaved mica orhighly oriented pyrolytic graphite for 5–10 min. Then, the sample waswashed twice in a drop of distilled water and air-dried.in vitro  assembly of PVX CP andthe RNAs of viruses belonging to various taxonomic groups. The RNAs of the followingviruses were used as heterologous RNA: four potexviruses , tobamovirus (TMV), bromovirus , and Mengo picornavirus . The homologous RNA of PVXwas used as a control.A series of vRNP were obtained by  )can be observed in a transmission electron microscope (TEM) after incubation of PVXCP with heterologous RNAs of various viruses at a RNA : CP ratio = 1 : 10 (w/w) . We have already demonstrated thathomologous vRNP “PVX RNA – PVX CP” are structurallyidentical to native PVX virions [The PVX CP is known not to form virus-like aggregates in the absence of RNA . Particl virions . ) are identical to those of homologous vRNP . The AFMdata showed that the average height of a homologous and heterologous complex was10.0 ± 0.6 and 9.9 ± 0.9 nm, respectively . These values coincide with each other within thelimits of error and correspond to the height of the native PVX (data not shown). Asmentioned above, the diameter of the PVX virion is 13.5 nm [The vRNP morphology was analyzed using high-resolution AFM imaging. The vRNPparticles containing homologous and heterologous RNAs were studied using thistechnique. The images of vRNP obtained upon the incubation of PVX CP withheterologous RNAs (  13.5 nm .However 13.5 nm .TEM and AFM were used previously to detect single-tailed particles (STPs) with the3’ terminus of PVX RNA that was vacant of CP, and rod-like heads thatresulted from the helical folding of the coat protein on the 5’ terminalRNA fragment .Figs. 2A, B, F ). It should be notedthat not all short vRNPs have unbound RNA tails . This may result from the hydrolysis ofthe 3’ RNA terminus not bound to CP due to the action of the ribonucleasesin the solution or upon loading the particle suspension on the mica surface prior tothe analysis.The 1 : 10 (w/w) RNA : coat protein ratio in the incubation mixture upon vRNPassembly ensures the absence of excessive unbound CP on the specimen surface. On theother hand, this amount of CP is insufficient for encapsulation of the entire RNA.As a result, AFM detects the particles in which a part of the RNA molecule withinvRNP remains unbound to CP . Particles containing a completely encapsidated RNA were notfound even when analyzing the homologous vRNP . The most completely reconstructed PVXvRNPs reached just 300 nm, whereas the modal length of the native virions is 515 nm.The decrease in vRNP length seems to be the result of the deficiency of the coatprotein in the incubation mixture (the RNA : CP ratio was 1 : 10 instead of the 1 :20 ratio that is used to reconstruct the full-size PVX particles).The self-assembly of RNA with the viral CP leads to the formation of a set of vRNPsheterogeneous in length . The size of the RNA of these viruses is comparable with that of PVX RNA. Whenusing shorter viral RNAs , the RNA : CP molar ratio decreased while thenumber of short particles (80–100 nm) increased . On the other hand, uponincubation of Mengo virus RNA (8400 n) with the PVX CP, the molar ratio increasedand the average size of the particles increased to 400–450 nm .The increase in the amount of protein within the incubation mixture  results in an increase in the length of “mixed”(heterologous) particles. Thus, at a RNA : CP ratio = 1 : 10, heterologous particlesformed after protein “coating” of the RNA of potexviruses NMV,PAMV, and tobamovirus TMV were characterized by an average length of 200 nm similarto that of PVX vRNP  is inaccessible fortranslation as opposed to TMV and a number of other viruses. However, RNAtranslation is activated upon phosphorylation of PVX CP or upon formation of thevirion or vRNP with PVX MP1 , 15, 19.In the present work, we studied the translation properties and specificity of thetranslation activation of “mixed” vRNPs usingMP1. ).The background translation level observed  may result from the presence of unbound RNAbecause of CP deficiency upon incubation [It was demonstrated in the control experiments that interaction between PVX RNA andPVX CP results in inhibition of RNA translation within vRNP compared to that ofunbound RNA ( cubation . The amocubation .).On the other hand, the interaction between MP1 and vRNP consisting of homologous coatproteins and RNA results in efficient translation activation of the encapsidated PVXRNA . The addition of PVX CP to theRNA at the 10 : 1 (w/w) ratio leads to a noticeable translation suppression of BMVRNA , PAMVRNA , NMV RNA, TMV RNA , andMengo virus RNA  within vRNP as compared with the same amount of unbound RNA. Almost completetranslation suppression of encapsidated RNA can be achieved by increasing the amountof CP to the RNA : CP ratio = 1 : 30.  ), NMV RNA , and TMV RNA . The addition of the MP1 to the“mixed” vRNP results in translation activation , the translational efficiency recovering to thelevel of unbound RNA . The set ofpeptides that are formed upon RNA translation within MP1-activated vRNP is identicalto the products of translation of unbound RNA as follows from It is worth mentioning that similar results were obtained by analyzing thetranslational activation of the heterologous RNAs within the vRNPs reconstructedfrom PVX CP  have beenidentified in the RNA molecules of a number of plant viruses  22–. Inpartet al . [in vitro the origin of assembly of PVX within the5’ terminus fragment of PVX RNA (51–84 nt) forming a stem-loopstructure (SL1). Moreover, the regulatory elements that are required for the bindingof RNA to the coat protein are shown to be located in the fragment1–107 nt of PVX RNA [in vitro assembly ofheterologous vRNP also starts at the 5’ terminus RNA fragment and proceedsin the 5’–3’ direction. This conclusion is appropriatefor potexvirus RNAs . However, the signal for specific assemby of TMV islocated in the 3’ terminus region; the tRNA-like 3’ terminalstructure and elements of the polymerase gene play a role in assembly initiation inBMV. Mengo virus RNA  with a length of 8400 nt contains thevirus-specific protein VPg [Kwon et al .  were abl PVX RNA . The dattein VPg  at its5tein VPg . It is ntein VPg ). The asIt can be assumed under the conditions of our experiment that PVX CP recognizes not aspecific nucleotide sequence, but a certain structure of the 5’ terminusfragment of the RNA, which initiates the formation of vRNP.in vitro has been shown to result in the formation of the vRNPshaving morphology and translational properties similar to those of homologous vRNPs.It can be assumed that a protein coat will be formed upon interaction of aheterologous RNA and PVX CP similar to the coat of homologous particles in terms ofits structure. The in vitro formation of heterologous vRNPs withthe participation of PVX CP seems to be initiated at the 5’ terminus of anRNA molecule and to be independent of the specific nucleotide sequence of the5’ terminus RNA fragment. As a result, PVX CP is capable of packagingforeign genetic material of different sizes into an artificial virus-like particle.Neither the heterologous nor homologous RNA within vRNP is accessible for ribosomes.However, it becomes translationally active upon incubation of the resulting vRNPswith PVX MP1. Binding of MP1 to one of the termini of the PVX virion inducesconformation changes in the terminal subunits of the coat protein, which results inthe destabilization (remodelling) and transition of the protein helix into ametastable state. The following MP1-dependent translational disassembly of PVXparticles occurs rapidly and, most likely, in cooperation with the release ofunbound RNA and protein subunits at early stages of the translation [The incubation of various foreign heterologous RNAs with PVX CPnslation . It is qin vivo disassembly under the control of variousfactors. Coat proteins of plant viruses with a helical structure could be used to design anddeliver into target organs artificial “hybrid” nanoparticles(vRNP) capable of


