Circular RNAs are the most recent addition in the non-coding RNA family, which has started to gain recognition after a decade of obscurity. The first couple of reports that emerged at the beginning of this decade and the amount of evidence that has accumulated thereafter has, however, encouraged RNA researchers to navigate further in the quest for the exploration of circular RNAs. The joining of 5′ and 3′ ends of RNA molecules through backsplicing forms circular RNAs during co-transcriptional or post-transcriptional processes. These molecules are capable of effectively sponging microRNAs, thereby regulating the cellular processes, as evidenced by numerous animal and plant systems. Preliminary studies have shown that circular RNA has an imperative role in transcriptional regulation and protein translation, and it also has significant therapeutic potential. The high stability of circular RNA is rendered by its closed ends; they are nevertheless prone to degradation by circulating endonucleases in serum or exosomes or by microRNA-mediated cleavage due to their high complementarity. However, the identification of circular RNAs involves diverse methodologies and the delineation of its possible role and mechanism in the regulation of cellular and molecular architecture has provided a new direction for the continuous research into circular RNA. In this review, we discuss the possible mechanism of circular RNA biogenesis, its structure, properties, degradation, and the growing amount of evidence regarding the detection methods and its role in animal and plant systems. Circular RNAs (CircRNAs) have recently spread into the non-coding RNA world. The circRNAs are formed by the covalent circularization of a 3′ downstream donor and the 5′ upstream acceptor in an alternate form of pre-mRNA splicing by a process called backsplicing . Flanking introns or exons are found to be much longer (~3 fold) in circularizing the intervening exonic or intronic sequences, respectively, than the normal corresponding sequences, which generally undergo linear splicing  are found to be important for circularization of exonic circRNAs, which have a conserved binding site in the flanking introns. Excess Muscleblind-like splicing regulator 1 (MBNL1) proteins bind to the conserved motifs present in the flanking introns of the second exon of their own gene and regulate circMBNL1 biogenesis  to Inosine (I) editing by adenosine deaminase acting on RNA1 (ADAR1) is frequent in the intronic sequences, which are responsible for circularizing intervening exons , Plasmacytoma Variant Translocation 1 (PVT1)  have a conserved K-K/S-L-N motif that is responsible for the export of long circRNA  to the cytoplasm through the nuclear pore complex. Similarly, another member of the human exportin family, URH49 (DDX39A) is accountable for the export of <400 nt short circRNA into the cytoplasm. URH49 is dependent on the R-S-F-S motif, but swapping with the K-K/S-L-N motif in the URH49 results in the alteration of its property and it begins to behave like Hel25E/UAP56. However, the nuclear export protein involved in short circRNA export in Drosophila is yet to be deciphered. Moreover, it has been hypothesized that long and short circRNAs may follow the NXF1–NXT1 and PHAX–CRM1 pathway, respectively, for cytoplasmic export; this is similar to mRNA, and this needs to be determined experimentally  or the highly structured double-stranded RNA, having 3′-overhangs that are shorter than 7 nt, are resistant to digestion by RNase R  to harness even low-expressed circRNAs from Based on the computational prediction, it has been determined that a specific gene locus could yield more than one circRNA of different lengths via alternative backsplicing. This mechanism is primarily attributed to association with the competition of putative RNA pairs across introns, which favor the circularization of exons  gene with 107 exons has been reported with around 59 RyR-specific circRNAs  alternative 5′ backsplicing and (b) alternative 3′ backsplicing  or by having high copy numbers of the sponging circRNA, preferably with large-sized circRNA . Studies have demonstrated a reduced level of circNfix in proliferating neonatal cardiomyocytes when compared to adult cardiomyocytes . Besides, 34–158 circRNAs derived from Drosophila, rats, and mice were found to be associated with polysomes, emphasizing that those circRNAs might be translated into proteins  is found to be differentially expressed during various stages of fruit ripening -domain with endoribonuclease activity. This may contribute to circRNA degradation, which is otherwise resistant to exonuclease activity due to its lack of linear ends (Schaeffer et al., The fourth pathway could be based on the known fact that m6A modified mRNAs that are recognized and guided by YTHDF2 into nuclear P-bodies for their degradation (Wang X. et al., In addition to the above mentioned pathway, the possible fifth pathway could be EV mediated, in which EVs are membrane-bound structures capable of encapsulating cellular components, including circRNAs as mentioned previously (Yang and Li, Significant reports in the field of circRNA have illuminated the RNA world recently. Novel circRNAs are reported to have diverse functions; this includes acting as biomarkers and having therapeutic potential in uses for cancer and other diseases, which could further pave the way for effective diagnosis, treatment, and prevention. As compared to the animal system, research on plant circRNAs is minimal and is in need of more attention, especially designing of exclusive bioinformatic tools. Nevertheless, some of the proposed findings on plant circRNAs, including the mechanism of their biogenesis, which deviates from animal circRNAs, are worth mentioning; putative circRNA has, for example, a regulatory role in metabolic pathways. In the coming years, more research has to be put forward into the translational potential of circRNA. It is important to unravel any differences in the function of proteins coded by circRNA from the canonical spliced mRNA. It is equally interesting to study the possibility of protein isoforms generated from alternative backsplicing and their possible function in gene regulatory networks. It is also intriguing to know whether the secondary structure of circRNA will affect its miRNA sponging ability. Similarly, in the absence of a terminator codon in circRNA, how the protein synthesis is regulated is another interesting question to be resolved. Nevertheless, the hunt is on across the scientific community to find answers to many more burgeoning questions, including in the field of plant circRNAs.AG and PS contributed equally in conceiving the review focus, conducting the literature review, summarizing the manuscript, reviewed literature, wrote the first draft, and finalized the manuscript. GP, SN, AG, and PS revised and made corrections to the manuscript. All authors approved the final version of manuscript.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.


Caliciviridae are viruses with a positive-sense, single-stranded RNA genome that is packaged into an icosahedral, environmentally stable protein capsid. The family contains five genera  that infect vertebrates including amphibians, reptiles, birds, and mammals. The RNA-dependent RNA polymerase (RdRp) replicates the genome of RNA viruses and can speed up evolution due to its error-prone nature. Studying calicivirus RdRps in the context of genuine virus replication is often hampered by a lack of suitable model systems. Enteric caliciviruses and RHDV in particular are notoriously difficult to propagate in cell culture; therefore, molecular studies of replication mechanisms are challenging. Nevertheless, research on recombinant proteins has revealed several unexpected characteristics of calicivirus RdRps. For example, the RdRps of RHDV and related lagoviruses possess the ability to expose a hydrophobic motif, to rearrange Golgi membranes, and to copy RNA at unusually high temperatures. This review is focused on the structural dynamics, biochemical properties, kinetics, and putative interaction partners of these RdRps. In addition, we discuss the possible existence of a conserved but as yet undescribed structural element that is shared amongst the RdRps of all caliciviruses.The  Caliciviridae family currently consists of five genera   has been proposed  and Vesicular exanthema of swine virus (VESV), are highly contagious in animals and can cause persistent infections. FCV causes fever and acute upper respiratory tract and oral cavity disease in all feline species and can lead to a virulent systemic disorder  causes acute necrotizing hepatitis and disseminated intravascular coagulation in European rabbits (Oryctolagus cuniculus), which leads to death 48–72 h post-infection, while the Rabbit calicivirus (RCV) causes only mild disease manifestations (The sivirus) . The estproposed . The expproposed . Virusesglobally . These vglobally . Despitedisorder . VESV afdisorder . The genstations . Since tstations . Even thCaliciviridae family share a number of features. The genome consists of positive-sense, single-stranded RNAs that contain coding sequences in two or more partially overlapping open reading frames (ORFs). The coding sequences are flanked by untranslated regions (UTRs) at both the 5′ and 3′ ends. Genomic RNAs are covalently linked at the 5′ end to a viral protein  and are polyadenylated at the 3′ end. Calicivirus particles contain two types of RNA, a genomic (full-length) RNA of about 7.5 kb and one or more copies of a subgenomic RNA of about 2 kb (Norovirus and Vesivirus) or five non-structural proteins, VPg, and the major capsid protein VP1  (Murine norovirus (MNV), there is an additional ORF in the VP1 coding region of both genomic and subgenomic RNAs that encodes the viral factor 1 (VF1), an antagonist of the innate antiviral immune response  . The secpovirus) . The subpovirus) . In Muriresponse .calyx” = cup). The basic VP2 protein has also been found associated with virus particles  and plays a role in RNA replication and the maturation of infectious virus particles   . The VPghoresis) .Picornaviridae family that counteract host defense mechanisms -regulator and is involved in vesicle transport  and unknown functions . The first one or two N-terminal proteins of the full-length genomic RNA  may have functions similar to the so-called “security proteins” of the chanisms . This hychanisms . The p48ransport . This inransport . Moreoveransport . The p22ransport . HoweverThe functions of the remaining non-structural proteins were deduced by comparing calicivirus and picornavirus sequences and by searching for conserved motifs. A 2C-like helicase . Remarkably, the calicivirus RdRp precursor protein is also an active polymerase enzyme . RdRps aThe shape of all RdRps resembles a right hand with fingers, palm, thumb, and an N-terminal domain that links the finger and thumb domains . The act2+ or Mn2+. Motif F contains the positively charged residues Arg and Lys that mediate interactions with the triphosphate moieties of incoming nucleoside triphosphates (NTPs)  . Motif Gs (NTPs) .The thumb domain of calicivirus and picornavirus RdRps is small compared with that of other RdRps and DNA-dependent DNA polymerases. The domain consists of only four helices and forms a relatively large, 15 Å-wide central cleft  that leads to the active site , 2006. T2+ or Mg2+) in the active site. The metal ions are coordinated by the Asp residues of motifs A and C. One of the ions interacts with the 3′-OH group of the primer, which reduces the affinity of this group for the hydrogen and enables a nucleophilic attack of the negatively charged 3′-O- on the α-phosphate residue of the incoming complementary nucleotide (i). As a result of