sRNA Species Research Articles

Long noncoding RNAs underlie multiple domestication traits and leafhopper resistance in soybean.

The origin and functionality of long noncoding RNA (lncRNA) remain poorly understood. Here, we show that multiple quantitative trait loci modulating distinct domestication traits in soybeans are pleiotropic effects of a locus composed of two tandem lncRNA genes. These lncRNA genes, each containing two inverted repeats, originating from coding sequences of the MYB genes, function in wild soybeans by generating clusters of small RNA (sRNA) species that inhibit the expression of their MYB gene relatives through post-transcriptional regulation. By contrast, the expression of lncRNA genes in cultivated soybeans is severely repressed, and, consequently, the corresponding MYB genes are highly expressed, shaping multiple distinct domestication traits as well as leafhopper resistance. The inverted repeats were formed before the divergence of the Glycine genus from the Phaseolus-Vigna lineage and exhibit strong structure-function constraints. This study exemplifies a type of target for selection during plant domestication and identifies mechanisms of lncRNA formation and action.

MRNA cleavage by 21-nucleotide phasiRNAs determines temperature-sensitive male sterility in rice.

Temperature-sensitive male sterility is one of the core components for hybrid rice (Oryza sativa) breeding based on the 2-line system. We previously found that knockout of ARGONAUTE 1d (AGO1d) causes temperature-sensitive male sterility in rice by influencing phased small interfering RNA (phasiRNA) biogenesis and function. However, the specific phasiRNAs and their targets underlying the temperature-sensitive male sterility in the ago1d mutant remain unknown. Here, we demonstrate that the ago1d mutant displays normal female fertility but complete male sterility at low temperature. Through a multiomics analysis of small RNA (sRNA), degradome, and transcriptome, we found that 21-nt phasiRNAs account for the greatest proportion of the 21-nt sRNA species in rice anthers and are sensitive to low temperature and markedly downregulated in the ago1d mutant. Moreover, we found that 21-nt phasiRNAs are essential for the mRNA cleavage of a set of fertility- and cold tolerance-associated genes, such as Earlier Degraded Tapetum 1 (EDT1), Tapetum Degeneration Retardation (TDR), OsPCF5, and OsTCP21, directly or indirectly determined by AGO1d-mediated gene silencing. The loss of function of 21-nt phasiRNAs can result in upregulation of their targets and causes varying degrees of defects in male fertility and grain setting. Our results highlight the essential functions of 21-nt phasiRNAs in temperature-sensitive male sterility in rice and suggest their promising application in 2-line hybrid rice breeding in the future.

A Non-Canonical Pathway Induced by Externally Applied Virus-Specific dsRNA in Potato Plants.

The external application of double-stranded RNA (dsRNA) has recently been developed as a non-transgenic approach for crop protection against pests and pathogens. This novel and emerging approach has come to prominence due to its safety and environmental benefits. It is generally assumed that the mechanism of dsRNA-mediated antivirus RNA silencing is similar to that of natural RNA interference (RNAi)-based defence against RNA-containing viruses. There is, however, no direct evidence to support this idea. Here, we provide data on the high-throughput sequencing (HTS) analysis of small non-coding RNAs (sRNA) as hallmarks of RNAi induced by infection with the RNA-containing potato virus Y (PVY) and also by exogenous application of dsRNA which corresponds to a fragment of the PVY genome. Intriguingly, in contrast to PVY-induced production of discrete 21 and 22 nt sRNA species, the externally administered PVY dsRNA fragment led to generation of a non-canonical pool of sRNAs, which were present as ladders of ~18-30 nt in length; suggestive of an unexpected sRNA biogenesis pathway. Interestingly, these non-canonical sRNAs are unable to move systemically and also do not induce transitive amplification. These findings may have significant implications for further developments in dsRNA-mediated crop protection.

Small RNA Profiling by Next-Generation Sequencing Using High-Definition Adapters.