Pinus taeda L.), contain high concentrations of phenolic compounds and polysaccharides that interfere with RNA purification.  Isolation of high-quality RNA from these species requires rigorous tissue collection procedures in the field and the employment of an RNA isolation protocol comprised of multiple organic extraction steps in order to isolate RNA of sufficient quality for microarray and other genomic analyses.  The isolation of high-quality RNA from field-collected loblolly pine samples can be challenging, but several modifications to standard tissue and RNA isolation procedures greatly improve results.  The extent of general RNA degradation increases if samples are not properly collected and transported from the field, especially during large-scale harvests.  Total RNA yields can be increased significantly by pulverizing samples in a liquid nitrogen freezer mill prior to RNA isolation, especially when samples come from woody tissues.  This is primarily due to the presence of oxidizing agents, such as phenolic compounds, and polysaccharides that are both present at high levels in extracts from the woody tissues of most conifer species.  If not removed, these contaminants can carry over leading to problems, such as RNA degradation, that result in low yields and a poor quality RNA sample.  Carryover of phenolic compounds, as well as polysaccharides, can also reduce or even completely eliminate the activity of reverse transcriptase or other polymerases commonly used for cDNA synthesis.  In particular, RNA destined to be used as template for double-stranded cDNA synthesis in the generation of cDNA libraries, single-stranded cDNA synthesis for PCR or qPCR's, or for the synthesis of microarray target materials must be of the highest quality if researchers expect to obtain optimal results.  RNA isolation techniques commonly employed for many other plant species are often insufficient in their ability to remove these contaminants from conifer samples and thus do not yield total RNA samples suitable for downstream manipulations.  In this video we demonstrate methods for field collection of conifer tissues, beginning with the felling of a forty year-old tree, to the harvesting of phloem, secondary xylem, and reaction wood xylem.  We also demonstrate an RNA isolation protocol that has consistently yielded high-quality RNA for subsequent enzymatic manipulations.Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine ( After the tree is selected and felled, it is important to work as carefully and as quickly as possible.  As each different tissue type is harvested, they should be placed immediately into their own liquid nitrogen vessels to avoid any cross contamination of sample material.  This RNA isolation protocol is scalable.                  Cut bolts into lengths of approximately 0.5-0.75 m and with a chainsaw score the bark in two or three places following the longitudinal axis of the bolt.Using a wood chisel, work the edge of the scored region until the bark begins to separate from the wood and continue working the chisel while pulling back on the section of bark until it separates completely from the stem.Secondary xylem is harvested with a straight edge, single sided razor by scraping along the longitudinal axis of the bolt that has been stripped of bark. Wear latex gloves to minimize contamination of the tissue.Phloem, which is located on the inner side of the bark sections, is readily harvested by manual peeling.Reaction wood is collected from the underside of limbs after marking them with spray paint to indicate their original orientation with respect the ground.Limbs are cut into bolts of 1-2 m and scored on opposite sides along the longitudinal axis according to the approximate locations of the two types of reaction wood: opposite wood (on the top side of the limb) and compression wood (on the bottom side of the limb).  The bark is separated from the limb as described above, except that only the bark covering each type of reaction wood is removed at one time.  Secondary xylem or phloem is then collected as described above and placed in liquid nitrogen.Other samples, such as needles, shoot tips and young cones, can be harvested by hand or using pruning shears as required.Collected samples frozen in liquid nitrogen should be placed in marked bags or suitable containers and packed in dry ice for shipment back to the laboratory.Fill the Spex 6850 freezer mill reservoir with liquid nitrogen and place the impact bar into one of the grinding vials and fill with liquid nitrogen to pre-chill.  Pour off liquid nitrogen and add sample to vial.Make sure that no excess liquid nitrogen remains in the milling chamber and that excess nitrogen gas is completely vented before the end cap is seated to seal the chamber. Insert the grinding vial into the carrier and close.Mill woody samples for two cycles of 1 minute/cycle at a rate setting of 14.Use a liquid nitrogen chilled spatula to remove the milled sample (wood flour) into a beaker that has been pre-chilled and contains a small amount of liquid nitrogen.Pour the liquid nitrogen/sample slurry into a storage container and place at -80° C until needed.  Place 20mL of RNA Isolation Buffer into a 50mL capped Falcon tube and add 400 μL β-mercaptoethanol, vortex briefly, then heat in a 60° C water bath.  Add 4 g of frozen tissue to each tube. Cap and shake vigorously by hand followed by vortexing for 10 seconds. Add an equal volume of chloroform to the tube and invert gently to mix. Place the tube on a rotating wheel and allow end-over-end mixing for 5 minutes. Decant the mixture into a 50 mL Oak Ridge tube and centrifuge at 12,000  rpm in a SS34 fixed-angle rotor for 5 minutes. Transfer the aqueous phase to a fresh Oak Ridge tube and add a half-volume of chloroform.  