the nucleophilic attack, a new phosphoester bond between the 3′-OH terminal group of the protein-linked primer and the α-phosphate of nucleoside monophosphate (NMP) is created and PPi is released  and the φ6 bacteriophage, in which C-terminal amino acids help to stabilize primers in the active site  RNA primer but in the presence of VPg, confirming the notion of a protein-primed initiation. For anti-subgenomic RNA replication to occur, a protein primer is probably not required, as an RNA product is yielded in the absence of exogenous primers  stretch of anti-subgenomic RNA, a scenario that is in line with the general observation that the de novo initiation on RNA genomes usually starts on pyrimidines  2A protease coding sequence ahead of the VP2-coding region. These reporter replicon variants were used to quantify subgenomic RNA synthesis. Replicons with mutations in the stem-loop produced less luciferase compared with wild type MNV replicons, but similar amounts to a replication-defective replicon. The amount of subgenomic RNA was determined using a primer extension assay, in which a radiolabeled primer complementary to the 5′ region of subgenomic RNA was used to generate a product corresponding to the start of the subgenomic RNA. Subgenomic RNA was detected in cells transfected with the wild type MNV genome but was absent in those transfected with a replicon bearing mutations in the stem-loop region. These results confirm the hypothesis that the stem-loop in the RdRp coding region is essential for the initiation of subgenomic RNA synthesis  slows at the point when VP1 starts to multimerize and assemble into new capsids, which prevents its interaction with RdRp and stops the stimulation of RNA synthesis  or exogenous promoters . Moreover, a domain was identified in the N-terminal region of the protease-polymerase precursor that is responsible for the observed inhibition of the RNA polymerase II . SimilarAnother cellular protein that affects FCV, MNV, and porcine enteric calicivirus (PEC) replication is the lysosomal endopeptidase cathepsin L, a protease that is involved in apoptosis and is primarily located in endosomes. Cathepsin L cleaves the structural protein VP1 of FCV and MNV, and VP2 of PEC. Its inhibition was shown to negatively affect the replication of FCV, MNV, and PEC in cell culture. The effect of cathepsin L inhibition is similar to the inhibition of endosomal acidification  and prevents MNV and PEC from endosomal escape. These and possibly other caliciviruses enter host cells via clathrin-mediated endocytosis, thus, it should not come as a surprise that any interference with the endosomal escape of incoming virus particles blocks the initiation of virus replication ,b.de novo GTP incorporation assay that can be used to analyze enzyme kinetics (-1) and had a lower affinity for the GTP substrate than the wild type, suggesting that phosphorylating Thr33 modulates the activity of the enzyme  . Akt is kinetics , the Thre enzyme .in vitro with increasing concentrations of RdRp, until a plateau phase is reached. Högbom and coworkers interpret the data as a shift from active monomeric RdRps to more active dimers. They analyzed the cooperativity between RdRp monomers by calculating the Hill coefficient that, in this case, was determined to have a value greater than one, which is indicative of a positive cooperativity  is 3.8 × 10-5, measured as substitutions per nucleotide per cycle of infection (s/n/c)  , and theleotides . Similarus GII.4 .in vitro fidelity assays) compared with those of the closely related but less frequently detected GII.b and GII.7 strains . Interestingly, the GII.4 lineage showed an approximately 1.7-fold higher rate of evolution of capsid sequences and a higher frequency of non-synonymous changes compared with non-pandemic norovirus strains  than the recombinant GII.4 RdRps from earlier outbreaks and the US95/96-like pandemic GII.4 strain although the error rates were very similar. The observed increase in the incorporation rate has been associated with the appearance of a mutation outside of the active site, i.e., a Lys291 to Thr substitution in the RdRp finger domain . Thus, h-3 substitutions/site/year), and has rapidly spread across the world  . For optnot Fe2+ . Sapovirofactors .46Pro-Ala-Asn-Leu49  and non-nucleoside inhibitors (NNIs) . NAs areDengue virus, Yellow fever virus, and West Nile virus (The active 5′-triphosphate form of 2′-C-methylcytidine (2CMC) is an HCV polymerase inhibitor that competes with the nucleotide cytidine triphosphate (CTP) for binding to the active site of RdRps. Incorporation of 2CMC into a nascent RNA strand leads to the termination of RNA synthesis. In cell culture, this compound is also active against le virus . 2CMC alle virus . Furtherle virus .Ribavirin  mimics the guanosine nucleotide and inhibits the replication of a broad range of DNA and RNA viruses . In cell50  , a purine nucleoside analog, was developed as an influenza virus inhibitor. T-705 is a prodrug which is turned into its active form (favipiravir-ribofuranosyl-5′-triphosphate) by cellular enzymes , 2013. Ttration) . The mectration) . This hytration) . Moreovetration) .50  value compared with NAF2  and pyrazole acetamide (NIC04) are novel compounds that inhibit GII.4 norovirus RdRps at relatively low IC complex . The inh complex .Norovirus, Sapovirus, and Lagovirus), as measured in a quantitative fluorescent de novo RdRp activity assay . All authors contributed to editing and revising the manuscript. All authors read and approved the final manuscript.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.


Thrips tabaci. The entire nucleotide sequence of the M and S RNA segments was elucidated by a conventional cloning and sequencing strategy, and contained 4797 respectively 2734 nucleotides (nt). Simultaneously, a next generation sequencing (NGS) approach (RNAseq) was employed and generated contigs covering the entire viral tripartite RNA genome. In addition to the M and S RNA nucleotide sequences, the L RNA (8865 nt) was obtained. The nucleocapsid (N) gene encoded by the S RNA of this virus consisted of 819 nucleotides with a deduced N protein of 272 amino acids and by comparative sequence alignments to other established orthotospovirus species showed highest homology (69.5% identity) to the N protein of polygonum ringspot virus. The data altogether support the proposal of AYSV as a new orthotospovirus species within a growing clade of orthotospoviruses that seem to share the Middle East basin as a region of origin.An orthotospovirus distinct from all other orthotospoviruses was isolated from naturally infected alstroemeria plants. Disease symptoms caused by this virus mainly consisted of yellow spots on the leaves based on which the name alstroemeria yellow spot virus (AYSV) was coined. A host range analysis was performed and a polyclonal antiserum was produced against purified AYSV ribonucleoproteins which only reacted with the homologous antigen and not with any other (established or tentative) orthotospovirus from a selection of American and Asian species. Upon thrips transmission assays the virus was successfully transmitted by a population of The online version of this article (10.1007/s00705-018-4027-z) contains supplementary material, which is available to authorized users. Tospoviridae continuously cause significant losses in both mono- and dicot crops worldwide ).The complete nucleotide sequence of AYSV M RNA consists of 4797 nt, and contains a 5’- (62 nt) and 3’-UTR (51 nt) that is able to form a panhandle structure due to sequence complementarity. The UTRs show almost entire sequence complementarity for 29 nt with only 3 mismatches (5’-AGAGCAAUCGGUGCAANAAUCAAAUNUNU-3’). The vORF starts with an AUG at nucleotide position 63 and terminates with an UAA codon at position 989, coding for the NSerfamily , 53, incThe vcORF codes for the glycoprotein precursor and starts with an AUG codon at nucleotide position 4746 and terminates with UGA at nucleotide position 1300. The M RNA encoding v- and vc-ORFs flank an IGR of 310 nt that runs from nucleotide position 990 to 1299 and, like the S RNA, consists of nucleotide stretches highly rich in A- and U residues. Folding predictions of the IGR sequence reveals the formation of a stable hairpin structure (data not shown).M was found with TYRV NSM (88.7%) whereas its glycoprotein precursor (GP) showed highest identity with that of TYRV GP (81.0%). Phylogenetic analysis based on the NSM protein and the glycoprotein precursor support a classification of AYSV within the Eurasian clade  transmembrane domains were found in the GN-GC precursor. Both of the predicted cleavage sites located within the first and fourth transmembrane domains, whereas none of the predicted glycosylation sites mapped within transmembrane domains  sequence, a motif earlier found in the TSWV precursor and putatively involved in virus cell attachment [The surface glycoprotein of AYSV was analyzed and compared to those of other Eurasian orthotospovirus species for the presence of N-, O- Glycosylation, peptide cleavage sites and transmembrane domains Figure . Two putThe entire sequence of AYSV L RNA consists of 8865 nt with a 5′ terminal (214 nt) and 3′ terminal (32 nt) UTR that contain the highly conserved 8 terminal nucleotides present in all orthotospoviral RNA segments. AYSV terminal sequences show almost entire base pair complementarity for the first 16 nt with only one base pair mismatch (5’-AGAGCAAUCNAGCAAC-3’).The L RNA contains a single ORF of 8619 nt in the vc-strand and coding for the putative viral RNA-dependent RNA polymerase  of 2873 aa with a predicted molecular mass of 316.0 kDa. The vcORF starts with an AUG at nucleotide position 8833 and terminates with an UAA stop codon at nucleotide position 212. The L gene of AYSV shares highest aa (87.3 %) identity with the one from TYRV. Analysis of the AYSV L protein revealed the presence of all conserved motifs earlier reported and indicative for the RdRp , 55. The478 vs. PolRSV-IGR183).A summary and comparative analysis of the AYSV L, M and S RNA sequence to those of other Eurasian species is shown in Table The complete L, M and S RNA sequences of AYSV are available under GenBank accession no. MF469033, MF469034 and MF469035 respectivelyE. sonchifolia plants exposed to T. tabaci, that in their larval stages were given access to AYSV-infected plants, revealed chlorotic spots similar to those observed earlier during the host range analyses  and contains 5 ORFs coding for six mature proteins i.e., the L protein , the cell-to-cell movement protein , the precursor  to the glycoproteins GN and GC, the suppressor of RNA silencing  and the nucleocapsid protein . The AYSV sequence shows all structural features typical for orthotospovirus RNA genomes with highest homology between its proteins and those from TYRV. Phylogenetic analysis indicates that AYSV, together with PolRSV, TYRV, HCRV and IYSV, belongs to a growing clade of orthotospoviruses for which the geographical origin seems to be based in the Middle-East [Here a tentative new orthotospovirus species has been identified and characterized from alstroemeria, and for which the name alstroemeria yellow spot virus (AYSV) has been proposed. The virus causes yellow spots on alstroemeria, has a narrow experimental host range limited to a few dicots and is transmitted by the vector dle-East . AlthougTospoviridae is still expanding and has currently reached the number of 30 tentative and established species   we have Table S1. Overview of viruses which have been reported from alstroemeria plants (DOCX 17 kb)Table S2. Accession numbers of orthotospoviral segments and/or corresponding genes, used in this study (DOCX 18 kb)Below is the link to the electronic supplementary material.