Next-generation sequencing (NGS) of small RNA (sRNA) cDNA libraries permits the identification and characterization of sRNA species de novo. However, the method through which these libraries are constructed can often introduce artifacts such as over- or underrepresentation of specific sequences or adapter oligonucleotides due to sequence biases held by the enzymes used. In this chapter we describe a protocol for sRNA library construction making use of high-definition (HD) adapters for the Illumina sequencing platform, which reduce ligation bias. This protocol leads to drastically reduced direct 5'/3' adapter ligation products and can be used for the synthesis of sRNA libraries from total RNA or sRNA of various plant, animal, and fungal samples. This protocol also includes a method for total RNA extraction from plant leaf and cultured cells or body fluids.

Serum-Exosome-Derived miRNAs Serve as Promising Biomarkers for HCC Diagnosis

Serum exosomes are emerging as key liquid biopsy biomarkers for the early diagnosis of cancer. However, the proportion and distribution of small RNA (sRNA) species from serum exosomes of hepatocellular carcinoma (HCC) patients remain unclear. Effective and reliable biomarkers for HCC diagnosis should be explored. In this study, we aimed to use sRNA sequencing to profile the sRNAs of serum exosomes in HCC and non-tumor donors. The serum exosomes of 124 HCC patients and 46 non-tumor donors were enrolled for detecting the values of the potential biomarkers for the diagnosis of HCC. We found that miRNAs accounted for the maximal percentage of all types of sRNAs both in the serum exosomes of HCC patients and non-tumor donors. This indicated that the serum-exosome-derived microRNAs (miRNAs) were the most valuable as potential biomarkers in HCC diagnosis. Then, miRNAs were set as research candidates. In our Chinese cohorts, three serum-exosome-derived miRNAs (miR-122-5p, let-7d-5p, and miR-425-5p) could be promising biomarkers for distinguishing HCC patients from non-tumor donors. In addition, they were preferred for the early diagnosis of HCC. We also presented the base distribution of some novel serum-exosome-derived miRNAs and described the potential values as biomarkers. The results suggested that the serum-exosome-derived miRNAs were the most crucial sRNA species and they highlighted the potential of serum-exosome-derived miRNAs as promising biomarkers for HCC diagnosis.

The Small RNA AmiL Regulates Quorum Sensing-Mediated Virulence in Pseudomonas aeruginosa PAO1.

ABSTRACTPseudomonas aeruginosa is an opportunistic and nosocomial pathogen of humans with hundreds of its virulence factors regulated by quorum sensing (QS) system. Small noncoding RNAs (sRNAs) are also key regulators of bacterial virulence. However, the QS regulatory sRNAs (Qrrs) that have been characterized in P. aeruginosa are still largely unknown. Here, sRNA AmiL (PA3366.1) in the amiEBCRS operon of PAO1 was identified as a novel Qrr by transcriptome sequencing (RNA-Seq). The expression of AmiL was negatively regulated by the las or rhl system, of which RhlR probably inhibited its transcription. AmiL deletion mutant and overexpressing strains were constructed in PAO1. Broad phenotypic changes were found, including reduced pyocyanin synthesis, elastase activity, biofilm formation, hemolytic activity, and cytotoxicity, as well as increased rhamnolipid production and swarming motility. AmiL appears to be a new regulator that influences diverse QS-mediated virulence. Furthermore, AmiL directly targeted PhzC, a key member of pyocyanin synthesis. AmiL also negatively regulated lasI expression in the early growth of PAO1, but predominantly increased rhlI expression and C4-HSL production in the middle and late stages. Therefore, a novel QS-sRNA signaling cascade of las/rhl (RhlR)-AmiL-PhzC/las/rhl was demonstrated, and it will help to shed new light on the virulence regulatory network of P. aeruginosa PAO1.IMPORTANCE P. aeruginosa is a common nosocomial pathogen that causes diverse opportunistic infections in humans. The virulence crucial for infection is mainly regulated by QS. Small noncoding RNAs (sRNAs) involved in virulence regulation have also been identified in many bacteria. Recently, there is a growing interest in the new sRNA species, QS regulatory sRNAs (Qrrs). Understanding Qrrs-mediated regulation in P. aeruginosa virulence is therefore important to combat infection. In this study, a previously uncharacterized sRNA AmiL in PAO1 has been identified as a novel Qrr. It has been found to influence diverse QS-mediated virulence factors including pyocyanin, elastase, rhamnolipid, and hemolysin, as well as biofilm formation, swarming motility, and cytotoxicity. Furthermore, PhzC essential for pyocyanin synthesis was a direct target of AmiL. QS gene expression and C4-HSL production were also regulated by AmiL. This study provides insights into the roles of Qrr AmiL in modulating P. aeruginosa virulence.