Vortex for 1 minute and continue mixing on a rotating wheel for 5 minutes. Centrifuge at 12,000rpm  in a SS34 fixed angle rotor for 5 minutes. Transfer the aqueous phase to fresh Oak Ridge tube and add a half-volume of chloroform. Vortex for 1 minute. Centrifuge at 12,000 rpm  in a SS34 fixed angle rotor for 10 minutes. Transfer the aqueous phase to a 50 mL high speed polypropylene tube. Centrifuge at 10,000 rpm  in a SS34 fixed angle rotor for 20 minutes. Decant supernatant into 50 mL Falcon tube and add a quarter-volume 10 M LiCl solution to supernatant. Briefly vortex and place at 4° C for overnight precipitation. Heat SSTE buffer in a 65° C water bath prior to starting. Pour the LiCl-precipitated sample into a polypropylene tube and pellet the RNA by centrifugation at 11,000rpm in a SS34 fixed angle rotor  for 30 minutes at 4° C. After centrifugation, pour off and discard the liquid, and invert the tubes on Kimwipes for approximately 1 minute.  Pipette off any remaining supernatant.  Resuspend each pellet in 800 μL of heated SSTE using a pipette to thoroughly mix the samples. Transfer the resuspended sample to 2 mL Ambion RNAse-free microfuge tubes. Heat samples at 65° C for 2 minutes followed by vigorous vortexing to ensure complete resuspension. Add an equal volume of phenol-chloroform, pH8, to each sample tube. Vortex each sample for 30 seconds and then spin in a microfuge at 14,000 rpm for 3 minutes Transfer the aqueous phase to a new 2mL Ambion RNAse-free microfuge tubes, and add an equal volume of chloroform.  Vortex each sample for 30 seconds and spin in the microfuge at 14,000rpm for 3 minutes. Transfer aqueous phase to Phase-Lock Gel tubes that have been previously prepared by microfuging at 11,000 rpm for 2 minutes. Add a half-volume of chloroform and gently pipette to mix.  Centrifuge at 11,000 rpm for 5 minutes and transfer the aqueous phase to fresh 2mL Ambion RNAse-free microfuge tubes. Add 50 μL 3.0 M sodium acetate, pH 4.8, and 1 mL 100% ethanol to the aqueous phase. Vortex and precipitate in -80° C for 30 minutes or overnight at -20° C. Microfuge samples at 14,000 rpm for 30 minutes at 4° C, and carefully aspirate off the supernatant. Do not disturb the pellet. Wash the RNA pellets with 1 mL -20° C 70% (v/v) ethanol.  Microfuge at 14,000 rpm for 1 minute and aspirate off the ethanol.  Repeat step 5.3. Uncap the tubes and air-dry pellets on the bench for 3-5 minutes. Resuspend each pellet in 100 μL DEPC-treated water. Mix by vortexing and incubate the tubes at 65° C for 2 minutes before placing on ice.  Repeat if necessary to ensure sample resuspension. Pool like samples if desired and make 50 μL of a 1:10 dilution in 10mM Tris, pH 7.5, for spectrophotometric quantitation.  Absorbance ratios for 260/280 and 260/230 are typically greater than 2.1 and 2.2, respectively.  RNA isolation yields range from 60-120μg/g frozen tissue. Analyze samples by running 1.5-2.5 μg RNA on a 1% agarose gel in TAE buffer.  For more critical samples, analyze the RNA using an Agilent 2100 Bioanalyzer as per the manufacturer's instructions. RNA isolation yields from different conifer tissues range from 60-120 μg/g frozen tissue with woody samples typically yielding around 70-80g/g frozen tissue.  Diagnostic ratios of 260/280 and 260/230 are both typically greater than 2.1, and are good indicators that the RNA sample is free of any significant phenolic or carbohydrate contamination, respectively.  Samples analyzed by agarose gel electrophoresis followed by EtBr staining should show three distinct bands for 25S, 18S, and 5S RNA .  Determining the stoichiometry of the 28S to 18S on agarose gels is somewhat subjective, and factors such as running conditions for the gel, staining, and sample load can all have an effect.  In general, the 28S:18S ratio will appear to be > 1.5.  However, some conifer samples have lower ratios.  For example, following Agilent 2100 analysis we have seen several instances of conifer samples from different tissue types where the stoichiometry is closer to 1.2:1.  Thus, an apparent low 28S:18S stoichiometry by agarose gel electrophoresis does not necessarily indicate a poor-quality sample. Leaf, shoot, and cone samples will often have additional distinct bands present due to chloroplastic ribosomal RNAs.  EtBr-stained materials that do not migrate from the sample well or high molecular mass bands (>8-10 kb) usually indicate genomic DNA contamination .  Low molecular mass smears in the vicinity of or below the 5S band and reduced ribosomal band staining or an apparent 18S > 28S stoichiometry is a good indicator that RNA degradation has occurred .  In the Agilent 2100 electropherogram analysis, the base line should be low and relatively smooth with major peaks corresponding to 18S and 28S ribosomal RNAs having 28S:18S ratios ranging anywhere from 1.2 to 2.0.  The Agilent RNA Integrity Number (RIN) value may be a better predictor of RNA quality and should be at least 7 or greater for RNA that is to be used for the preparation of microarray targets.  Figure 4 shows a typical Agilent 2100 electropherogram for xylem RNA with a 28S:18S ratio equal to 1.4 and a RIN value of 8.6 while Figure 5 shows a poor xylem RNA prep having a very high baseline and noticeable degradation as revealed by the 28S:18S ratio equal to 0.65 and a RIN value of 3.4.          Figure 1.  Agarose gel analysis of high-quality pine RNA. Lane 1, NEB 1 kb standard, Lane 2 shows a typical total RNA isolation from loblolly pine secondary xylem with characteristic ribosomal 28S, 18S, and 5S bands and apparent 28S:18S ratio >1.                    Figure 2.Agarose gel analysis of pine RNA sample contaminated with genomic DNA. Lane 1, NEB 1 kb standard, Lane 2, xylem RNA sample showing genomic DNA contamination.                    Figure 3.Agarose gel analysis of pine sample showing RNA degradation and contamination with genomic DNA. Lane 1, NEB 1 kb standard, Lane2, xylem RNA sample showing genomic DNA contamination and severe RNA degradation.                    Figure 4.Agilent 2100 electropherogram of xylem total RNA isolated using the described method on loblolly pine xylem.  28S:18S ratio equals 1.4, RIN value equals 8.6                     Figure 5.Agilent 2100 electropherogram of apical tip total RNA showing severe degradation and genomic contamination.   28S:18S ratio equals 0.65, RIN value equals 3.4           Obtaining high-quality RNA from conifer species can be a difficult task given the high levels of phenolic and polysaccharide compounds found in woody tissues.  Starting with the protocol developed by Chang et al. (1), we have found that more rigorous extraction and clean up steps lead to the consistent isolation of very high-quality total RNA from various woody and non-woody tissues sampled from a wide variety of conifer species.  This video has demonstrated not only our modified RNA isolation protocol, but also the steps taken in field collection and processing techniques used for loblolly pine prior to RNA isolation.  These harvesting and preparation steps are equally important to minimize RNA degradation in field-collected samples, as well as to increase both RNA quality and total yield.  Most significantly, we have found that RNA prepared using this method has led to increased cDNA synthesis yields compared to other methods used to prepare microarray targets for hybridization against the PtGen2 loblolly pine microarray (2).


