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Key Points Small RNAs with roles in the nucleus include those that are generated mainly by Dicer proteins and loaded into Argonaute proteins (small interfering RNAs (siRNAs)) and also those that are Dicer-independent (largely generated by the ping-pong cycle) and are loaded into PIWI proteins (PIWI-interacting RNAs (piRNAs)). The subcellular localization where siRNA biogenesis occurs is variable among organisms. However, cytoplasmic Argonaute loading might be conserved. Nuclear RNA interference (RNAi) directs heterochromatic modifications at target loci including methylation of histone H3 at lysine 9 (H3K9; in Schizosaccharomyces pombe ) and DNA methylation (in Arabidopsis thaliana ). These reduce transcription, facilitating transcriptional gene silencing (TGS). Examples in A. thaliana and S. pombe show that transcription by RNA polymerase is required to produce nascent RNA that is targeted by nuclear RNAi; this process is termed co-transcriptional gene silencing (CTGS). There is evidence in the somatic cells of metazoans that endogenous siRNA pathways are involved in co-transcriptional regulation and heterochromatin formation, suggesting a conserved nuclear role for RNAi. RNAi has a crucial germline role in silencing transposons both post-transcriptionally and transcriptionally. In both metazoans and plants, transposons are revealed for transcription and produce small RNAs that target transposons in the germ cells to maintain silencing through nuclear RNAi. In mammals, piRNAs can direct de novo cytosine methylation in the germ line and have been shown to do so at an imprinted locus. This suggests that nuclear RNAi might have another conserved role in parent-of-origin imprinting. The piRNAs of Caenorhabditis elegans (21U small RNAs) can direct transcriptional silencing by H3K9 methylation in the germ line that is heritable and dependent on nuclear RNAi (NRDE) pathway members. Roles are emerging for small RNAs in DNA repair and genome maintenance, through both the maintenance and regulation of heterochromatic domains (such as centromeres and telomeres) and through direct involvement in DNA repair: for example, at double-strand breaks. In addition to well-known roles in the cytoplasm, a growing number of functions for small RNAs in the nucleus are being discovered. These include roles in transcriptional repression, epigenetic modifications and genome stability. This Review considers examples from animals, plants and fungi. A growing number of functions are emerging for RNA interference (RNAi) in the nucleus, in addition to well-characterized roles in post-transcriptional gene silencing in the cytoplasm. Epigenetic modifications directed by small RNAs have been shown to cause transcriptional repression in plants, fungi and animals. Additionally, increasing evidence indicates that RNAi regulates transcription through interaction with transcriptional machinery. Nuclear small RNAs include small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) and are implicated in nuclear processes such as transposon regulation, heterochromatin formation, developmental gene regulation and genome stability.

Endogenous small interfering RNAs (endo-siRNAs) regulate diverse gene expression programs in eukaryotes by either binding and cleaving mRNA targets or mediating heterochromatin formation; however, the mechanisms of endo-siRNA biogenesis, sorting, and target regulation remain poorly understood. Here we report the identification and function of a specific class of germline-generated endo-siRNAs in Caenorhabditis elegans that are 26 nt in length and contain a guanine at the first nucleotide position (i.e., 26G RNAs). 26G RNAs regulate gene expression during spermatogenesis and zygotic development, and their biogenesis requires the ERI-1 exonuclease and the RRF-3 RNA-dependent RNA polymerase (RdRP). Remarkably, we identified two nonoverlapping subclasses of 26G RNAs that sort into specific RNA-induced silencing complexes (RISCs) and differentially regulate distinct mRNA targets. Class I 26G RNAs target genes are expressed during spermatogenesis, whereas class II 26G RNAs are maternally inherited and silence gene expression during zygotic development. These findings implicate a class of endo-siRNAs in the global regulation of transcriptional programs required for fertility and development.