Levels of seminal tRNA-derived fragments from normozoospermic men correlate with the success rate of ART.

Decreased fertility is becoming an important social and medical problem and the male factor is involved in at least half of infertility cases. Since conventional semen analysis provides limited prediction of male fertility; in this work, we evaluated the potential use of seminal small RNAs (sRNA) as markers of semen quality in ART. Our bioinformatic analyses of available sRNA-seq databases showed that the most abundant sRNA species in seminal plasma of normozoospermic men are tRNA-derived fragments (tRFs), a novel class of regulatory sRNAs. These molecules not only exert their function within cells but also are released into the extracellular environment where they could carry out signaling functions. To evaluate whether the assessment of seminal tRFs in normozoospermic men has a predictive value for the clinical outcome in ART, we performed a prospective study with couples who underwent ICSI cycles with donated oocytes. The results obtained demonstrated that levels of 5'tRF-Glu-CTC, 5'tRF-Lys-CTT, and 5'tRF-Gly-GCC are significantly elevated in seminal samples from cases with repeated failed ICSI cycles, suggesting a potential association between increased seminal tRFs and unexplained male infertility. Interestingly, these tRFs showed a negative association with seminal testosterone, highlighting their involvement in male endocrinology. Our findings also suggest that tRFs could play a role in modulating male reproductive function in response to physiological stress since they showed significant associations with the levels of sperm DNA fragmentation in couples that achieved pregnancy but not in cases with failed ICSI cycles where seminal cortisol levels correlate with sperm quality.

Huntington's disease brain-derived small RNAs recapitulate associated neuropathology in mice.

Progressive motor alterations and selective death of striatal medium spiny neurons (MSNs) are key pathological hallmarks of Huntington's disease (HD), a neurodegenerative condition caused by a CAG trinucleotide repeat expansion in the coding region of the huntingtin (HTT) gene. Most research has focused on the pathogenic effects of the resultant protein product(s); however, growing evidence indicates that expanded CAG repeats within mutant HTT mRNA and derived small CAG repeat RNAs (sCAG) participate in HD pathophysiology. The individual contribution of protein versus RNA toxicity to HD pathophysiology remains largely uncharacterized and the role of other classes of small RNAs (sRNA) that are strongly perturbed in HD is uncertain. Here, we demonstrate that sRNA produced in the putamen of HD patients (HD-sRNA-PT) are sufficient to induce HD pathology in vivo. Mice injected with HD-sRNA-PT show motor abnormalities, decreased levels of striatal HD-related proteins, disruption of the indirect pathway, and strong transcriptional abnormalities, paralleling human HD pathology. Importantly, we show that the specific blockage of sCAG mitigates HD-sRNA-PT neurotoxicity only to a limited extent. This observation prompted us to identify other sRNA species enriched in HD putamen with neurotoxic potential. We detected high levels of tRNA fragments (tRFs) in HD putamen, and we validated the neurotoxic potential of an Alanine derived tRF in vitro. These results highlight that HD-sRNA-PT are neurotoxic, and suggest that multiple sRNA species contribute to striatal dysfunction and general transcriptomic changes, favoring therapeutic strategies based on the blockage of sRNA-mediated toxicity.

Comparative Analysis of Transcriptome and sRNAs Expression Patterns in the Brachypodium distachyon- Magnaporthe oryzae Pathosystems.