The preparation of small RNA cDNA sequencing libraries depends on the unbiased ligation of adapters to the RNA ends. Small RNA with 5’ recessed ends are poor substrates for enzymatic adapter ligation, but this 5’ adapter ligation problem can go undetected if the library preparation steps are not monitored. Here we illustrate the severity of the 5’ RNA end ligation problem using several pre-miRNA-like hairpins that allow us to expand the definition of the problem to include 5’ ends close to a hairpin stem, whether recessed or in a short extension. The ribosome profiling method can avoid a difficult 5’ adapter ligation, but the enzyme typically used to circularize the cDNA has been reported to be biased, calling into question the benefit of this workaround. Using the TS2126 RNA ligase 1 (a.k.a. CircLigase) as the circularizing enzyme, we devised a bias test for the circularization of first strand cDNA. All possible dinucleotides were circle-ligated with similar efficiency. To re-linearize the first strand cDNA in the ribosome profiling approach, we introduce an improved method wherein a single ribonucleotide is placed between the sequencing primer binding sites in the reverse transcriptase primer, which later serves as the point of re-linearization by RNase A. We incorporate this step into the ribosomal profiling method and describe a complete improved library preparation method, Coligo-seq, for the sequencing of small RNA with secondary structure close to the 5’ end. This method accepts a variety of 5’ modified RNA, including 5’ monophosphorylated RNA, as demonstrated by the construction of a HeLa cell microRNA cDNA library. Fig. 1A) -ATP by T4 polynucleotide kinase (NEB) according to the manufacturer’s protocol.Custom sequencing adapters and primer oligonucleotides  was immunoprecipitated from HEK293/POLR3F stable cell line  lysates,escribed . Transcrin vitro  from a coligo template by immunoprecipitated FLAG-RNAP III were ligated to the 3’ adapter in a 20 μl ligation reaction that included the following components: 1 mM ATP, T4 RNA ligase buffer (NEB), 1 μl of T4 Rnl1 (NEB M0204S), 15% PEG 8000 (NEB), 20% DMSO, and 30 μM Adapter1 (Table 1). Ligation reactions were incubated for 2 h at 37°C. Nucleic acids were phenol/chloroform/isoamyl alcohol (PCI) extracted, ethanol precipitated and resuspended in RNase-free water for subsequent use. Note that to avoid undesirable side-reactions when using this method on 5’-monophosphorylated RNA, like miRNA and pre-miRNA, the 3’ adapter should first be 5’-pre-adenylylated, then ligated using the truncated T4 Rnl2(1-249) K227Q mutant without ATP -UTP incorporation. To visualize first strand cDNA synthesis, the RT primer, which includes the single riboC, was labeled at the 5’ end using T4 polynucleotide kinase and [γ-32P]-ATP. In Figure 2B, the reverse transcript  can be seen, and the RNase H-mediated removal of the transcript that served as the RT template is evident by comparing lanes 2 and 3 (species II). In this particular experiment, sub-optimal 3’ ligation conditions had been used, and some of the initial transcript was not ligated to the adapter . The unligated RNA, though complementary to the cDNA produced, was not degraded by RNase H. This observation shows that RT produced a stable RNA:cDNA hybrid, and underscores the importance of optimizing the 3’ adapter ligation efficiency since sequence information in unligated transcripts will be lost at this step. The reverse transcript (species III), was extracted from the gel and circularized using TS2126 Rnl1 . The circularization reaction produced trace amounts of higher molecular weight concatamers. These products undergo digestion by RNase A to the same final PCR template . In our experience, the TS2126 Rnl1 ligase consistently circularizes with about 90% efficiency regardless of the DNA sequence (see later for a bias test for this step). The 10% of unligated species III  will not interfere with the PCR because RNase A will cut it between the P1 and P2 sites in the next step . The circularized cDNA (species IV) was cleanly re-linearized by RNase A (lane 9) and used in a PCR reaction, which yielded a PCR product in the expected size range . This DNA was recovered from the gel and cloned into a T/A cloning plasmid [Fig. 2E). This result demonstrated that the Coligo-seq approach could be used to sequence the cDNA of coligo transcripts without a 5’ adapter ligation step.In a preliminary experiment ending with Sanger sequencing, we subjected the  plasmid . Five cl plasmid  . We also included the P1 and P2 PCR primer binding sites and, between them, a single riboC. Thus, this library uses a variation of the Coligo-seq strategy to identify sequences that have undergone successful ligation. Circularization by TS2126 Rnl1 at increasing substrate library concentration showed the typical ~90% circularization up to about 1 μM substrate . After isolation from the gel, the circularized library was re-linearized by RNase A and adapted for high throughput sequencing. The frequency of each nucleotide found at the fourteen positions is shown in Figure 3C. Throughout the randomized region, where each nucleotide should be represented at about 25% during DNA synthesis, there was a slight enrichment above 25% for A/T, and a slight reduction below 25% for G/C. However, throughout the randomized sequence the deviation from 25% at individual nucleotide positions was small, ranging from 23% to 26%. The 3’ terminal nucleotide showed almost equal frequency for G, C and T, and a slight preference for A. Importantly, all sixteen possible dinucleotide combinations were found to have been ligated and deviations from the predicted 6.25% (100%/16 dinucleotides) were small . A complete biological repeat confirming these results is described in the Supplemental Information, including Figure S2, Figure S3, Figure S4 and Table S1. This result demonstrated that in the context of circularization, the TS2126 Rnl1 has no significant bias, and we conclude that for RNA with structured 5’ ends, exchanging the inefficient 5’ adapter ligation step of RNA-seq for the TS2126 Rnl1 circularization step of Coligo-seq offers a significant improvement. As noted above, the TS2126 Rnl1 is reported to be the same protein as the commercially available CircLigase [The construction of a cDNA library using Coligo-seq and other ribosome profiling-based methods replaces the unfavorable 5’ adapter ligation step with a cDNA first-strand circularization step. We have consistently observed intramolecular ligation yields of ~90% when circularizing ss DNA substrates of defined sequence ,23, but rcLigase , so thesFig. 4). The Coligo-seq steps and species leading to the library PCR template are outlined in Figure 4A. Dcr3 was transcribed in vitro by Pol III, and the 3’ ends of the unpurified transcripts were ligated to the 3’ sequencing adapter under the optimized conditions . Note that all transcripts, including the higher molecular weight tandem repeat transcripts (formed by failure to terminate on the first or second circumtranscription), were shifted by efficient addition of the 3’ adapter. The 3’ ligation products corresponding to the two main circumtranscription products  were gel purified , extracted from the gel, reverse transcribed  and, after a second gel purification to remove excess RT primer (lane 5), circularized (lane 6), re-linearized by RNase A (lane 7) and amplified by PCR using primers containing sequences to adapt the cDNA library to the Illumina HTS platform . During sequencing, 2107614 reads were obtained. After removal of the primer sequences, 89.41% of the reads fell within the 60–71 nt size range of the original transcripts, and did so in two peaks  corresponding to the size of the excised transcript bands. This result demonstrated that about 90% of the Coligo-seq reads contained information from the size-selected Pol III transcripts.A second pre-miRNA-encoding coligo, Dcr3, based on the human pre-miRNA-19a cDNA template strand, was used to test adaptation of Coligo-seq to the Illumina HTS platform, again with careful monitoring at each step . These changes, and the use of the modified T4 RNA ligase  [Figure 6 steps to make a cDNA library of mature miRNA isolated from HeLa cells. Mature miRNA carries a 5’ phosphate. We split a sample of total RNA and made a cDNA library from one half using Coligo-seq, and a cDNA library from the other half using the standard RNA-seq method [Figure S5 and the miRNAs found in both cDNA libraries are tabulated in a searchable spreadsheet in Table S2. Out of the 186 most abundant miRNA cDNA reads found in the standard RNA-seq library, 184 were also found in the Coligo-seq library, and cases where a microRNA was found in one library but not the other were mainly limited to very low percentage reads, reflecting low abundance miRNA. These results validate the Coligo-seq variation shown in Figure 6. Thus, Coligo-seq can be used on small RNA regardless of the 5’ end modification, and provides a general method to circumvent the 5’ adapter ligation problem.Our method for small RNA cDNA library preparation can be easily adapted to small RNA from sources other than coligo transcription. If the RNA carries any modification besides a 5’ monophosphate, then no changes need be made to the procedure. However, during 3’ adapter ligation, a 5’ monophosphate on the source RNA can lead to transcript self-ligation side reactions such as multimerization or circularization . To avoiq method . The reset al. found that the 5’ adapter could not be ligated to cellular pre-miRNA hairpins using the standard RNA-seq protocol. They traced this ligation difficulty to the secondary structure and, in particular, the recessed 5’ ends common to pre-miRNA [The sequencing of small RNA is usually accomplished through construction and sequencing of the RNA’s cDNA library. While every step in the cDNA library preparation is a potential source of bias, it is the nucleic acid ligases needed to add the sequencing adapters that have generated the most concern ,10,13,25et al. [Like Liu et al. , we founet al. , among oet al. . If leftet al. . In our et al. [et al. [RNA-seq methods that include a cDNA circularization step, like those of Ingolia et al.  and Liu  [et al.  have rel [et al. , though  [et al. ,28. We t [et al. . The dif [et al.  that theet al. [et al. [Though the Coligo-seq method we describe here is closely related to the earlier Ingolia et al.  and Liu  [et al.  library et al., fragmentation methods that tend to leave a 5’ ss extension near a ds region should be avoided because 5’ adapter ligation will be impaired and bias introduced. RNase III has been widely adopted, though it is not without bias itself [RNA-seq protocols that profile mRNA typically begin with a fragmentation step to produce smaller RNA using RNase III or sonication . Based os itself . Since iSupplementary informationFigure S1. Examples of 5’ adapter ligation optimization attempts using coligo 122 transcript as test case.Figure S2. TS2126 Rnl1 (CircLigase) circularization bias experiments and sequencing fragment size.Figure S3. TS2126 Rnl1 circularization bias experiment; library preparation.Figure S4. TS2126 Rnl1 circularization bias analysis results.Figure S5. Application of Coligo-seq to HeLa cell miRNA.Table S1. Oligonucleotide sequences used in TS2126 Rnl1 bias experiments.Table S2. Comparison of miRNA reads obtained using standard RNA seq method (Method1) versus Coligo-seq method (Method 2).Supplementary information of this article can be found online athttp://www.jbmethods.org/jbm/rt/suppFiles/269.