RNA-dependent processes are essential to determine when, where, and how much of a protein is synthesized. In eukaryotes, these processes start with transcription in the nucleus and end with mRNA translation at distinct cytoplasmic sites followed by mRNA degradation Ustilago maydisDue to the central importance of RNA biology in regulating protein synthesis, it is not surprising that fungal pathogens intensely rely on RNA-dependent processes to control infection. Here, we focus on RNA biology in fungal plant pathogenesis, which is best studied in the corn smut U. maydis, a major pathogenicity determinant is the homeodomain transcription factor bW/bE that triggers a cascade of transcriptional responses essential for full virulence clp1 is a direct target gene of bW/bE, which is induced early during formation of infectious filaments. However, the Clp1 protein is only synthesized later at the specific stage of plant penetration when it acts as an antagonist of bW/bE clp1 mRNA by a currently unknown mechanism  protein RBP35 causes defects in virulence and development. Protein interaction studies revealed that RBP35 functions as a novel auxiliary protein of the polyadenylation machinery of plant pathogens  is an important mechanism to control endogenous and foreign RNA in most organisms. Noteworthily, genome comparison with U. maydis the ELAV -type protein Rrm4 functions in long-distance mRNA transport, a process that is specifically important during formation of infectious unipolar filaments In higher eukaryotes, active microtubule-dependent transport of mRNAs by molecular motors functions in spatial protein expression during developmental and neuronal processes ubi1 and rho3, encoding ubiquitin fused to ribosomal protein Rpl40 and the small GTPase Rho3, respectively rrm4 deletion strains are disturbed in septum formation. Hence, microtubule-dependent transport of rho3 mRNA might act in correct septal localization of Rho3, which could promote septum insertion in infectious filaments Rrm4 transports specific mRNAs such as C. albicansIn summary, endosomal mRNA transport along microtubules is a novel aspect of RNA biology and this posttranscriptional process is particularly important during early infection. Since the RNA-binding protein She3 functions in actin-dependent mRNA transport during infection of U. maydis, which is usually localized in the cytoplasm. However, 10% of its mRNA is alternatively spliced, generating an enzyme with a C-terminal peroxisomal targeting sequence (PTS1 type). This results in peroxisomal localization of GAPDH, a process termed cryptic peroxisomal targeting  of argeting  [21]. Siargeting . The appThe importance of RNA biology in fungal infection is further underlined by the fact that posttranscriptional regulation is targeted during host defense. Plants react to infection by producing antimicrobial peptides (AMPs) that are proposed to degrade the fungal cell wall or to permeabilize the membrane. Some AMPs also enter the cell to exert their function U. maydis secretes ribonucleases that might function as virulence factors in degrading host RNAs Pseudomonas syringae that is essential for virulence on Arabidopsis thalianaIntriguingly, the opposite is true as well: pathogens interfere with posttranscriptional control of the host to promote infection. Although this has so far only been shown for bacterial and oomycete phytopathogens vice versa pathogen effectors suppress host RNA silencing during plant protection.Moreover, microbial effectors interfere with plant microRNA activity. miR393, for instance, targets the F-box auxin receptor TIR1 and thereby represses auxin signaling, a process involved in plant immunity The different levels of RNA biology such as alternative splicing, 3′ end processing, endosomal mRNA transport, and translational regulation are essential to orchestrate plant infection of fungal pathogens. RNA-binding proteins are key components in executing these functions. After improving transcriptome-wide approaches such as iCLIP


Caenorhabditis elegans genome contains 21 T-box genes, and their characterized functions include cell fate specification in a variety of tissues. The C. elegans Tbx1 sub-family member MLS-1 functions during larval development to specify the fate of non-striated uterine muscles; and, in mls-1 mutants, uterine muscles are transformed to a vulval muscle fate. Here we demonstrate that MLS-1 function depends on binding to the Groucho-family co-repressor UNC-37. MLS-1 interacts with UNC-37 via a conserved eh1 motif, and the MLS-1 eh1 motif is necessary for MLS-1 to specify uterine muscle fate. Moreover, unc-37 loss-of-function produces uterine muscle to vulval muscle fate transformation similar to those observed in mls-1 mutants. Based on these results, we conclude that MLS-1 specifies uterine muscle fate by repressing target gene expression, and this function depends on interaction with UNC-37. Moreover, we suggest that MLS-1 shares a common mechanism for transcriptional repression with related T-box factors in other animal phyla.T-box proteins are conserved transcription factors that play crucial roles in development of all metazoans; and, in humans, mutations affecting T-box genes are associated with a variety of congenital diseases and cancers. Despite the importance of this transcription factor family, very little is known regarding how T-box factors regulate gene expression. The  C. elegans to explore the mechanism(s) of the muscle-specifying T-box factor MLS-1. We show that MLS-1 physically interacts with another conserved protein called Groucho to repress gene expression and that this interaction is necessary for normal MLS-1 function. C. elegans MLS-1 is highly related to T-box factors in humans and other organisms, and we suggest Groucho interaction may be a common mechanism for T-box factor function. These insights may lead to therapies that modulate the activity of T-box factors in disease.T-box proteins are a conserved family of transcription factors that play important roles in development of all animals. A variety of human congenital diseases and cancers have been associated with mutations that either decrease or increase T-box factor activity. By learning more about how T-box transcription factors function, we can better understand and treat these disorders. Here we use genetic and molecular techniques in the roundworm  T-box transcription factors play essential roles in the development of all multicellular organisms, where their functions include the specification of primary germ layers and the specification of cell fates during organogenesis Caenorhabditis elegans T-box factors MLS-1 and MAB-9 in vivo.Groucho family (Gro/TLE) proteins are conserved transcriptional co-repressors that interact with distinct Engrailed homology 1 (eh1) or WRPW/Y motifs in a wide variety of transcription factors and, in many cases, recruit histone deacetylases to target gene promoters . Thal cells , and traFSI→ASA::vn173mls-1 and unc-37::vc155 would present a BiFC signal. After a one hour heat shock, transgenic animals co-expressing these proteins displayed no BiFC signal, whereas animals co-expressing wild-type mls-1::vn173 with unc-37::vc155 exhibited abundant BiFC signal  mutants in five independent lines, but at reduced percentages (FSI→ASAmls-1 transgene did not rescue mls-1(cc569) in seven of seven lines  on egl-15::gfp animals. These RNAi affected animals displayed egl-15::gfp expression in the four vm1 cells but also in up to four additional cells (unc-37(RNAi) experiments on animals with an integrated rgs-2::gfp transgene. In wild-type adults rgs-2::gfp is expressed in the pharynx, the ventral nerve cord, lumbar ganglia, and uterine muscles unc-37(RNAi) greatly reduced the number of and intensity of rgs-2::gfp expressing uterine muscles, while having no effect on rgs-2::gfp expression in other