The hemibiotrophic fungus Magnaporthe oryzae (Mo) is the causative agent of rice blast and can infect aerial and root tissues of a variety of Poaceae, including the model Brachypodium distachyon (Bd). To gain insight in gene regulation processes occurring at early disease stages, we comparatively analyzed fungal and plant mRNA and sRNA expression in leaves and roots. A total of 310 Mo genes were detected consistently and differentially expressed in both leaves and roots. Contrary to Mo, only minor overlaps were observed in plant differentially expressed genes (DEGs), with 233 Bd-DEGs in infected leaves at 2 days post inoculation (DPI), compared to 4978 at 4 DPI, and 138 in infected roots. sRNA sequencing revealed a broad spectrum of Mo-sRNAs that accumulated in infected tissues, including candidates predicted to target Bd mRNAs. Conversely, we identified a subset of potential Bd-sRNAs directed against fungal cell wall components, virulence genes and transcription factors. We also show a requirement of operable RNAi genes from the DICER-like (DCL) and ARGONAUTE (AGO) families for fungal virulence. Overall, our work elucidates the extensive reprogramming of transcriptomes and sRNAs in both plant host (Bd) and fungal pathogen (Mo), further corroborating the critical role played by sRNA species in the establishment of the interaction and its outcome.

Methodologies for Discovery and Quantitative Profiling of sRNAs in Potato.

Small RNAs (sRNAs) are short noncoding RNAs involved in the regulation of a wide range of biological processes in plants. Advances in high-throughput sequencing and development of new computational tools had facilitated the discovery of different classes of sRNAs, their quantification, and elucidation of their functional role in gene expression regulation by target transcript predictions. The workflow presented here allows identification of different sRNA species: known and novel potato miRNAs, and their sequence variants (isomiRs), as well as identification of phased small interfering RNAs (phasiRNAs). Moreover, it includes steps for differential expression analysis to search for regulated sRNAs across different tested biological conditions. In addition, it describes two different methods for predicting sRNA targets, in silico prediction, and degradome sequencing data analysis. All steps of the workflow are written in a clear and user-friendly way; thus they can be followed also by the users with minimal bioinformatics knowledge. We also included several in-house scripts together with valuable notes to facilitate data (pre)processing steps and to reduce the analysis time.

Identification of oligo-adenylated small RNAs in the parasite Entamoeba and a potential role for small RNA control

BackgroundThe RNA interference (RNAi) pathway is a gene regulation mechanism that utilizes small RNA (sRNA) and Argonaute (Ago) proteins to silence target genes. Our previous work identified a functional RNAi pathway in the protozoan parasite Entamoeba histolytica, including abundant 27 nt antisense sRNA populations which associate with EhAgo2–2 protein. However, there is lack of understanding about the sRNAs that are bound to two other EhAgos (EhAgo2–1 and 2–3), and the mechanism of sRNA regulation itself is unclear in this parasite. Therefore, identification of the entire pool of sRNA species and their sub-populations that associate with each individual EhAgo protein would be a major step forward.ResultsIn the present study, we sequenced sRNA libraries from both total RNAs and EhAgo bound RNAs. We identified a new population of 31 nt sRNAs that results from the addition of a non-templated 3–4 adenosine nucleotides at the 3′-end of the 27 nt sRNAs, indicating a non-templated RNA-tailing event in the parasite. The relative abundance of these two sRNA populations is linked to the efficacy of gene silencing for the target gene when parasites are transfected with an RNAi-trigger construct, indicating that non-templated sRNA-tailing likely play a role in sRNA regulation in this parasite. We found that both sRNA populations (27 nt and 31 nt) are present in the related parasite Entamoeba invadens, and are unchanged during the development. In sequencing the sRNAs associating with the three EhAgo proteins, we observed that despite distinct cellular localization, all three EhAgo sRNA libraries contain 27 nt sRNAs with 5′-polyphosphate (5′-polyP) structure and share a largely overlapping sRNA repertoire. In addition, our data showed that a fraction of 31 nt sRNAs associate with EhAgo2–2 but not with its mutant protein (C-terminal deletion), nor other two EhAgos, indicating a specific EhAgo site may be required for sRNA modification process in the parasite.ConclusionWe identified a new population of sRNA with non-templated oligo-adenylation modification, which is the first such observation amongst single celled protozoan parasites. Our sRNA sequencing libraries provide the first comprehensive sRNA dataset for all three Entamoeba Ago proteins, which can serve as a useful database for the amoeba community.