P = 0.003); however, no group differences were found in lying time (P = 0.551). During the daytime (between 8:00 and 20:00), the frequency of lying down was 50, 33, and 41% higher, respectively, than during the nighttime on Days 2, 3 and 4 . On the heat stress day, non-shaded calves changed posture 88.4 and 76.6% more often than shaded ones between 8:00 and 12:00 and 12:00 and 16:00, respectively . Similar group differences were observed for Day 3 between 8:00 and 12:00 (71.2%) and Day 4 between 12:00 and 16:00 (76.6%), respectively . On Day 5, there was no difference between groups (P = 0.732). As indicated by our results, heat stress causes changes in lying down frequency and lying time in dairy calves. Supplemental shading reduces discomfort as indicated by lying down frequency, but not by lying time.Changes in lying behaviour in response to extreme ambient temperatures have not been examined in dairy calves so far. In this study, lying time, and frequency of lying down were investigated in shaded (n = 8) and non-shaded (n = 8) Holstein bull calves during a 5-d period . The thermal environment around the calves was characterized by the temperature–humidity index (THI). A three-dimension accelerometer was used to record posture of the calves and lying time and lying down frequency were analysed with 4-h sampling intervals. On Day 1 no differences were found in THI between the shaded and non-shaded environments. On Days 2, 3 and 4 maximal and average THI were higher in the shaded than those recorded for the non-shaded environment. On Day5 no significant differences in THI were observed between calf environments. A similar diurnal pattern of lying time and lying down frequency was observed in both groups. Lying times were shorter during the afternoon ( Hutch-reared dairy calves are vulnerable to warm episodes during summer because their natural heat-dissipating behaviour (e.g. finding shade) is hindered. As these are all parts of complex comfort behaviour, total time spent lying down, the number of lying bouts, and the frequency of lying down are often studied in response to environmental factors in adult cattle3. Although the effect of in utero heat stress exposure on calf activity patterns4 and on overall well-being6 have been recently evaluated during the neonatal period, there is a paucity of information regarding biobehavioral responses to acute heat stress experienced by calves during the pre-weaning period. Commercial automatic data loggers overcome the limitation of time-consuming video-based behavioural observations in calves;7 however, no research has evaluated lying behaviour in dairy calves during extreme heat stress events.Global warming and related weather patterns are associated with unprecedentedly high ambient temperatures, which can increase morbidity and mortality in livestock animals, including dairy cattleDue to the common ‘row-feeding’ method of hutch-reared calves, hutches are often positioned in a face-off-arrangement (‘bull row’ vs. ‘heifer row’). This way, hutches of bull calves are often exposed to direct sunlight as farmers would like to protect heifer calves from heat load, therefore, bull calves are often at risk of heat stress. The aim of the present study was to assess lying behaviour of shaded (S) and non-shaded (NS) bull calves to determine the effect of heat stress on calf comfort. We hypothesized changes in lying behaviour in response to heat stress, and that calves in shaded hutches would show less discomfort (increased lying times and lower lying down frequencies) than calves housed in hutches without supplemental shade.P = 0.930) and daily average values for THI . The THI recordings indicated an extreme heat load on Day 2 (heat stress day) and Day 3. On Day 2, maximal THI was observed at 16:00 in the NS hutch environment that was higher than those recorded for the S one . Reduced heat load in the S hutch environment compared to the NS one was reflected by daily averages of THI as well on the heat stress day . Similar reductions were found in ambient temperature9 and THI10 in shaded commercial calf hutches during short-term warm episodes in summer. Like on Day 2, maximal  and average THI  were higher in the NS than the S environment for Day 3 (P = 0.003 and P = 0.014). Similar, but moderate differences were found on Day 4 for maximal  and average THI . On Day 5 maximal and average THI for the NS and S hutch environments were 71.0 ± 0.1 vs. 69.5 ± 0.1 and 64.2 ± 0.1 vs. 63.7 ± 0.1 respectively, without significant differences (P = 0.526 and P = 0.545).On Day 1 (control) no group differences were found in maximal . On Days 4 and 5 similar lengths of lying time were observed to Days 1 and 2. The frequency of lying down showed similar patterns between groups during Day 1 (control), with 32% higher values for daytime compared to nighttime (P = 0.003). During the daytime (between 8:00 and 20:00), the frequency of lying down was 50, 33, and 41% higher, respectively, than during the nighttime on Days 2, 3 and 4 . Besides discomfort caused by heat stress, diurnal changes in ambient temperature and anticipation around the time of afternoon feeding11 are possibly reflected in the observed activity patterns.Diurnal patterns of lying time and lying down frequency were observed in both groups throughout the experimental period Fig. . Calves P = 0.551). On the heat stress day, NS calves changed posture 88.4 and 76.6% more often than S ones between 8:00 and 12:00 and 12:00 and 16:00, respectively . Similar group differences were observed also for Day 3 between 8:00 and 12:00 (71.2%) and Day 4 between 12:00 and 16:00 (76.6%), respectively . Increased daytime lying down frequency in NS calves during the most heat straining periods illustrates short-term acclimatory responses12 initiated to compensate for increased heat stress. On Day 5, calves changed posture 44% more often during the daytime compared to nighttime (P < 0.001); however, we found no group difference in lying down frequency (P = 0.732). No study evaluated lying behaviour of calves exposed to heat stress; however, in accordance to the present findings, it was observed that in case of thermal discomfort adult dairy cattle change posture more often to maximize heat dissipation from the body surface14.It was hypothesized that calves in a S environment would show less discomfort than calves exposed to a NS environment. Our hypothesis was supported by significant increases in the frequency of lying down in NS calves during the daytime periods on Days 2, 3, and 4. On Day 1 no effect of shading was observed on the frequency of lying down  that approved the study. The experiment was carried out on a large-scale dairy farm in Hungary (N47°18′191′′ E18°48′336′′), which has a herd of over 1000 lactating Holstein cows. The farm was visited for a 6-day period in the month of August . Preweaned Holstein bull calves (n = 16) at one month of age with a mean body weight of 74 (±3.2) kg were used for the study. Calves were housed in 1.65 × 1.20 m individual hutches with a 1.60 m16. Water from a plastic bucket (7.6 liters), filled twice a day, was provided throughout the study. The diet did not change throughout the experiment.The experiment covered 6 days and was designed and timed based on the expected temperature patterns of the regional weather forecast. Day 0 (habituation to the study environment) was followed by Day 1 : 20.7/26.8), Day 2 : 27.8/38.8) and Days 3–5: ; Day 3: 26.2/33.5, Day 4: 23.7/28.7, Day 5: 21.2/24.7). Between 8:00 and 14:00 on Day 0, a shading structure was prepared over 16 adjacent hutches and pens where experimental calves were kept. A sun shade net with shade rate of 85% was used as shading material and was located 1.9 m above the ground. The following 10 h served as a habituation period for the calves to get used to the novel environment. At 24:00 on Day 1, the shading net was removed from 8 adjacent hutches that remained exposed to direct sunlight throughout the experiment , while the other 8 adjacent hutches remained shaded  for the reminder of the study period. Both S and NS calves were fed the same diet, which did not change throughout the experiment. Calves received one feeding of 2 L of milk replacer at 6:00 and 18:00 and had ad libitum access to chopped alfalfa hay and the starter grain diet , which met the requirements for preweaned Holstein calves17db = dry bulb temperature and Twb = wet bulb temperature.Thermal environment around the animals was assessed by recording the ambient temperature and relative humidity at 10-min intervals. Data loggers were fitted inside the hutches . Daily maxima and averages of the temperature-humidity index (THI) were used to characterize the microclimatic environment:et al.18 for “low-relative-humidity”  climatic regions.This THI was recommended by Bohmanova 7. Data was downloaded using Onset HOBOware Software  and exported to Microsoft Excel. The data logger recorded the g force on the x, y, and z-axes on a scale of −1 to 1. The cut-off values used to categorize logger readings as a specific behaviour (lying vs. standing) were determined based on preliminary observation from video recordings. Information from the x-axis was used to evaluate lying down (g < 0.75) and standing (g ≥ 0.75) with a 30-s sampling interval, considering that lying down and standing bouts are short19. The time spent lying down (lying time) and the frequency of lying down were calculated for 4-h recording intervals and presented as means plus standard error of the mean (SEM).A three-dimension accelerometer  was used to record body posture. The logger was attached to the right hind leg of the calves20. To evaluate the effect of shading on the thermal environment, daily maximal and average values of THI were calculated for all experimental days using all recorded meteorological data with 10-min sampling frequency. Comparisons between S and NS environments were made with the t-test at the significance level of 0.05. Daily variations in lying time were compared using linear mixed models, with group (S vs. NS) and 4-h sampling periods as fixed factors. Due to the Poisson distribution of data and the relatively low sample size, lying down frequency was compared between groups by fitting Poisson regression to GLM models. The model included group and time of day  as fixed factors. All models analyzed behavioural data included calf within treatment as a random factor. Comparison of S and NS groups were made with the Fisher-type z-transformation-based z-test in all models. A P-value < 0.05 was considered significant. All results are expressed as mean plus SEM values.Statistical analyses were performed in the R–3.3.1 statistical environment and language