Argonaute-associated siRNAs and Piwi-associated piRNAs have overlapping roles in silencing mobile genetic elements in animals. In Caenorhabditis elegans, mutator (mut) class genes mediate siRNA-guided repression of transposons as well as exogenous RNAi, but their roles in endogenous RNA silencing pathways are not well-understood. To characterize the endogenous small RNAs dependent on mut class genes, small RNA populations from a null allele of mut-16 as well as a regulatory mut-16{mg461) allele that disables only somatic RNAi were subjected to deep sequencing. Additionally, each of the mut class genes was tested for a requirement in 26G siRNA pathways. The results indicate that mut-16 is an essential factor in multiple endogenous germline and somatic siRNA pathways involving several distinct Argonautes and RNAdependent RNA polymerases. The results also reveal essential roles for mut-2 and mut-7 in the ERGO-1 class 26G siRNA pathway and less critical roles for mut-8, mut-14, and mut-15. We show that transposons are hypersusceptible to mut-16-dependent silencing and identify a requirement for the siRNA machinery in piRNA biogenesis from Tel transposons. We also show that the somaspecific mut-16(mg461) mutant allele is present in multiple G elegans laboratory strains.

In eukaryotes, RNA silencing pathways utilize 20-30-nucleotide small RNAs to regulate gene expression, specify and maintain chromatin structure, and repress viruses and mobile genetic elements. RNA silencing was likely present in the common ancestor of modern eukaryotes, but most research has focused on plant and animal RNA silencing systems. Phytophthora species belong to a phylogenetically distinct group of economically important plant pathogens that cause billions of dollars in yield losses annually as well as ecologically devastating outbreaks. We analyzed the small RNA-generating components of the genomes of P. infestans, P. sojae and P. ramorum using bioinformatics, genetic, phylogenetic and high-throughput sequencing-based methods. Each species produces two distinct populations of small RNAs that are predominantly 21- or 25-nucleotides long. The 25-nucleotide small RNAs were primarily derived from loci encoding transposable elements and we propose that these small RNAs define a pathway of short-interfering RNAs that silence repetitive genetic elements. The 21-nucleotide small RNAs were primarily derived from inverted repeats, including a novel microRNA family that is conserved among the three species, and several gene families, including Crinkler effectors and type III fibronectins. The Phytophthora microRNA is predicted to target a family of amino acid/auxin permeases, and we propose that 21-nucleotide small RNAs function at the post-transcriptional level. The functional significance of microRNA-guided regulation of amino acid/auxin permeases and the association of 21-nucleotide small RNAs with Crinkler effectors remains unclear, but this work provides a framework for testing the role of small RNAs in Phytophthora biology and pathogenesis in future work.

Many plant small RNAs are sequence-specific negative regulators of target mRNAs and/or chromatin. In angiosperms, the two most abundant endogenous small RNA populations are usually 21-nucleotide microRNAs (miRNAs) and 24-nucleotide heterochromatic short interfering RNAs (siRNAs). Heterochromatic siRNAs are derived from repetitive regions and reinforce DNA methylation at targeted loci. The existence and extent of heterochromatic siRNAs in other land plant lineages has been unclear. Using small RNA-sequencing (RNA-seq) of the moss Physcomitrella patens, we identified 1090 loci that produce mostly 23- to 24-nucleotide siRNAs. These loci are mostly in intergenic regions with dense DNA methylation. Accumulation of siRNAs from these loci depends upon P. patens homologs of DICER-LIKE3 (DCL3), RNA-DEPENDENT RNA POLYMERASE2, and the largest subunit of DNA-DEPENDENT RNA POLYMERASE IV, with the largest subunit of a Pol V homolog contributing to expression at a smaller subset of the loci. A MINIMAL DICER-LIKE (mDCL) gene, which lacks the N-terminal helicase domain typical of DCL proteins, is specifically required for 23-nucleotide siRNA accumulation. We conclude that heterochromatic siRNAs, and their biogenesis pathways, are largely identical between angiosperms and P. patens, with the notable exception of the P. patens-specific use of mDCL to produce 23-nucleotide siRNAs.

RNA silencing with inverted repeat (IR) constructs has been used to suppress gene expression in various organisms. However, the transitive RNA-silencing effect described in plants may preclude the use of RNA silencing for a gene family. Here, we show that, in rice (Oryza sativa), transitive RNA silencing (spreading of double-stranded RNA along the target mRNA) occurred with the green fluorescent protein transgene but not with the endogenous phytoene desaturase gene. We fused IR copies of unique 3' untranslated regions derived from the rice OsRac gene family to a strong promoter and stably introduced them into rice. Each of the seven members of the OsRac gene family was specifically suppressed by its respective IR construct. We also examined IR constructs in which multiple 3' untranslated regions were fused and showed that three members of the OsRac gene family were effectively suppressed by a single construct. Using highly conserved regions of the two members of the OsRac gene family, we also suppressed the expression of all members of the gene family with variable efficiencies. These results suggest that RNA silencing is a useful method for the functional analysis of gene families in rice and other plants.