The abundance of a transfer RNA-derived RNA fragment small RNA subpopulation is enriched in cauda spermatozoa

The small RNA (sRNA) landscape of mammalian spermatozoa is considerably altered as these gametic cells migrate through the segment specific microenvironments of the epididymis. More specifically, the microRNA (miRNA) species of sRNA dominates the sRNA landscape of spermatozoa of the proximal caput segment of the epididymis. However, in sperm cells sourced from the distal cauda epididymal segment, the transfer RNA (tRNA)-derived RNA fragment (tRF) sRNA species is the most abundant. Here we show that the 5′ halves of fifteen mature tRNAs were used as processing substrates for the production of a specific subpopulation of tRF sRNAs, 30 to 33 nucleotides (30–33-nt) in length. A quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) approach was used to experimentally validate the sRNA sequencing identified trend of enriched abundance of this specific 30–33-nt tRF subpopulation in cauda spermatozoa. The length, and exclusive alignment of the cauda spermatozoa enriched tRF subpopulation to the 5′ half of each processed tRNA precursor, identified ANGIOGENIN (ANG) as the endonuclease likely responsible for tRF production in the mouse epididymis: a prediction confirmed via immunoblotting assessment of ANG abundance in spermatozoa sourced from the caput, corpus and cauda epididymal segments. When taken together with our previous profiling of miRNA and Piwi-interacting RNA (piRNA) sRNA abundance in spermatozoa sourced from the three segments of physiologically normal mouse epididymides, the tRF profile reported here adds greater depth of coverage to the global sRNA landscape of the mouse epididymis; a roadmap constructed to assist with the future molecular characterization of sRNA-directed responses to a wide range of imposed environmental stressors.

Effects of Host-Adaptive Mutations on Hop Stunt Viroid Pathogenicity and Small RNA Biogenesis.

Accidental transmission of hop stunt viroid (HSVd) from grapevine to hop has led to several epidemics of hop stunt disease with convergent evolution of HSVd-g(rape) into HSVd-h(op) containing five mutations. However, the biological function of these five mutations remains unknown. In this study, we compare the biological property of HSVd-g and HSVd-h by bioassay and analyze HSVd-specific small RNA (HSVd-sRNA) using high-throughput sequencing. The bioassay indicated an association of these five mutations with differences in infectivity, replication capacity, and pathogenicity between HSVd-g and HSVd-h, e.g., HSVd-g induced more severe symptoms than HSVd-h in cucumber. Site-directed mutagenesis of HSVd-g showed that the mutation at position 54 increased pathogenicity. HSVd-sRNA analysis of cucumber and hop plants infected with different HSVd variants showed that several sRNA species containing adaptive nucleotides were specifically down-regulated in plants infected with HSVd-h. Several HSVd-sRNAs containing adaptive mutations were predicted to target cucumber genes, but changes in the levels of these genes were not directly correlated with changes in symptom expression. Furthermore, expression levels of two other cucumber genes targeted by HSVd-RNAs, encoding ethylene-responsive transcription factor ERF011, and trihelix transcription factor GTL2, were altered by HSVd infection. The possible relationship between these two genes to HSVd pathogenicity is discussed.

Manatee: detection and quantification of small non-coding RNAs from next-generation sequencing data

Small non-coding RNAs (sncRNAs) play important roles in health and disease. Next Generation Sequencing (NGS) technologies are considered as the most powerful and versatile methodologies to explore small RNA (sRNA) transcriptomes in diverse experimental and clinical studies. Small RNA-Seq (sRNA-Seq) data analysis proved to be challenging due to non-unique genomic origin, short length, and abundant post-transcriptional modifications of sRNA species. Here, we present Manatee, an algorithm for the quantification of sRNA classes and the detection of novel expressed non-coding loci. Manatee combines prior annotation of sRNAs with reliable alignment density information and extensive rescue of usually neglected multimapped reads to provide accurate transcriptome-wide sRNA expression quantification. Comparison of Manatee against state-of-the-art implementations using real and simulated data demonstrates its high accuracy across diverse sRNA classes. Manatee also goes beyond common pipelines by identifying and quantifying expression from unannotated loci and microRNA isoforms (isomiRs). It is user-friendly, can be easily incorporated in pipelines, and provides a simplified output suitable for direct usage in downstream analyses and functional studies.