tissues  animals we examined had no rgs-2::gfp expressing uterine muscles.Because the MLS-1 eh1 motif mediates interaction with UNC-37 and is necessary for rescue of al cells , Table 2 tissues , Table 2egl-15::gfp expression in two unc-37 mutants: unc-37(e262) and unc-37(wd17wd21). unc-37(e262) is a hypomorphic allele that can be maintained as a homozygous strain, and it encodes a missense mutation affecting a conserved residue in the WD repeat domain that specifically affects UNC-37 function in VA motor neurons unc-37(wd17wd21) is a putative null allele containing a splice acceptor site mutation upstream of the WD repeats unc-37(wd17wd21) mutants exhibit a maternal effect embryonic lethal phenotype, but unc-37(wd17wd21) homozygotes produced from heterozygous mutant hermaphrodites grow to adulthood and exhibit uncoordinated and protruding vulva phenotypes. Most unc-37(e262) mutants exhibited a wild-type pattern of egl-15::gfp expression, but approximately 5% of these animals expressed egl-15::gfp in extra cells. A much higher percentage of unc-37(wd17wd21) adult animals  contained supernumerary egl-15::gfp expressing vulval muscles. In both cases GFP-expressing cells were near the vulva similar to mls-1(cc569) animals (egl-15::gfp expressing cells has previously been observed in mls-2 mutants unc-37(e262) may affect the activity of both MLS-1 and MLS-2 in specifying cell fates in the M lineage.We next examined  animals , but in  animals . A similC. elegans depends on this interaction. These results indicate that MLS-1 specifies uterine muscle fate at least in part by repressing target gene transcription.MLS-1 is a selector gene that is necessary and sufficient to specify uterine muscle development in the M lineage mls-1 loss-of-function results in a transformation of uterine muscle to vulval muscle, whereas ectopic mls-1 expression results in production of ectopic uterine muscles mls-1 in the M lineage can convert many M lineage cells to a uterine muscle fate egl-15 promoter is active in the supernumerary vulval muscles in mls-1 mutants, and we asked if it might be directly repressed by MLS-1. However mutation of predicted T-box binding sites in this promoter did not lead to expanded expression egl-15::gfp reporter , suggesting MLS-1 regulation of the egl-15 promoter is indirect.What types of genes does MLS-1 regulate? We suggest that MLS-1 represses expression of regulatory genes that themselves encode inhibitors of uterine muscle development and activators of vulval muscle development. This model is consistent with previous observations that mls-1 in these cell types is unknown MLS-1 may also function with UNC-37/Groucho in other MLS-1 expressing cells. When expressed with its own promoter, MLS-1 interacts with UNC-37 in BiFC assays in the vm2 vulval muscles and one to two intestinal/enteric muscles. The function of unc-37(RNAi) and some mutant alleles produce a protruding vulva (Pvl) phenotype that is not observed in mls-1 mutants egl-15::gfp expressing cells that looked like body wall muscles in unc-37(e262) and more frequently in unc-37(wd17wd21) mutants, while we never observed this phenotype in mls-1 mutants. At least two other transcription factors expressed in the M lineage contain high scoring eh1 motifs  I; ayIs2 [egl-15::gfp] IVlin-15(n765ts); vsIs5 unc-37(e262) Iunc-37(wd17wd21)/dpy-14(e188) Iunc-37(e262) I; ayIs2 [egl-15::gfp] IV, and OK0787 unc-37(wd17wd21)/dpy-14(e188) I; ayIs2 [egl-15::gfp] IV. The following transgenic strains were constructed for this work.mls-1(+) genomic fragment  or FSI→ASAmls-1 (pOK257.02) at 2.5 ng/µl and pRF4 at 100 ng/µl mls-1(cc569); ayIs2; cuEx575[mls-1(+)], OK0721 mls-1(cc569); ayIs2; cuEx576[mls-1(+)], OK0722 mls-1(cc569); ayIs2; cuEx577[mls-1(+)], OK0723 mls-1(cc569); ayIs2; cuEx578[mls-1(+)], OK0724 mls-1(cc569); ayIs2; cuEx579[mls-1(+)], OK0725 mls-1(cc569); ayIs2; cuEx580[mls-1(+)], OK0683 FSI→ASA]mls-1(cc569); ayIs2; cuEx568mls-1(cc569); ayIs2; cuEx596mls-1(cc569); ayIs2; cuEx597mls-1(cc569); ayIs2; cuEx598mls-1(cc569); ayIs2; cuEx599mls-1(cc569); ayIs2; cuEx600mls-1(cc569); ayIs2; cuEx601, OK0689 cuEx573[mls-1::vn173+unc-37::vc155], OK0690 cuEx574[mls-1::vn173+unc-37::vc155], OK0691 cuEx575[mls-1::vn173+unc-37::vc155], OK0709 FSI→ASA::VN173+unc-37::vc155]cuEx567cuEx568, OK0743 cuEx594[genomic mls-1::VN173+genomic unc-37::VC155], OK0744 cuEx595[genomic mls-1::VN173+genomic unc-37::VC155].Transgenic lines were constructed by injecting N2 with pRF4 at 100 ng/µl and the BiFC plasmids pCE-BiFC-VN173, pCE-BiFC-VC155, pOK257.05 (mls-1 (Open Biosystems) and unc-37  cDNA were cloned into pCE-BiFC-VN173 and pCE-BiFC-VC155 plasmids  to generate pOK257.05 (mls-1::vn173) and pOK257.06 (unc-37::vc155). FSI→ASAmls-1 cDNA was generated by site directed mutagenesis of mls-1 cDNA , and cloned into pCE-BiFC-VN173 producing pOK258.06 (FSI→ASA::vn173mls-1). Transgenic adults were picked to OP50 seeded plates, heat shocked for one hour at 33°C as described in Results, allowed to recover for one hour at 20°C, and examined for BiFC signal at 63× magnification.mls-1::vn173 and unc-37::vc155 constructs, with gene expression under the control of their respective endogenous promoters, were produced by PCR of promoter+gene fragments using pSAK244.13 and N2 genomic DNA templates, respectively. hsp-16.41 promoter fragments were removed from pCE-BiFC-VN173 and pCE-BiFC-VC155 and replaced with these mls-1 and unc-37 promoter+gene cassettes generating pOK263.03 and pOK266.02. Transgenic animals from various stages (L1 to Adult) were examined at 63× for BiFC signal.Genomic mls-1 bait plasmid (pOK248.03) was constructed by inserting the full-length mls-1 cDNA in to pLexA-NLS, and the unc-37 prey plasmid was previously isolated from the pACT-RB1 cDNA library (provided by R. Barstead).Yeast two-hybrid assays were performed as previously described mls-1(cc569); ayIs2 strains with mls-1(+) or FSI→ASAmls-1 extrachromosomal arrays were generated as above. Adult transgenic animals from these lines and animals from NH2447 and PD4285 were examined and scored at 40× and 63× magnification for the number of cells expressing egl-15::gfp.unc-37 cDNA was cloned into the L4440 (double T7 promoter) vector and the resulting plasmid (pOK247.03) was used to transform HT115(DE3) E. coli cells. unc-37 “feeding” RNAi was performed as before with some modifications ayIs2 [egl-15::gfp] IV (NH2447) or lin-15(n765ts) X; vsIs5 [rgs-2::gfp] (LX354)  adults and embryos were bleached and embryos were transferred to unseeded NGM plates and placed at 25°C overnight to allow embryos to hatch and synchronize as L1s. The next day synchronized L1s were transferred to unc-37(RNAi) seeded plates and placed at 20°C for 48 hours to 72 hours. Adult animals were examined and scored at 40× and 63× magnification for egl-15::gfp or rgs-2::gfp (mis)expression. Note that it is only possible to qualitatively evaluate rgs-2::gfp expression due to the unusual morphology of uterine muscles.Figure S1pmls-1::Venus expression in enteric and sex muscles of L4s and adults. A) L4 animal showing expression of pmls-1::Venus in left intestinal (Int) and anal depressor (AnDep) muscles. B) L4 animal (top) showing expression of pmls-1::Venus in vulval muscles (VM). A young adult (bottom) displays pmls-1::Venus expression in expanded uterine muscles. The pmls-1::Venus plasmid contains bp 1–1308 of cosmid H14A12 (Accession AF025459) cloned into pPD95.79-Venus (kindly provided by D. Byrd and J. Kimble).(TIF)Click here for additional data file.Figure S2egl-15::gfp in body wall muscle cells of unc-37(e262) animals. Image of two unc-37(e262); ayIs2[egl-15::gfp] adults shown at 40× and 63× magnification. Arrowheads point to supernumerary vulval muscles. Arrows point to cells with body wall muscle morphology that ectopically express egl-15::gfp.Ectopic expression of (TIF)Click here for additional data file.