Drosophila melanogaster development, enriched in neurons and testes, often localized within heterochromatic regions, and important for viability. Strikingly, we find AAGAG transcripts are necessary for male fertility, and that AAGAG RNA depletion results in defective histone-protamine exchange, sperm maturation and chromatin organization. Since these events happen late in spermatogenesis when the transcripts are not detected, we speculate that AAGAG RNA in primary spermatocytes ‘primes’ post-meiosis steps for sperm maturation. In addition to demonstrating essential functions for AAGAG RNAs, comparisons between closely related Drosophila species suggest that satellites and their transcription evolve quickly to generate new functions.Tandemly-repeated DNAs, or satellites, are enriched in heterochromatic regions of eukaryotic genomes and contribute to nuclear structure and function. Some satellites are transcribed, but we lack direct evidence that specific satellite RNAs are required for normal organismal functions. Here, we show satellite RNAs derived from AAGAG tandem repeats are transcribed in many cells throughout  Long arrays of tandemly repeated short DNA sequences (known as satellites) are abundant in centromeres  and periD. melanogaster, simple, tandemly repeated satellite DNAs, such as AAGAG(n) and AATAT(n), comprise ~15–20% of the genome   and RNA-To determine where these transcripts localize within the nucleus, we simultaneously performed antibody staining (IF) for a histone post-translational modification enriched in heterochromatin (H3K9me3), and FISH for both AAGAG RNA and DNA. In cycle 12 embryos, AAGAG RNA is distributed randomly throughout the nucleus  and doesThe presence of AAGAG RNA throughout development suggested a potential role in development or viability. This hypothesis was tested by depleting AAGAG RNA in somatic cells, using actin-GAL4-driven AAGAG shRNA expression . DepletiSurviving act-GAL4-driven AAGAG RNAi adults exhibited partial sterility, prompting further investigation into the distribution and potential functions of AAGAG RNA in the germ line see , A to C.To deplete AAGAG RNA in 4–16 cell spermatogonial cysts, we used the Bag of marbles (Bam)-GAL4  driver tThese results suggested that male infertility upon AAGAG RNA depletion would be caused by defects at stages where AAGAG RNA is expressed. Surprisingly, Bam-GAL4-driven depletion of AAGAG RNA resulted in no gross morphological defects prior to or during meiosis I or II in pupal or adult (0–6 hr and 4–7 days post-eclosion) testes. However, individualized mature sperm DNA was completely absent from the seminal vesicles (SV), in contrast to their abundance in controls , demonstDrosophila melanogaster, at least in part by promoting the histone-protamine transition and/or other post-meiotic steps in sperm maturation.Importantly, fertility defects resulting from AAGAG RNA depletion are partially rescued by simultaneously expressing AAGAG RNA , when both are controlled by the Bam-GAL4 driver. Under these conditions we observe a 2-fold increase in AAGAG RNA signal compared to AAGAG RNAi alone , which iHere, we demonstrate that AAGAG(n) satellite RNAs are transcribed from heterochromatic regions on multiple chromosomes, cluster into nuclear foci, associate with the earliest forms of heterochromatin in embryos, and persist throughout fly development. AAGAG RNA is important for viability, though further investigations are necessary to determine its functions in early development. Most strikingly, we observe that AAGAG RNA is expressed in the male germ-line and is absolutely essential for male fertility.It is surprising that AAGAG RNA is expressed only in primary spermatocytes yet is critical for completing much later stages of sperm development, when AAGAG RNA is not detected. Specifically, defects in late spermatogenesis, including canoe, individualization and maturation stages and the histone-protamine exchange, were observed when AAGAG RNA was depleted in primary spermatocytes, and expression of AAGAG RNA at the same stage partially restored these fertility and sperm defects. It is interesting that aberrant histone-protamine transition and sperm individualization are also observed in Segregation Distorter (SD) testes, where the affected sperm contain abnormally high numbers of another satellite repeat  . We suggAlthough the molecular mechanisms directly impacted by AAGAG RNA are currently unknown, the spatial and temporal disconnect between its expression and depletion phenotypes limit the possibilities. We speculate that proper histone:protamine exchange and post-meiotic chromatin organization require AAGAG RNA in primary spermatocytes to sequester or exclude factors that regulate localization of late-acting proteins or ncRNAs , form esD. melanogaster, comprising ~5% of the genome , and H20 to 40 μl were combined. Reactions were heated to 80°C, 3 min to denature probes, iced 2 min., 4 μl (or 200Units) of T3 (or T7) RNA polymerase (NEB cat. M0378S) added and incubated at 37°C overnight. 2 μl Turbo DNAse (ThermoFisher Cat. AM2238) was then added to degrade DNA templates, incubated at 37°C for 15 min and the reaction stopped by adding 1.6 μl of 500 mM EDTA. Probes were then purified using standard sodium acetate/ethanol purification. Probe concentration was then assessed using Qubit RNA high sensitivity protocols and reagents and stored at −80°C.RNA probes were made by using oligo templates with antisense T3 promoters on the 3’ends, hybridizing an oligo composed of sense T3 promoter so as to create a double stranded 3’ end, or in the case of 359 bp repeat, amplification with oligos containing T3 and T7 promoter ends on genomic DNA using standard protocols. Probe templates were then transcribed with T3 RNA polymerase (or T7 for one strand of 359 bp repeat) and either UTP-biotin or UTP-digoxigenin labels, or in the case of RNA without Uracil, biotin-ATP. Oligos are listed in PBT solution: 1X PBS and 0.1% Tween-20.Western Blocking Reagent 10X: 10% casein in 100 mM maleic acid; 150 mM NaCl; pH 7.5. heated at 60°C for 1 hr to dissolve.PBT block: 1:1 PBT/2X WBR;Hybridization buffer: 50% formamide, 5X SSC, 100 μg/mL heparin, 100 μg/mL sonicated salmon sperm DNA, and 0.1% Tween-20, filtered through a 0.2 μm filter.For clarity, the methods for RNA-FISH probe hybridization and detection are numbered below.RNA probe hybridization for all tissues was carried about according to For ‘non-TSA amplification’, samples were incubated with either mouse anti-digoxigenin-Cy5 (source unknown) or rabbit anti-digoxigenin A488 (Invitrogen cat# 700772) in PBT block at 1/200 dilution for 1 hr at room temperature. Afterwards, samples were incubated 6x’s 10 min each in PBT block, stained with DAPI 10 min, washed 3’xs 10 min. each in PBS and mounted in Prolong-Gold antifade mountant .For samples undergoing ‘TSA amplification for RNA-FISH probe detection,’ samples were incubated with primary antibody ), with 0.2 U/μl protector RNAse inhibitor and incubated overnight at 4°C. Next, samples were washed 6x’s 10 min each in PBT block. The next steps are essentially as per ‘tyramide signal amplification kit’ protocols (ThermoFisher) but with reagents purchased separately: Samples were incubated with 1:100 streptavidin-HRP  in PBT block for 1 hr at room temperature. Samples were then washed in 1:1 PBT/2XWBR 6x’s 10 min each, once with PBT, and 2x’s with PBS. Samples were then incubated with Alexa 647 tyramide  according to company protocols. Essentially, this consisted of adding 1 μl of 30% hydrogen peroxide to 200 μl tyramide signal kit amplification buffer, then diluting this solution 1/100 in tyramide signal amplification buffer for a final hydrogen peroxide concentration of 0.0015%. This solution was then added to the sample and incubated at room temperature for 1 hr in the dark. Samples were then washed 1x with PBS for 10 min, stained with DAPI for 10 min, washed 4x’s with PBS 10 min. each, and mounted in Prolong Gold Antifade mountant.For RNA-FISH of repeat RNAs, 0–8 hr Oregon R embryos were collected on apple juice plates, dechorionated and processed according to . Co-IF RThis protocol is essentially as described in Third instar larvae were dissected in PBS supplemented with 0.2 U/μl Protector RNase Inhibitor. The posterior end of the larvae was removed, then the remaining L3 inverted inside out. The inverted larvae were then transferred to ice cold PBS with 0.2 U/μl RNAse inhibitor. Larvae were then fixed in PBT with 4% formaldehyde for 15 min, then washed 3x, 5 min each with PBT. Larvae were then incubated with 0.1%(vol/vol) DEPC in PBT for 5 min to deactivate endogenous RNAses. Samples were then rinsed 2x’s with PBS.2O2 in PBS 15 min at room temperature and the tube kept open to prevent gas buildup. Samples were then rinsed 2x with PBT 10 min. each.Use of TSA amplification in L3 requires removal of endogenous peroxidases and requires the following protocol after DEPC treatment above and rinsing in PBS: In order to quench endogenous peroxidases, samples were incubated in 350 μl  of 3% HTo all larval samples: Larvae were then permeabilized by incubation in 500 μl cold 80% acetone in water at −20°C 10 min. Samples were then washed 2x, 5 min. per wash with PBT, then post fixed with 4% formaldehyde in PBT for 5 min. Samples were then washed 5x’s with PBT 2 min each. Samples were then rinsed with 1:1 PBT/RNA hybridization solution, then with 100% RNA hybridization solution, and then stored in hybridization solution at −20°C until needed. Samples were then processed according to RNA-FISH protocol  for probe hybridization and either  for non-TSA probe or  for TSA amplification. Larvae w2; RNAseH treatment: 1X RNAseH buffer, 1.5 μl RNAseH ; RNAse one treatment: 1X RNAseH buffer, 1.5 μl RNAse1 (Ambion cat. AM2294); RNAse A treatment: 1x RNAseH buffer, 15 μg RNAseA- at 37°C for 5 hr. Samples were then washed 5x’s in PBT 2 min each, treated with 0.1% DEPC to deactivate any remaining RNAse, then washed in PBT. Samples were then rinsed in 1:1 mixture of PBT:RNA hybridization solution for 2 min and resuspended in 100% hybridization solution. Samples were then processed as per ‘RNA-FISH probe hybridization and primary antibody incubation’ (protocol one above) and protocol ‘TSA amplification for RNA-FISH probe detection’ (protocol three above). For each condition, at least 10 entire embryos were imaged. For RNAs2 or RNAseH treatment:  at 37°C for 2 hr. Samples were then blocked with 2x PBT:WBR 1 hr then processed as per protocol ‘TSA amplification for RNA-FISH probe detection’ (protocol three above). Three embryos treated with RNaseH were imaged, while six treated with RNAseIII were imaged.. After p1 progeny grown at 29°C to mimic conditions used to assess sperm morphological defects. AAGAG RNA was also visualized in RNAi testes grown at 25°C to rule out that temperature affected levels and distribution of AAGAG RNA (not shown). For analysis of AAGAG RNA in Oregon R and XO/XY testes  charged slides, the contents spilled by poking with sharp forceps, a RainX-treated coverslip placed over the testes and both snap frozen in LiN2. The coverslip was then immediately removed with a razor blade and slides stored at −80°C until needed. When ready to process, slides were fixed for 20 min in 4% formaldehyde in PBT, washed three times, 5 min. each wash, in PBT. Samples were then incubated in 80% cold acetone in PBT for 10 min at −20°C and processed as per RNA-FISH for ‘all larval samples’ using protocol two above for detection without TSA amplification. For determination of average AAGAG(n) intensity levels, for each condition at least three testes were imaged, and at least 5 S5 spermatocytes derived from each of these testes were imaged.For analysis of AAGAG RNA in RNAi adult testes , flies wY testes , flies wFlies were grown at 29°C and processed as above in ‘RNA-FISH in adult testes’ up until −80°C storage. Samples were then fixed 20 min in 4% formaldehyde in PBT, passed through an ethanol series (75–85–95%) at −20°C and dried prior to permeabilisation in 1X PBS-0.4% Triton X-100 (0.4 PBT). Samples were then blocked in 0.1PBT with 1% BSA for 1 hr at room temperature, incubated with primary antibodies overnight at 4°C and with secondary antibodies for 1 hr at room temperature . Essentially, purified RNA was denatured for 3 min at 70°C in NorthernMax formaldehyde loading dye. Samples were then run on denaturing agarose gels with 6.9% formaldehyde in MOPS buffer. RNA was transferred to (+) charged nylon membranes in an electroblotter  using 200mA for 30 min. The membrane was then UVC crosslinked and prehybridized with ULTRAhyb Ultrasensitive Hybridization Buffer  at 68°C for 30 min. Biotinylated probes at a concentration of 30 ng/ml were then added to UltraHyb buffer, pre-hybridization solution replaced with solution containing probe and hybridized overnight at 68°C with rotation. The next day, membranes were washed and processed according to Chemiluminescent Nuclei Acid Detection kit manual. For Northern blots shown in D. melanogaster transcriptome data  (−5) to identify potential genomic locations in release 6 of D. melanogaster genome   for AAGAr genome   to AAGAG RNA driven with the UAS/GAL4 system, or in the case of control, a scrambled RNA sequence, using genomic insertion of the pValium20 vector used for the Transgenic RNAi project (TRiP) at Harvard . ImportaE. coli cells.Sense and antisense strands were annealed and ligated into digested pValium20 vector . For annealing, in a 50 μl final volume, 1.5 μl each of 100 μM stock oligos were added to 1X NEBuffer, incubated 4 min at 95°C, then slowly cooled to RT in a 1L beaker filled with 70°C water. Samples were blunt ended with klenow using standard procedures, purified with min-elute PCR purification kit, run on agarose gel, and appropriate size bands removed and purified. Purified bands were digested with Nhe1 and EcoR1 HF enzymes and purified with min-elute PCR purification kit. For cloning, 1 μl of annealed and purified oligo pair complement was added to 30 ng of digested pValium20 vector and ligated with T4 DNA ligase (not quick ligase) at 16°C overnight, and transformed into dh5alpha y[1] v[1]:UAS-AAGAGshRNA:: (shRNA to AAGAG), y[1] v[1]:UAS-scramble shRNA:: (shRNA to scrambled) y[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=VALIUM20-mCherry}attP2 (dsRNA to mCherry) males were crossed to y[1] w[*]:: P{w[+mC]=Act5 C-GAL4}17bFO1/TM6B, Tb[1] (actin-GAL4/Tubby) female virgins see , the num0) sets. From each parental set, individual F1 male progeny  were then allowed to mate with two female Oregon R virgins for 10 days at 25°C. Male flies were counted as sterile if, after 10 days, the male and at least one female were still alive and no larvae, pupae or adult F2 progeny present. Female fertility was calculated as above, with one female RNAi and two Oregon R males. For Bam-GAL4-driven RNAi, female fertility was calculated as above from a minimum of three parental (F0) sets using a minimum of 10 F1 progeny for each. Scrambled RNAi male fertility for this cross was calculated as above from a minimum of four parental (F0) sets, using a minimum of 11 F1 progeny.Flies containing shRNA to AAGAG or scrambled control were mated to different testes GAL4 drivers  at 25°C,0) parental sets, hundreds of F1 individual males, and at both 25°C and 29°C. For rescue experiments, triplicate parental sets were used, where one F1 male  was mated to three Oregon R virgin females for 10 days and fertility assayed as above.AAGAG Bam-GAL4-driven RNAi male fertility of 0% was calculated from >>10 RM, y[1] v[1]/0 females (Bloomington stock # 2494) at 25°C and 0–6 hr testes from FRNA was extracted and cDNA made by established methods. For qPCR, 10 μl 2X Absolute Blue qPCR SYBR low Rox mix  was added, forward and reverse oligos each to 0.15 μM, 0.5 μl cDNA, and water to 20 μl. qPCR conditions were as follows: 95°C, 15 min; 40cycles ; 72°C 30 s performed on AB 7500 Fast Real Time PCR System. Performed in biological triplicates. See  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Drosophila male germ line development, and is essential for male fertility. Although the mechanism of action is still unclear, these results highlight novel roles of nuclear noncoding RNA in chromatin organization and provided insights about in vivo functions that depends on lncRNA.Transcription from repetitive DNA can nucleate heterochromatin formation, a structural role using RNA to decorate chromatin. Mills et al. show a surprising and focused requirement of a simple repeat RNA in development. This work shows that the AAGAG tandem repeat long noncoding RNA localizes in nuclear foci, primes Decision letter after peer review:Drosophila melanogaster" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by James Manley as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Yukiko M Yamashita (Reviewer #3).Thank you for submitting your article "RNA from a simple-tandem repeat is required for male fertility and histone-protamine exchange in The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Essential revisions:The role of satellite RNA in spermatogenesis as presented in this study is extremely interesting, but there are major concerns, which must be addressed prior to publication.1) The authors use one short hairpin RNA (shRNA) construct to knockdown AAGAG RNAs and observe several phenotypes. To their credit, the authors show that the knockdown phenotype on fertility can be partially reversed by ectopic overexpression of the RNA. However, it was surprising that shRNA against mCherry also greatly reduces the AAGAG RNA . This raises concerns about the specificity of the experiment. Moreover, if the AAGAG RNA can be reduced but without phenotype in the mCherry shRNA experiments, then the concern is that the observed phenotypes are due to an off-target or non-specific effect of the particular shRNA. Independent RNAi lines or orthogonal methods to deplete or block AAGAG RNA are necessary to confirm the phenotype. Given that this study focuses on spermatogenesis, RNAi validation (AAGAG RNA-FISH) on AAGAG RNAi testes must also be shown.2) Whereas AAGAG RNAi phenotype is consistent with histone-protamine transition defects, many other upstream causes could lead to histone-protamine transition defects. Accordingly, the observed phenotype does not necessarily pinpoint AAGAG's function in histone-protamine exchange, and thus AAGAG RNA's function remains undefined. But the manuscript is written in a way to strongly indicate AAGAG is directly involved in a 'chromatin based' mechanism that promotes histone-protamine exchange. We suggest that the authors be more cautious in writing (we are not asking to provide 'mechanism' for AAGAG function in histone-protamine transition).3) The Northerns need clarification. The authors show that a 1.5-kb maternal product is loaded, and then RNA levels increase throughout development. There are several technical questions . Regarding strands and satellite-derived RNA, it's not really clear what (+) and (–) mean. While it is ok to arbitrarily assign (+) and (–) with respect to the sequence 'AAGAG', it would be surprising if the orientation within an AAGAG satellite block didn't occasionally flip. For example, a structural rearrangement of AAGAG tandem repeats could generate both types of transcripts (AAGAG and CUCUU) off of one strand. The presence of CUCUU in the spermatogenesis doesn't necessarily mean that the same repeats are transcribed off of both strands. Essential revisions:The role of satellite RNA in spermatogenesis as presented in this study is extremely interesting, but there are major concerns, which must be addressed prior to publication.1) The authors use one short hairpin RNA (shRNA) construct to knockdown AAGAG RNAs and observe several phenotypes. To their credit, the authors show that the knockdown phenotype on fertility can be partially reversed by ectopic overexpression of the RNA. However, it was surprising that shRNA against mCherry also greatly reduces the AAGAG RNA . This raises concerns about the specificity of the experiment. Moreover, if the AAGAG RNA can be reduced but without phenotype in the mCherry shRNA experiments, then the concern is that the observed phenotypes are due to an off-target or non-specific effect of the particular shRNA. Independent RNAi lines or orthogonal methods to deplete or block AAGAG RNA are necessary to confirm the phenotype.We are sorry the way we presented this data caused confusion about the specificity of RNAi. Although the AAGAG RNA signal in mCherry RNAi control lane appears weak, as stated in the legend “The AAGAG RNAi L3 AAGAG RNA top band signal is approximately 86% and 75% reduced compared to either scrambled or mCherry controls, respectively, when normalized to the actin-5c loading control.” Thus, quantitation with proper normalization shows that AAGAG RNA is greatly reduced after AAGAG RNAi compared to either control (though there is a bit less depletion for mCherry vs. Scrambled). Yes, the Northern loading is not optimal, but producing a new Northern is greatly hampered by the departure of the lead and second authors from the lab.More importantly, there are multiple lines of evidence supporting the specificity of the RNAi with respect to the observed phenotypes;1) The Northern results are validated by the AAGAG RNA-FISH  The highly penetrant phenotypes are only observed after AAGAG RNAi, and not with either control.3) The phenotypes are partially (but significantly) rescued by addition of AAGAG RNA.Thus, we believe that concerns about the phenotypes and off-target effects are not warranted. Note that we did try ‘gapmers’, but they did not knockdown AAGAG RNA and exhibited high toxicity after injection into syncytial embryos. We are not aware of other methods, including all current versions of CRISPR-Cas9, etc., that would, without significant development and optimization in flies, be useful for specific knockdown of this type of RNA without affecting underlying DNA or without producing unexpected phenotypes.Given that this study focuses on spermatogenesis, RNAi validation (AAGAG RNA-FISH) on AAGAG RNAi testes must also be shown.RNAi validation of AAGAG RNAi knockdown of AAGAG in testes is shown in Figure 2—figure supplement 3.2) Whereas AAGAG RNAi phenotype is consistent with histone-protamine transition defects, many other upstream causes could lead to histone-protamine transition defects. Accordingly, the observed phenotype does not necessarily pinpoint AAGAG's function in histone-protamine exchange, and thus AAGAG RNA's function remains undefined. But the manuscript is written in a way to strongly indicate AAGAG is directly involved in a 'chromatin based' mechanism that promotes histone-protamine exchange. We suggest that the authors be more cautious in writing (we are not asking to provide 'mechanism' for AAGAG function in histone-protamine transition).not visible in wild-type, well after the primary spermatocyte stage. We have tried to alter the text to