Features of sRNA biogenesis in rice revealed by genetic dissection of sRNA expression level

We previously conducted a QTL analysis of small RNA (sRNA) abundance in flag leaves of an immortalized rice F2 (IMF2) population by aligning sRNA reads to the reference genome to quantify the expression levels of sRNAs. However, this approach missed about half of the sRNAs as only 50% of all sRNA reads could be uniquely aligned to the reference genome. Here, we quantified the expression levels of sRNAs and sRNA clusters without the use of a reference genome. QTL analysis of the expression levels of sRNAs and sRNA clusters confirmed the feasibility of this approach. sRNAs and sRNA clusters with identified QTLs were then aligned to the high-quality parental genomes of the IMF2 population to resolve the identified QTLs into local vs. distant regulation mode. We were able to detect new QTL hotspots by considering sRNAs aligned to multiple positions of the parental genomes and sRNAs unaligned to the parental genomes. We found that several local-QTL hotspots were caused by sequence variations in long inverted repeats, which probably function as precursors of sRNAs, between the two parental genomes. The expression levels of these sRNAs were significantly associated with the presence/absence of the long inverted repeats in the IMF2 population. Moreover, we found that the variations in whole-genome sRNA species composition among different IMF2s were attributed to sRNA biogenesis genes including OsDCL2b and OsRDR2. Our results highlight that genetic dissection of sRNA expression is a promising approach to disclose new components functioning in sRNA biogenesis and new mechanisms of sRNA biogenesis.

Profiling of epididymal small non-protein-coding RNAs.

Our understanding of epididymal physiology and function has been transformed over the three decades in which the International Meeting Series on the Epididymis has been hosted. This transformation has occurred along many fronts, but among the most significant advances has been the unexpected discovery of the diversity of small non-protein-coding RNAs (sRNAs) expressed in the epididymal epithelium and differentially accumulated in the luminal population of spermatozoa. Here we survey recent literature pertaining to profiling the sRNA landscape of the mammalian epididymis with the goal of demonstrating the contribution that these key regulatory elements, and their associated pathways, make to epididymal physiology and sperm maturation. High throughput sequencing strategies have fueled an unprecedented advance in our understanding of RNA biology. In the last decade, such high throughput profiling tools have been increasingly applied to study the mammalian epididymis, presaging the discovery of diverse classes of sRNA expressed along the length of the tract. Among the best studied sRNA classes are the microRNAs (miRNA), a sRNA species shown to act in concert with endocrine signals to fine-tune the segmental patterning of epididymal gene expression. In addition to performing this homeostatic role, epithelial cell-derived sRNAs also selectively accumulate into the epididymosomes and spermatozoa that occupy the duct lumen. This exciting discovery alludes to a novel form of intracellular communication that contributes to the establishment of the sperm epigenome and its modification under conditions of paternal stress. Compelling literature has identified sRNAs as a crucial regulatory tier that allows the epididymis to fulfill its combined roles of sperm transport, maturation, and storage. Continued research in this emerging field will contribute to our growing understanding of the etiology of male factor infertility and potentially allow for the future design of rational therapeutic options for these individuals.

PmiRDiscVali: an integrated pipeline for plant microRNA discovery and validation

BackgroundMicroRNAs (miRNAs) constitute a well-known small RNA (sRNA) species with important regulatory roles. To date, several bioinformatics tools have been developed for large-scale prediction of miRNAs based on high-throughput sequencing data. However, some of these tools become invalid without reference genomes, while some tools cannot supply user-friendly outputs. Besides, most of the current tools focus on the importance of secondary structures and sRNA expression patterns for miRNA prediction, while they do not pay attention to miRNA processing for reliability check.ResultsHere, we reported a pipeline PmiRDiscVali for plant miRNA discovery and partial validation. This pipeline integrated the popular tool miRDeep-P for plant miRNA prediction, making PmiRDiscVali compatible for both reference-based and de novo predictions. To check the prediction reliability, we adopted the concept that the miRNA processing intermediates could be tracked by degradome sequencing (degradome-seq) during the development of PmiRDiscVali. A case study was performed by using the public sequencing data of Dendrobium officinale, in order to show the clear and concise presentation of the prediction results.ConclusionSummarily, the integrated pipeline PmiRDiscVali, featured with degradome-seq data-based validation and vivid result presentation, should be useful for large-scale identification of plant miRNA candidates.