The proteins LMO4 and DEAF1 contribute to the proliferation of mammary epithelial cells. During breast cancer LMO4 is upregulated, affecting its interaction with other protein partners. This may set cells on a path to tumour formation. LMO4 and DEAF1 interact, but it is unknown how they cooperate to regulate cell proliferation. In this study, we identify a specific LMO4-binding domain in DEAF1. This domain contains an unstructured region that directly contacts LMO4, and a coiled coil that contains the DEAF1 nuclear export signal (NES). The coiled coil region can form tetramers and has the typical properties of a coiled coil domain. Using a simple cell-based assay, we show that LMO4 modulates the activity of the DEAF NES, causing nuclear accumulation of a construct containing the LMO4-interaction region of DEAF1. Both proteins are widely expressed but show high levels of expression in epithelial cells and the central nervous system e.g., , and mice.g., ex vivo, increases mammary cell populations in a transgenic mouse model, and promotes cell invasion and tumour formation in human cell lines e.g., LMO4 is present in all human breast tumour subtypes, with >50% of primary tumours showing increased levels of expression e.g., , with a 404–438) and (2) the majority of a coiled coil domain (DEAF1454–479) encompassing the nuclear export signal (NES) of DEAF1. Further, we show that LMO4 can regulate the subcellular localisation of a DEAF1 construct incorporating the new LMO4-binding region. Together these results support the idea that high levels of LMO4 in the nucleus, which is a hallmark of sporadic breast cancers, may upset the delicate balance between interactions with partner proteins such as DEAF1.LMO4 and DEAF1 are co-expressed in breast tissue and were shown to interact in mammalian two-hybrid assays 334–545) could bind LMO4 His3, Ade2 and Mel1 reporter genes are activated by a positive interaction. Although MYND domains are considered to be protein-binding motifs 404–479, which includes 50 residues from the C-terminal end of the predicted unstructured region and the majority of the putative coiled coil.DEAF1 encodes a 566 residue (∼60 kDa) protein containing DNA-targetting SAND (Sp100/AIRE1/NucP41/75/DEAF1) domain and Helix-Loop-Helix (HLH) motifs at the N-terminus, a protein-binding MYND (Myeloid translocation protein 8/Nervy/DEAF1) domain at the C-terminus, a nuclear localisation sequence (NLS) and an NES . Previou454–479) might stabilise a shorter region near the N-terminus of DEAF1404–479 for interaction with LMO4 we generated an additional set of internal deletions using the DEAF1404–479 template (439–456) could be deleted without apparently affecting the interaction in the yeast two-hybrid assay. Fourteen of these residues were from the unstructured domain, and three from the putative coiled coil. Thus, within the contiguous DEAF1404–479 region, we identified DEAF1404–438/457–479 as the new binding motif for LMO4.Several partners of LMO and related LIM-homeodomain proteins interact with LIM domains through intrinsically unstructured domains of ∼30 residues template . A 17-re404–479), and a chimera, in which the DEAF1 coiled coil was replaced by the leucine zipper domain from GCN4, using yeast two-hybrid analysis (454–487) (data not shown), nor isolated the GCN4 leucine zipper 404–479, which resulted in yeast growth under both moderate and high stringency conditions. The ability of the chimera to promote yeast growth under any condition suggested to us that the coiled coil was stabilising the rest of the construct rather than making specific interactions with LMO4, and that reduction in yeast growth was a result of less effective stabilisation by the GCN4 leucine zipper. However, it is also possible that the coiled coil domain from DEAF1 does make contacts with LMO4, which can be partially replaced by interactions with the leucine zipper domain of GCN4, or that self-association of DEAF1404–438 is required for binding. Although the leucine zipper domain of GCN4 is a highly characterised dimer  could associate with both itself and full length DEAF1 , was not able to interact with full length DEAF in this assay; however, only five residues separates the GAL4 activation domain and the beginning of the coiled coil in the construct. We postulate that the structured GAL4 activation domain may sterically prevent formation of the coiled coil.To characterise the self-association of DEAF1 we first used yeast two-hybrid analysis to establish that DEAF1th DEAF1 . The intin vitro, we made a series of DEAF1 constructs as recombinant proteins fused to the Maltose Binding Protein (MBP) or the β subunit of the Streptococcal G protein (GB1) tag. These proteins were subjected to size exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALLS). This technique supplies information about weight average molecular weight in solution and requires relatively large quantities of essentially pure protein that cannot normally be obtained from mammalian cellular lysates. DEAF1 contains multiple protein domains, including the DNA-binding SAND and HLH domains that may also mediate self-association 335–485, which lacks the SAND, HLH and MYND domains; the minimal LMO4-binding domain DEAF1404–479; and, the complete putative coiled-coil domain, DEAF1454–487. The larger constructs all showed evidence of self association, as indicated by weight average molecular weights corresponding to tetramers or higher order species , but sloping MALLS data indicating a mixture of species  to probe the secondary structure of the isolated coiled coil .Eukaryotic NESs are often part of coiled coil domains due to their leucine-rich nature 404–479 could be delivered to the nucleus using a well-characterised strong NLS from simian virus 40 large T-antigen (SV40) with an enhanced Green Fluorescent Protein (EGFP) tag. Transient transfection with EGFP-SV40-DEAF1404–479 showed essentially complete localisation of the construct to the nucleus, whereas EGFP-DEAF1404–479 (no NLS) was largely excluded from the nucleus , whereas NLS2 showed a reduced efficiency (48% nuclear) and NLS4 a markedly reduced efficiency (36%) compared to NLS1 and NLS3 . We established that a construct comprising an enhanced Green Fluorescent Protein (EGFP) tag, the short NLS from DEAF1 with a glutamine in place of lysine at position 4 (NLS4), and DEAF1404–479 (EGFP-NLS4-DEAF1404–479), resulted in ∼40% nuclear localisation of the expressed protein  with and without pCMV-LMO4 and showed that LMO4 had no effect on the nuclear localisation of DEAF1404–479 without an NLS; the EGFP protein appeared to be excluded from the nucleus (404–479 (no NLS) in the presence of LMO4 we noted that DEAF1404–479 was concentrated to distinct foci around the periphery of the nucleus  in yeast two-hybrid assays. The native tetrameric coiled coil can be replaced by a non-native dimeric coiled coil with only a moderate loss of apparent affinity in this assay. Our current model for binding is that DEAF1404–438 makes direct contacts with LMO4 in a manner similar to other well characterised LMO and LIM-homeodomain binding domains 457–479 either stabilises the construct, or provides an appropriate self-association state for the interaction with LMO4.Our data indicate that the DEAF1 coiled coil forms a tetramer in vivo, binding by LMO4 could prevent nuclear export of DEAF1 by stabilising DEAF1 tetramer (or higher order oligomers) through mass action effects, or by recruiting other protein partners that form oligomers and/or promote molecular clustering. Alternatively, LMO4 binding to DEAF1 may inhibit tetramerisation and prevent binding of an exportin to the DEAF NES, through direct steric inhibition, or by recruiting other partners that block binding. These hypotheses remain to be tested, although similar ‘masking effects’ of NESs have been shown for other proteins e.g., LIM-domain binding protein 1 e.g.,The presence of an NES in the coiled coil domain is not uncommon; NESs can be found in leucine rich segments of proteins, including coiled coil domains, located proximal to disordered regions Our results begin to shed light on the mechanism of some breast cancers where high levels of LMO4 are present. Under these circumstances DEAF1 may become sequestered into nuclear LMO4-DEAF1 complexes, perturbing the normal functions of DEAF1. Forming aberrant transcription complexes can prevent the formation of normal complexes. For example Rac3 GTPase, which is linked