Background PIWI-interacting RNA (piRNA) is a novel and emerging class of small non-coding RNA (sncRNA). Ranging in length from 26-32 nucleotides, this sncRNA is a potent player in guiding the vital regulatory processes within a cellular system. Inspite of having such a wide role within cellular systems, piRNAs are not well organized and classified, so that a researcher can pool out the biologically relevant information concerning this class. Description Here we present piRNAQuest- a unified and comprehensive database of 41749 human, 890078 mouse and 66758 rat piRNAs obtained from NCBI and different small RNA sequence experiments. This database provides piRNA annotation based on their localization in gene, intron, intergenic, CDS, 5 / UTR, 3 / UTR and repetitive regions which has not been done so far. We have also annotated piRNA clusters and have elucidated characteristic motifs within them. We have looked for the presence of piRNAs and piRNA clusters in pseudogenes, which are known to regulate the expression of protein coding transcripts by generating small RNAs. All these will help researchers progress towards solving the unanswered queries on piRNA biogenesis and their mode of action. Further, expression profile for piRNA in different tissues and from different developmental stages has been provided. In addition, we have provided several tools like 'homology search’, 'dynamic cluster search’ and 'pattern search’. Overall, piRNAQuest will serve as a useful resource for exploring human, mouse and rat piRNAome. The database is freely accessible and available at http://bicresources.jcbose.ac.in/zhumur/pirnaquest/ . Conclusion piRNAs play a remarkable role in stem cell self-renewal and various vital processes of developmental biology. Although researchers are mining different features on piRNAs, the exact regulatory mechanism is still fuzzy. Thus, understanding the true potential of these small regulatory molecules with respect to their origin, localization and mode of biogenesis is crucial. piRNAQuest will provide us with a better insight on piRNA origin and function which will help to explore the true potential of these sncRNAs.

Phased small interfering RNA (phasiRNA) generating loci (briefly as PHAS) in plants are a novel class of genes that are normally regulated by microRNAs (miRNAs). Similar to miRNAs, phasiRNAs encoded by PHAS play important regulatory roles by targeting protein coding transcripts in plant species. We performed a genome-wide discovery of PHAS loci in Chinese sacred lotus and identified a total of 106 PHAS loci. Of these, 47 loci generate 21 nucleotide (nt) phasiRNAs and 59 loci generate 24 nt phasiRNAs, respectively. We have also identified a new putative TAS3 and a putative TAS4 loci in the lotus genome. Our results show that some of the nucleotide-binding, leucine-rich repeat (NB-LRR) disease resistance proteins and MYB transcription factors potentially generate phasiRNAs. Furthermore, our results suggest that some large subunit (LSU) rRNAs can derive putative phasiRNAs, which is potentially resulted from crosstalk between small RNA biogenesis pathways that are employed to process rRNAs and PHAS loci, respectively. Some of the identified phasiRNAs have putative trans-targets with less than 4 mismatches, suggesting that the identified PHAS are involved in many different pathways. Finally, the discovery of 24 nt PHAS in lotus suggests that there are 24 nt PHAS in dicots.

Background Short interfering RNAs (siRNAs) can be used to knockdown gene expression in functional genomics. For a target gene of interest, many siRNA molecules may be designed, whereas their efficiency of expression inhibition often varies. Results To facilitate gene functional studies, we have developed a new machine learning method to predict siRNA potency based on random forests and support vector machines. Since there were many potential sequence features, random forests were used to select the most relevant features affecting gene expression inhibition. Support vector machine classifiers were then constructed using the selected sequence features for predicting siRNA potency. Interestingly, gene expression inhibition is significantly affected by nucleotide dimer and trimer compositions of siRNA sequence. Conclusions The findings in this study should help design potent siRNAs for functional genomics, and might also provide further insights into the molecular mechanism of RNA interference.

Textbook descriptions of RNA interference (RNAi) may have to be rewritten in the wake of recent studies. The new research indicates that RNAi is in fact a two-step process involving primary short-interfering RNAs (siRNAs), which function as triggers, and secondary siRNAs, which act as executors.