GTSF-1 is required for formation of a functional RNA-dependent RNA Polymerase complex in Caenorhabditis elegans.

Argonaute proteins and their associated small RNAs (sRNAs) are evolutionarily conserved regulators of gene expression. Gametocyte-specific factor 1 (Gtsf1) proteins, characterized by two tandem CHHC zinc fingers and an unstructured C-terminal tail, are conserved in animals and have been shown to interact with Piwi clade Argonautes, thereby assisting their activity. We identified the Caenorhabditis elegans Gtsf1 homolog, named it gtsf-1 and characterized it in the context of the sRNA pathways of C.elegans We report that GTSF-1 is not required for Piwi-mediated gene silencing. Instead, gtsf-1 mutants show a striking depletion of 26G-RNAs, a class of endogenous sRNAs, fully phenocopying rrf-3 mutants. We show, both invivo and invitro, that GTSF-1 interacts with RRF-3 via its CHHC zinc fingers. Furthermore, we demonstrate that GTSF-1 is required for the assembly of a larger RRF-3 and DCR-1-containing complex (ERIC), thereby allowing for 26G-RNA generation. We propose that GTSF-1 homologs may act to drive the assembly of larger complexes that act in sRNA production and/or in imposing sRNA-mediated silencing activities.

SPORTS1.0: A Tool for Annotating and Profiling Non-coding RNAs Optimized for rRNA- and tRNA-derived Small RNAs

High-throughput RNA-seq has revolutionized the process of small RNA (sRNA) discovery, leading to a rapid expansion of sRNA categories. In addition to the previously well-characterized sRNAs such as microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and small nucleolar RNA (snoRNAs), recent emerging studies have spotlighted on tRNA-derived sRNAs (tsRNAs) and rRNA-derived sRNAs (rsRNAs) as new categories of sRNAs that bear versatile functions. Since existing software and pipelines for sRNA annotation are mostly focused on analyzing miRNAs or piRNAs, here we developed the sRNA annotation pipelineoptimized for rRNA- and tRNA-derived sRNAs (SPORTS1.0). SPORTS1.0 is optimized for analyzing tsRNAs and rsRNAs from sRNA-seq data, in addition to its capacity to annotate canonical sRNAs such as miRNAs and piRNAs. Moreover, SPORTS1.0 can predict potential RNA modification sites based on nucleotide mismatches within sRNAs. SPORTS1.0 is precompiled to annotate sRNAs for a wide range of 68 species across bacteria, yeast, plant, and animal kingdoms, while additional species for analyses could be readily expanded upon end users’ input. For demonstration, by analyzing sRNA datasets using SPORTS1.0, we reveal that distinct signatures are present in tsRNAs and rsRNAs from different mouse cell types. We also find that compared to other sRNA species, tsRNAs bear the highest mismatch rate, which is consistent with their highly modified nature. SPORTS1.0 is an open-source software and can be publically accessed at https://github.com/junchaoshi/sports1.0.

Target-enrichment sequencing for detailed characterization of small RNAs.

Identification of important, functional small RNA (sRNA) species is currently hampered by the lack of reliable and sensitive methods to isolate and characterize them. We have developed a method, termed target-enrichment of sRNAs (TEsR), that enables targeted sequencing of rare sRNAs and diverse precursor and mature forms of sRNAs not detectable by current standard sRNA sequencing methods. It is based on the amplification of full-length sRNA molecules, production of biotinylated RNA probes, hybridization to one or multiple targeted RNAs, removal of nontargeted sRNAs and sequencing. By this approach, target sRNAs can be enriched by a factor of 500-30,000 while maintaining strand specificity. TEsR enriches for sRNAs irrespective of length or different molecular features, such as the presence or absence of a 5' cap or of secondary structures or abundance levels. Moreover, TEsR allows the detection of the complete sequence (including sequence variants, and 5' and 3' ends) of precursors, as well as intermediate and mature forms, in a quantitative manner. A well-trained molecular biologist can complete the TEsR procedure, from RNA extraction to sequencing library preparation, within 4-6 d.