to breast cancer, cellular migration and adhesion, is transcriptionally upregulated by DEAF1 in immortalised mammary epithelial cells 600nm = 0.2, diluted serially 2×1-in-10) and spotted on selection plates for growth (−L/−W), medium ) or high (−L/−W/−H/−A) stringency selection.Mouse LMO4 (NCBI accession: NP_001155241) and DEAF1 (NM_016874) were used throughout. Full length DEAF1 was a gift from Jane Visvader. DEAF1 mutants and fusion constructs were generated by standard or optimised PCR methods 335–485 were cloned into pMALC2 to produce an N-terminal maltose-binding protein (MBP) fusion when expressed in Escherichia coli Rosetta 2 strain (Novagen) induced with 0.1 mM isopropylthiogalactoside (IPTG), 20°C, 16 h. The MBP fusion was purified by amylose affinity chromatography in 20 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM dithiothreitol (DTT) and eluted in 50 mM maltose. MBP fusion proteins were further purified by size exclusion chromatography (SEC) on a Superdex 200 column . The DEAF1454–485 construct was expressed in Rosetta 2 cells  with an N-terminal His-GB1  tag 454–487 using TEV protease . Tag was removed by Ni-NTA affinity chromatography prior to SEC using a Superose 12 column  to yield a 95% pure sample (as judged by SDS-PAGE).Full length DEAF1 and DEAF1454–487 was dialysed into 20 mM Trisma pH 8.0, 50 mM NaF and 0.5 mM TCEP-HCl. Data were collected on a 20 µM sample using a Jasco J-720 spectropolarimeter at 20°C. The spectrum in Size exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALLS) was carried out as described previously 2 atmosphere at 37°C. For transfection experiments, HEK293 cells were grown on glass cover slips in 6-well plates. Transfection (4 µg of plasmid DNA) was carried out for 24 h using GeneJuice (Novagen) according to the manufacturer’s instructions. Adherent cells were washed with phosphate buffered saline pH 8 (PBS), fixed with 3% (wt/vol) paraformaldehyde, 2% sucrose in PBS, stained with Hoechst dye H33342 (which stains nuclei by binding AT-rich regions of DNA) and mounted onto class slides in ProLong Gold antifade medium (Invitrogen). Bright field and GFP fluorescent (FITC) images were obtained using a 100×objective on an Olympus BX51 System microscope and analySIS LS software (Olympus).pEGFP-C2 and pCMV plasmids were from Clontech. The pEGFP-SV40 plasmid ) was a gift from Richard Grant. The SV40 NLS was replaced with synthetic DEAF1 NLS sequences to make pEGFP-NLS1–4. HEK293 cells (from ATCC) were cultured without antibiotics in high-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% foetal bovine serum (Invitrogen) in a humidified 5% COn  = 8 fields of view measured. Cell images were analysed using ImageJ image processing and analysis software, and the amount of EGFP protein in the cytoplasm and the nucleus was quantified as previously described t-test with p<0.05 considered significant.All data were derived from at least three independent experiments, with at least Figure S1Choosing an appropriate NLS for DEAF1 nuclear localization experiments. A. EGFP-DEAF1404–479 was first fused to the SV40 NLS. This strong NLS targeted all EGFP-DEAF1404–479 to the nucleus. B. Four EGFP-DEAF1404–479 constructs were made using variations to the native DEAF1 NLS to find a weaker NLS that did not target all DEAF1 to the nucleus. C. These EGFP-NLS-DEAF1404–479 constructs (4 µg) were transfected into HEK293 cells grown on cover slips in 6-well plates. After 24 h transfection, cells were fixed with paraformaldehyde and the nuclei stained with the Hoechst dye. Cells were imaged for EGFP fluorescence and nuclear staining by fluorescence microscopy. D. Quantification of percent nuclear accumulation of n = 6 fields of cells for each EGFP-DEAF-NLS construct used in C.(TIF)Click here for additional data file.Figure S2404–479 nuclear retention.Specificity of the LMO4 effect on EGFP-NLS4-DEAF1 EGFP-DEAF1404–479  with and without LMO4 (2 µg) was transfected into HEK293 cells as stated previously. LMO4 has no effect on the nuclear localisation of DEAF1404–479 without an NLS. Although, in the presence of LMO4 this DEAF1404–479 (no NLS) construct appeared to concentrate to distinct foci around the periphery of the nucleus. This may indicate an LMO4-DEAF1404–479 interaction in the cytoplasm concentrated within these foci.(TIF)Click here for additional data file.


The hepatitis delta virus genome is a small circular RNA, similar to viroids. Although HDV contains a gene, the protein produced (HDAg) is encoded by less than half the genome and possesses no RNA polymerase activity. Because of this limited coding capacity, HDV relies heavily on host functions and on structural features of the viral RNA—very much like viroids. The virus’ use of host RNA editing activity to produce two functionally distinct forms of HDAg is a particularly good example of this reliance. This review covers the mechanisms and control of RNA editing in the HDV replication cycle. HDV is C-terminus of HDAg-L. The 195 aa short form (HDAg-S) is required for RNA replication; the 214 aa long form (HDAg-L) is not only required for RNA packaging, but also inhibits replication [HDV differs from viroids in that the genome is larger ∼1,700 nt) and encodes a protein, hepatitis delta antigen (HDAg). HDAg serves at least two critical functions in HDV infection: participation in replication of the RNA genome, and packaging of the viral RNA with the envelope protein of hepatitis B virus. These two functions depend on two forms of HDAg, HDAg-S and HDAg-L, that differ by the presence of an additional 19–20 aa at the lication . Infecti00 nt andBecause inosine base pairs preferentially with C rather than U, upon subsequent replication and transcription, the amber stop codon in the hepatitis delta antigen (HDAg) open reading frame is changed to a tryptophan codon, the reading frame is extended by 19 or 20 codons and HDAg-L is produced. Thus, HDV uses the host RNA editing activity to switch the mode of replication from RNA synthesis to packaging.From the scheme depicted in We have found that editing is tightly controlled in three ways: it is highly specific for the particular adenosine at the amber/W site; and both the rate at which editing occurs and the fraction of RNAs edited are modulated. HDV RNA editing requires highly specific interactions between the host RNA editing enzyme, ADAR1, and structural features in the HDV RNA. Analysis of RNA editing in two HDV genotypes, types 1 and 3, has indicated that editing is essential for both, yet, the RNA structures involved and the mechanisms by which editing is controlled differ substantially. Comparison of editing in these two genotypes has revealed remarkably consistent functional requirements despite widely divergent structural characteristics, underscoring the critical role of this process, and its control, in the HDV RNA replication cycle. This review covers the mechanisms of RNA editing in the HDV replication cycle and the regulatory mechanisms by which HDV controls editing.2.Xenopus homolog of ADAR1 on dsRNA substrates and on analysis of interactions between the closely related enzyme ADAR2 and its substrates. For example, mutational analysis has indicated that the presence of a 6 nt internal loop strongly diminished editing of a dsRNA substrate by Xenopus ADAR1 [The host enzyme ADAR1 is responsible for editing the HDV amber/W site ,8. No adus ADAR1 . In the us ADAR1 –27, the Xenopus ADAR1 or the few substrates of ADAR2 editing that have been characterized.As described below, the secondary structures required for amber/W site editing have been analyzed for two of the 8 HDV genotypes—type 1, which is the most common, and type 3, which is the most distantly related. The structures involved share the first feature common to other editing sites—the 5′ base is not G, and at least some elements of the second, the amber/W site is present in a base-paired context. However, the overall size of the structure required for type 1 and type 3 HDV editing is considerably larger than for either dsRNA substrates of 2.1.The unbranched rod structure is a characteristic feature of HDV RNAs. This structure, in which about 70% of nucleotides are paired, is required for HDV RNA replication ,28. Siteet al. concluded that no additional secondary structures were required beyond the 8bp and the A-C mismatch [via interactions with the DRBMs and appear to be consistent with the conclusion of Sato et al. that structures outside the immediate vicinity