state that AAGAG RNA expression in primary spermatocytes is necessary for normal spermatogenesis, loss of AAGAG RNA causes the phenotypes, and direct vs. indirect effects cannot be distinguished.We agree that it is unclear if AAGAG functions directly in histone-protamine exchange and did not intend to strongly convey that conclusion. This is a striking phenotype, but there are many defects observed during sperm maturation. Further, direct effects  are unlikely given that all phenotypes are only visible when the RNA is That said, we have changed the title to emphasize effects on sperm maturation and fertility, rather than histone-protamine exchange.We also included more information showing that AAGAG(37) RNA expression in primary spermatocytes partially rescues the sperm defects seen with AAGAG RNA depletion in primary spermatocytes. Specifically, we added new data to Figure 3E, confirming that rescue with AAGAG(37) RNA expression in primary spermatocytes in AAGAG RNAi background partially rescues individualization defects (only reported% fertile in previous version).Additionally, we added a new figure,  showing that histones retained in AAGAG RNAi testes up to late canoe stage, but not in the control. This is an interesting finding considering that histones are generally removed just prior to late stage canoe and lends more support that AAGAG RNA affects histone:protamine transition (of course without addressing direct vs. indirect mechanism).3) The Northerns need clarification. The authors show that a 1.5-kb maternal product is loaded, and then RNA levels increase throughout development.We do not claim that RNA levels increase throughout development, although it is likely given the increased signal in the Northerns throughout development. Similar levels of RNA were loaded per well (according to Qubit RNA high sensitivity measurements) and similar exposure times were used, but it is impossible to quantify levels between 0-24hr, larval vs. maternal and 2-4hr due to the absence of loading controls and separate northerns for later individual stages. We do not want to say anything at this time about the levels of RNA during development or in different tissues, so if any text suggests changes remains please point them out so we can remove them.There are several technical questions .Figure 1—figure supplement 2 Northern blots now have a larger cutoff and show that the 1,500 nt band is the largest band present. We speculate that this signal is coming from the AAGAG(n) regions adjacent chr3R:3,169,758-3,169,820, chr2R:1,826,691-1,826,740 and/or chrX:22,453,019-22,453,076 regions, as northern blotting to each of these regions produces a 1,500 nt band . It is unclear why they all present as a 1,500 nt band, although it is possible they originate in different sizes and are processed to 1,500 nt or smaller.Regarding strands and satellite-derived RNA, it's not really clear what (+) and (–) mean. While it is ok to arbitrarily assign (+) and (–) with respect to the sequence 'AAGAG', it would be surprising if the orientation within an AAGAG satellite block didn't occasionally flip. For example, a structural rearrangement of AAGAG tandem repeats could generate both types of transcripts (AAGAG and CUCUU) off of one strand.We agree that there could be ‘flipping’ and other structural changes, and we struggled with how to present this complex experiment and data. We intend to use the (+) and (–) only in reference to the probes and annotated DNA sequence, not putative adjacent sequences that may have ‘flipped’ or changed. Figure 1—figure supplement 4A has been changed to provide more clarification and indicate where the probes will bind when designated (+) and (-). Text has also been changed to indicate that orientation of an AAGAG satellite block may occasionally flip (though note it is striking that in embryos we cannot find the complementary RNA foci…if flipping occurred we’d expect to see some).The presence of CUCUU in the spermatogenesis doesn't necessarily mean that the same repeats are transcribed off of both strands.We agree, and did not intend to state they are transcribed off both strands. Again, we want to know if specific text remains that suggests this is the case.


Guanine-rich RNA sequences can fold into four-stranded structures, termed G-quadruplexes (G4-RNAs), whose biological roles are poorly understood, and in vivo existence is debated. To profile biologically relevant G4-RNA in the human transcriptome, we report here on G4RP-seq, which combines G4-RNA-specific precipitation (G4RP) with sequencing. This protocol comprises a chemical crosslinking step, followed by affinity capture with the G4-specific small-molecule ligand/probe BioTASQ, and target identification by sequencing, allowing for capturing global snapshots of transiently folded G4-RNAs. We detect widespread G4-RNA targets within the transcriptome, indicative of transient G4 formation in living human cells. Using G4RP-seq, we also demonstrate that G4-stabilizing ligands (BRACO-19 and RHPS4) can change the G4 transcriptomic landscape, most notably in long non-coding RNAs. G4RP-seq thus provides a method for studying the G4-RNA landscape, as well as ways of considering the mechanisms underlying G4-RNA formation, and the activity of G4-stabilizing ligands. In vivo existence of guanine-rich four-stranded RNA structures (G4-RNAs) has been a matter of debate. Here the authors developed a protocol, G4RP-seq, to capture and identify transcriptome-wide G4-RNA, providing insights into the formation of transient G4-RNA in live human cells. RNAs can adopt intricate bulged, stem–loop structures involving duplex-, triplex-, and quadruplex-RNA motifs4. G-quadruplexes (G4s) are structures formed by Hoogsteen bonding of four guanines to form planar guanine quartet (G-quartet) units, which π-stack on each other, to assemble into columnar four-stranded structures with the central cavity stabilized by monovalent cations . G4 folding is spontaneous in vitro and results in a highly stable structure. While both single-stranded DNA and RNA can fold into G4s, the latter is less studied6, even though G4 formation in RNA molecules is generally more stable, and RNA molecules can fold more readily due to their predominant single-stranded nature in vivo.The biological functions and cellular regulations of RNAs are dependent on their secondary and tertiary structures8. To better understand the roles of G4-RNA in cell biology, there has been a strong interest in mapping the distribution of G4-RNAs within the human transcriptome. However, to date, in vitro and in vivo evidence appear to be contradictory, limiting interpretation of the relevance of G4-RNAs12. In vitro transcription experiments suggest that opportunities exist for G4-RNA formation during co-transcriptional folding of nascent RNA13. Kwok et al.9 used reverse transcription (RT)-stalling coupled with next-generation sequencing to map thousands of G4-RNA sites in vitro, showing widespread potential G4-forming sites within the human transcriptome. In contrast, Guo and Bartel10 showed through in vivo DMS-mediated RNA modification with RT-stall-sequencing that G4s are nearly entirely in an unfolded state in vivo in mammalian cells. This observation is surprising since it has widely been assumed that G4-RNAs are formed in vivo, at least transiently. Conversely, evidence for in vivo G4 formation has been provided by cellular imaging studies using G4-specific antibodies and probes12. To reconcile these results, we hypothesized that G4-RNAs must be able to form, at least transiently, in live human cells, and that the identity of these G4-RNAs may provide valuable insights into their regulatory mechanisms and functions. However, because none of the above methods are suitable for capturing transient G4s, an alternative approach was needed.Formation of G4-RNA has recently been implicated in key RNA metabolism events, including the regulation of RNA processing and translationHere, we report on a small-molecule-based approach to assessing the existence and the identity of transient G4-RNAs in the human transcriptome. We designed a biotinylated version of a previously characterized G4-specific ligand/probe, template-assembled synthetic G-quartet (TASQ), which self-assembles into a synthetic G-quartet upon association with a G4 target through end-quartet stacking. We developed a protocol, G4RP-seq (G4-RNA-specific precipitation and sequencing) using BioTASQ to capture G4-RNAs from human breast cancer cells in log-phase growth. Using this protocol to characterize in vivo transcriptomic landscapes, we showed that more G4s are present in gene transcripts that are GC-rich and have higher densities of predicted G4 motifs. We also evaluated G4 ligand-induced changes to the G4-RNA landscape following treatments with the G4 ligand, BRACO-19 or RHPS4, showing both similarities and differences in their respective induction profiles. Our data show that G4-RNAs can be ligand-induced in diverse RNA entities that include long non-coding RNAs; further, differential G4-RNAs induced by G4 ligands suggest that specific G4 structure–ligand interactions could be exploited.15, known for their high G4-selectivity and, for some of them, their ability to track G4-RNAs in live cells (N-TASQ)16. Detailed synthesis and characterization of the biotinylated TASQ, or BioTASQ, can be found in Supplementary Figure 17 and electrospray ionization mass spectrometry (ESI-MS) analyses  three G4-DNAs ;20 (b) one G4-RNA ;22 and (c) one duplex-DNA as a control (F-DS-T) -labeled nucleic acids since fluorescence signal measurements allow for convenient and sensitive detection of the ligand/G4 assemblies. We performed these experiments with: (a) three G4-DNAs ; (b) three G4-RNAs ; and (c) one duplex-DNA as control (F-Duplex). Labeled oligonucleotides (1 μM) were incubated with BioTASQ (20 μM) and streptavidin-coated magnetic beads . After overnight incubation at 25 °C, streptavidin beads were precipitated, the supernatant removed, and the beads resuspended in denaturing buffer before thermal denaturation (8 min at 90 °C). After separation from the beads (via centrifugation and magnet immobilization), fluorescent signals from the supernatant solutions were measured to quantify nucleic acid capture. Our results confirmed the efficiency of BioTASQ-mediated G4 capture  G4s . The preference of BioTASQ for type I G4, which displays accessible external G-quartets, is expected, given that TASQs are sterically demanding ligands that require accessible, loopless G-quartets for binding G4 targets efficiently. This property represents a limitation to the use of BioTASQ for G4 detection, especially for G4-DNAs, which have higher conformational diversity than G4-RNAs. We have not yet tested the affinity of BioTASQ on the recently reported antiparallel G4-RNA29. While the cellular prevalence of G4-RNA with antiparallel topology remains to be established, they may provide key insights into the topological preference of TASQ ligands. We confirmed that the streptavidin bead/BioTASQ system did not extract duplex-DNA (0.7-fold). We further confirmed G4 selectivity of BioTASQ via competitive pull-down experiments, which we performed with F-SRC and F-NRAS (1 μM) in the