of the amber/W site are not required for editing.The role of base-paired regions outside the 8 bp immediately surrounding the type 1 amber/W site is not settled. Sato mismatch . Inspectmismatch . Mutatiomismatch ,12. Thesvia a long range interaction that could involve bending of the RNA.In contrast to the above conclusion, more recent work in our lab suggests that sequences even further removed from the type 1 amber/W site play an important role in editing. While the previous mutational analysis indicated that base-pairing within ∼10–15 nt 3′ of the editing site might not be required for editing , analysiet al. [The apparent contradiction between our recent results and those of Sato et al.  could beet al. . Moreoveet al. . We have2.2.HDV type 3 also forms an unbranched rod structure that is required for replication. However, inspection of this structure indicates that the base pairing in the immediate vicinity of the amber/W adenosine is much more disrupted than in type 1 and, in fact, the type 3 amber/W adenosine is not edited when the RNA is in this conformation ,36. Nevein vitro, even at 37 °C. Perhaps HDAg, or even cellular factors, play a role in facilitating the conformational change.Because editing occurs on the antigenome, which is a replication intermediate, and replication requires the unbranched rod structure a consequence of this model is that the RNA must undergo a conformational change after editing has occurred. This conformational change is energetically favored because the branched structure required for editing is less stable than the unbranched rod structure, which is the lowest energy structure formed by the RNA . Neverthin vitro has indicated that SL2, which is 3′ of the site, is essential for editing, but SL1 is required for neither editing nor ADAR1 binding (as long as the remainder of the structure is formed). Thus, the role of SL1 appears to be simply to help stabilize the structure required for editing; once the structure is formed, SL1 does not participate in the editing reaction. The requirement for SL2 is consistent with the model for ADAR1 binding to regions with double-stranded RNA character that are 3′ of the editing site. Indeed, analysis of ADAR1 binding indicates that SL2 is required. However, deletion analysis has indicated that nearly all of SL2 is required for binding (and editing); thus the linear distance along the RNA of sequence required for editing is about 40 bp—not as long as we have observed for the type 1 amber/W site  minimizing non-specific editing at sites other than the amber/W site; (2) maintaining the optimal rate of amber/W site editing to fit the timing of the viral replication cycle; and (3) preventing over-accumulation of editing.3.1.Given the important role of dsRNA secondary structure in editing by ADAR1 and the high degree of base pairing in the HDV RNA unbranched rod structure, it is clear that potential exists for editing of sites other than the amber/W site. However, such non-specific editing could be harmful to the virus. Because HDAg functions as a multimer , deleterThere are three likely explanations for how HDV prevents editing from occurring at non-amber/W sites: (1) as indicated by the analysis of the type 1 amber/W site, although about 70% of bases are paired in the unbranched rod structure, this pairing is frequently disrupted by internal bulges and loops that limit ADAR binding; (2) the predicted secondary structure of type 1 RNA contains just 2 A-C mismatch pairs, which have been shown to be edited with the highest efficiency; (3) the frequency of GA dinucleotides, which are strongly disfavored for editing, is 60% higher than predicted based on a random distribution. Indeed, G is the 5′ neighbor of 48% of adenosines and both of the adenosines present as A-C mismatch pairs have G as the 5′ neighbor. In addition to the above effects that restrict the amount of editing at non-amber/W sites, HDV appears to be able to limit propagation of genomes that have been edited at these sites. We observed that editing of non-amber/W adenosines was strongly correlated with editing at the amber/W site on the same RNA . Becauseet al. were able to observe the susceptibility of additional sites to editing by using a viroid-like model in which replication of an HDAg-defective HDV genome was supported for one year by HDAg provided in trans [It is important to note that, although the amount of editing that occurs at non-amber/W sites is very low relative to the amber/W site and does not likely contribute either positively or negatively to the replication cycle, such editing does occur at low levels during replication  and may in trans . Becausein trans , in the in trans . It rema3.2.The rate and level of amber/W site editing achieved during replication are critical because HDAg-L is the limiting factor for virus particle production, but inhibits viral RNA replication. Comparison of HDV editing with other editing substrates indicates that amber/W site editing occurs much more slowly and at lower levels. Most cellular substrates for editing are pre-mRNAs that are not only frequently edited at high levels (∼100% in some cases), but must be edited prior to splicing, which occurs within minutes following transcription. By contrast, only about 4% of HDV RNAs are edited at the amber/W site four days post transfection ,12, and 3.3.As mentioned above, previous mutational analyses had shown that increased base pairing 3′ of the type 1 editing site increased editing and substantially decreased HDV RNA replication ,12. Thes3.4.In addition to limiting editing by forming sub-optimal editing sites, we have found that HDV controls editing levels by limiting the availability of this sub-optimal substrate to ADAR1. Perhaps not surprisingly, the mechanisms employed by types 1 and 3 to limit substrate availability are as different as the structures used for editing. Just as the use of different structures by these genotypes underscores the important functional role of editing in the HDV replication cycle, the use of different control mechanisms emphasizes the essential need to manage the process.in vitro. Editing of the K12 RNA is not affected , indicating that the inhibition occurs by a direct interaction between HDAg and the RNA, rather than binding to ADAR1. Thus, our model for the down-modulation of editing during HDV type 1 replication is that the availability of the RNA for editing by ADAR1 is limited by binding of HDAg to the RNA  post-transfection . The low the RNA . This binuclease . Both HDin vitro [in vitro, ADAR1 edits the amber/W site of HDV-3P RNA more effectively than that of HDV-3E RNA. However, the converse is true when editing levels attained during replication are compared. Computational analysis of the secondary structures formed by HDV-3E and HDV-3P [in vitro HDV-3E and HDV-3P RNAs formed both the branched structure required for editing and the unbranched rod (which is not edited), but HDV-3E RNA formed the branched editing structure 3–4 fold more efficiently than did HDV-3P [The mechanism employed by HDV type 3 to control editing is quite different. For HDV type 3, editing is not inhibited by HDAg-S, either in transfected cells or in vitro . The mecin vitro . The posin vitro . We explin vitro . As showd HDV-3P  suggested HDV-3P . Based o3.5.HDV must regulate both the rate and the extent of editing at the amber/W site because, as shown in Section 4, levels of viral RNA replication and virion production are sensitive to the kinetics and amount of HDAg-L produced. Moreover, as shown in et al. showed that, although HDAg-S does not effectively reduce editing, HDAg-L strongly inhibits editing of a non-replicating HDV type 3 RNA [Two studies have indicated that editing can be regulated by negative feedback ,46, but et al., who suggested that HDAg-L can indirectly regulate amber/W editing by inhibiting HDV RNA replication [We currently favor the model proposed by Sato lication . As can lication ; thus, olication . The mec4.Editing at the HDV amber/W site expands the very limited coding capacity of this small human pathogen and plays a central role in its replication cycle. Our recent analysis of the secondary structure requirements for both the HDV type 1 and 3 amber/W sites broadens the scope of structures involved in editing to include those in which structures more than 20–25 nt away from the editing site are required. As additional cellular RNA editing substrates are identified and characterized [