presence of an excess of duplex-DNA  or DNA extracts . The capture efficiencies of the fluorescently labeled G4-RNA were not significantly affected by an excess of synthetic duplexes  or with DNA extracts . We also tested BioTASQ/streptavidin association with an excess of either biotin  or PNADOTASQ   Fig. . Togethe30. MCF7 cells were first crosslinked with formaldehyde to halt biological processes and stabilize transient structural interactions. Harvested cells were then sonicated briefly to release cellular content. Cell lysates were incubated with a high concentration of BioTASQ (100 µM) overnight  protocol, a modified version of the commonly used RNA-immunoprecipitation (RIP) protocol31. G4RP-qPCR analysis of RNAs extracted from the BioTASQ-enriched fractions showed that non-specific binding was negligible (black bars)   with gene-specific primers to confirm the efficiency of the G4RP protocolrs) Fig.  while ders) Fig. .Fig. 2Is33. We chose the treatment ligand doses to be between the respective IC15 and IC25 , as determined by growth kinetics profiling of MCF7 in the presence of G4 ligands, compared with vehicle controls . BioTASQ enrichment of these targets was quantified using RT-qPCR and compared between the non-crosslinked and crosslinked samples , not only harbor but contribute to a substantial proportion of cellular G4s37. For the sequencing analysis, two comparisons were required: BioTASQ versus input ), and G4 ligand-treated versus untreated cells (given as Enrichment Score Change (ΔES))  in human breast cancer cells harvested at log-phase growth. Due to the crosslinking step in the G4RP protocol, we expected to capture global levels of transient G4s in the transcriptome. Notably, as G4 ligand treatment resulted in gene expression changes in human cellsWe observed BioTASQ enrichment of many gene transcripts, suggesting the existence of widespread G4-RNAs, at least transiently, in live human cells. This observation was expected since we were capturing a snapshot of the G4-RNA landscape in which some G4-rich sequences were in folded states, while others were in unfolded states. While the sequencing depth was not high enough to detect subtle changes in individual G4-forming sequences, we were able to confidently determine gene-level changes by focusing on a subset of highly abundant transcripts, filtered by high normalized mean read counts (as an estimate of relative expression level). We compared ES for each condition to generate initial lists of gene transcripts filtered by a minimal abundance threshold  (Supplementary Data p < 0.0001)   motifs  in each gene transcript. The ratios of the number of pG4s to gene length were calculated and then plotted against the transcripts ranked by their ES  Fig. . ES was 89) Fig. . To eval ES Fig. . CompariMALAT1, RPPH1, RMRP, and XIST. The presence of MALAT1 on this list was unexpected, as it has been previously reported as a G4-forming lncRNA in vitro10. Interestingly, residual rRNAs (that escaped from the ribodepletion step during library preparation) were also among the most depleted within this list.We also found that highly expressed lncRNAs had some of the lowest relative G4 levels, as shown by their low ES Fig. . These b43.We evaluated ligand-induced changes in the G4-RNA landscapes by applying G4RP-seq to samples treated with G4 ligands and calculating an Enrichment Score Change (ΔES) from the ratio of treated versus non-treated samples. By filtering the initial list ΔES > 1.75, we found BioTASQ enrichment in 251 and 463 gene transcripts to be highly induced by BRACO-19 and RHPS4, respectively   and at least 500 mean read counts, the lncRNAs sts Fig. . Some RN44.When we compared the absolute number of pG4 motifs  between the three treatment conditions ranked by their respective ES, we observed differential changes in pG4 profiles between the two G4 ligand treatments Fig. . pG4 scoMALAT145, XIST46, and RPPH147. Fold change differences between G4RP-qPCR and G4RP-seq were seen and expected due to the small qPCR region amplified. Nevertheless, the qPCR results were consistent with those obtained from sequencing, confirming that these lncRNAs were targets of G4 ligands 10, but that G4-formation is actively counteracted by the actions of helicases and/or other RNA-binding proteins52. We anticipate that there may be a window of opportunity for ligand-induced G4-formation/stabilization between the initial creation of nascent RNA and the RNA–RNA/RNA–protein interactions that are part of the co-transcriptional maturation stage of the ribonucleoprotein complex13 . As dysregulation of lncRNAs has been implicated in various human diseases, including cancers, cardiovascular, and neurodegenerative diseases, we anticipate that targeting G4s within lncRNAs may present a valuable therapeutic strategy to alter the functions of these RNA entities59.While BRACO-19 and RHPS4 G4-profiles are distinct in several aspects, they also share common targets within the in vivo MCF7 transcriptome, most notably in the abundant lncRNAs. We anticipate that these targets are of interest, as they contain G4 targets that can be accessed and stabilized by both tested G4 ligands, suggesting that these G4s may be druggable. We propose that lncRNAs can spontaneously form G4s  played more important roles at specific sites, data from G4RP-seq could be studied in combination with other functional genomic strategies  to better characterize the interactions between G4 genome and transcriptome (collectively the G4ome).The G4RP-seq protocol has certain limitations and, we anticipate, can be further improved. One concern is that BioTASQ is itself a G4 ligand and thus may alter the G4 landscape. However, as demonstrated, the chemical crosslinking step before BioTASQ binding serves to minimize the effects of BioTASQ-induced stabilization  in 100 μL  of 10 mM lithium cacodylate buffer (pH 7.2) plus 10 mM KCl/90 mM LiCl  or plus 1 mM KCl/99 mM LiCl  with 0.2 μM of labeled oligonucleotide and 0, 1, 5, or 10 µM of BioTASQ. Competitive experiments were carried out with labeled oligonucleotide (0.2 μM), 10 µM BioTASQ, and increasing amounts  of the unlabeled competitor ds26. After a first equilibration step , a stepwise increase of 1 °C every 30 s for 65 cycles to reach 90 °C was performed, and measurements were made after each cycle. Final data were analyzed with Excel (Microsoft Corp.) and OriginPro®9.1 (OriginLab Corp.). The emission of FAM was normalized (0 to 1), and T1/2 was defined as the temperature for which the normalized emission is 0.5; ΔT1/2 values are means of triplicates.The sequences of the oligonucleotides used herein are listed in Supplementary Table λex = 492 nm; λem = 516 nm). Data were analyzed with Excel and OriginPro®9.1. FAM emission was normalized as follows: the FAM emission of the three control wells (without BioTASQ) was collected and normalized to 1; then, the FAM emission of the three wells comprising labeled oligonucleotides, BioTASQ, and beads were collected and compared to the control experiments. This allowed for a direct quantification of the BioTASQ capture efficiency. Competitive experiments were performed with BioTASQ (20 µM), labeled oligonucleotides (1 µM), and MagneSphere® beads (25 µL) along with the either nucleic acid competitors, ds17 (20 µM), ds26 (20 µM) or calf thymus DNA , or small-molecule competitors, biotin (80 µM) or PNADOTASQ (10 µM). All experiments were performed in triplicates.The sequences of the oligonucleotides used herein are listed in Supplementary Table 2 atmosphere-controlled incubator (HERAcell). Standard cell-culturing procedures were employed including aspiration and washing with phosphate-buffered saline . Cells were trypsinized using Trypsin-EDTA (0.25%) (Gibco). Cell counting was performed using a Coulter Counter (Beckman Coulter).Human breast cancer cells MCF7 was obtained from American Type Culture Collection (ATCC). The cells were culture in Dulbecco's modified Eagle's medium (Gibco) supplemented with 5% synthetic fetal bovine serum  and 100 U penicillin–streptomycin mixture (Gibco). Cells were incubated at 37 °C in a humidified, 5% CO63. Phase confluency was used to measure cellular proliferation under a range of doses. The maximum and minimum achievable confluency values under these conditions were used for normalization. Three biological replicates were used to produce the dose–response curve. Doses between LD15 to LD25 were calculated from dose–response curve profiles , or RHPS4 1.5 µM (LD25) for 72h. Cells were then crosslinked using 1% formaldehyde/PBS for 5 min at 25 °C and the crosslinking was then quenched with 0.125 M glycine for 5 min. Cells were scraped and resuspended in G4RP buffer , homebrew protease inhibitor cocktail). Cells were then sonicated using a Covaris m220 Ultrasonicator using default settings at 10% duty for 2 min. The sonicated fractions were then incubated with 100 µM BioTASQ (or 100 µM biotin for negative controls) overnight at 4 °C. Five percent of the sonicate was collected as input control. Ten micrograms of streptavidin-magnetic beads (Promega) was added and the extract was incubated for 2 h at 4 °C. Magnetic beads were then washed four times in G4RP buffer for 5 min. The beads were then incubated at 70 °C for 1 h to reverse crosslink. TRIZOL was then used to extract the RNA from the beads using the manufacturer’s instructions.MCF7 cells were seeded at 3.5 × 10[5] cells per 10-cm dish before treatment with either vehicle (PBS), BRACO 5 µg/mL (LDC(t) values of pull-down samples were normalized to the input control. Three biological replicates were used for all qPCR-based quantifications. Exon-spanning primers for quantifying mRNA levels were derived from Primerbank31.The primer sets used for RT-qPCR are listed in Supplementary Table 37. Exon feature count and annotation to genes were performed using HTSeq38. DESeq239 was used for normalization and differential gene expression analyses. Further filtering and analyses were performed on Excel. The analysis workflow is outlined in Supplementary Figure 42. Detailed list of gene transcripts can be found in Supplementary Data G4RP samples were DNAase-treated, briefly thermally fragmented and ribo-depleted using the Illumina Ribo-Zero rRNA removal kit. RNA library preparation was performed using the Illumina TruSeq RNA Library Prep kit by following the manufacturer’s instructions. Two replicates  along with an input for each condition were paired end sequenced at 2 × 75 bp using NextSeq 500. The sequenced reads were mapped to the human reference genome hg19 assembly using HISAT2t-test. Linear regression goodness of fit was determined from coefficient of correlation (R2) or Pearson correlation (R), and non-zero slope significance was given as p-value <0.05. All p-values <0.05 were considered significant unless specified otherwise.Graphs were produced by Microsoft Excel or GraphPad Prism. Statistical testing for multiple groups of dataset were performed using one-way or two-way ANOVA, and multiple comparisons corrected by the Bonferroni’s method. Statistical comparisons of the average of two groups were performed using two-tailed Student's Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary ItemsSupplementary Data 1Reporting Summary