Canonical small interfering RNAs (siRNAs) are processed from double-stranded RNA (dsRNA) by Dicer and associate with Argonautes to direct RNA silencing. In , 22G-RNAs and 26G-RNAs are often referred to as siRNAs but display distinct characteristics. For example, 22G-RNAs do not originate from dsRNA and do not depend on Dicer, whereas 26G-RNAs require Dicer but derive from an atypical RNA duplex and are produced exclusively antisense to their messenger RNA (mRNA) templates.
View Article and Find Full Text PDFSmall RNA pathways regulate eukaryotic antiviral defense. Many of the mutations that were identified based on their enhanced RNAi, the synMuv B genes, also emerged from unrelated genetic screens for increased growth factor signaling. The dozen synMuv B genes encode homologues of the mammalian dREAM complex found in nearly all animals and plants, which includes the /retinoblastoma oncogene.
View Article and Find Full Text PDFPrimary microRNA (pri-miRNA) transcripts are processed by the Microprocessor, a protein complex that includes the ribonuclease Drosha and its RNA binding partner DGCR8/Pasha. We developed a live, whole animal, fluorescence-based sensor that reliably monitors pri-miRNA processing with high sensitivity in . Through a forward genetic selection for alleles that desilence the sensor, we identified a mutation in the conserved G residue adjacent to the namesake W residue of Pasha's WW domain.
View Article and Find Full Text PDFSummary: tiny-count is a highly flexible counting tool that allows for hierarchical classification and quantification of small RNA reads from high-throughput sequencing data. Selection rules can be used to filter reads by 5' nucleotide, length, position of alignments in relation to reference features, and by the number of mismatches to reference sequences. tiny-count can quantify reads aligned to a genome or directly to small RNA or transcript sequences.
View Article and Find Full Text PDFA major challenge to our understanding of translational control has been deconvolving the individual impact specific regulatory factors have on the complex dynamics of mRNA translation. MicroRNAs (miRNAs), for example, guide Argonaute and associated proteins to target mRNAs, where they direct gene silencing in multiple ways that are not well understood. To better deconvolve these dynamics, we have developed technology to directly visualize and quantify the impact of human Argonaute2 (Ago2) on the translation and subcellular localization of individual reporter mRNAs in living cells.
View Article and Find Full Text PDFPiwi-interacting RNAs (piRNAs) regulate many biological processes through mechanisms that are not fully understood. In Caenorhabditis elegans, piRNAs intersect the endogenous RNA interference (RNAi) pathway, involving a distinct class of small RNAs called 22G-RNAs, to regulate gene expression in the germline. In the absence of piRNAs, 22G-RNA production from many genes is reduced, pointing to a role for piRNAs in facilitating endogenous RNAi.
View Article and Find Full Text PDFPiwi-interacting RNAs (piRNAs) play essential roles in silencing repetitive elements to promote fertility in metazoans. Studies in worms, flies, and mammals reveal that piRNAs are expressed in a sex-specific manner. However, the mechanisms underlying this sex-specific regulation are unknown.
View Article and Find Full Text PDFpiRNAs play a critical role in the regulation of transposons and other germline genes. In , regulation of piRNA target genes is mediated by the complex, which synthesizes high levels of siRNAs through the activity of an RNA-dependent RNA polymerase. However, the steps between mRNA recognition by the piRNA pathway and siRNA amplification by the complex are unknown.
View Article and Find Full Text PDFPiwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) are distinct classes of small RNAs required for proper germline development. To identify the roles of piRNAs and siRNAs in regulating gene expression in Caenorhabditis elegans, we subjected small RNAs and mRNAs from the gonads of piRNA and siRNA defective mutants to high-throughput sequencing. We show that piRNAs and an abundant class of siRNAs known as WAGO-class 22G-RNAs are required for proper expression of spermatogenic and oogenic genes.
View Article and Find Full Text PDFThe germline contains an immortal cell lineage that ensures the faithful transmission of genetic and, in some instances, epigenetic information from one generation to the next. Here, we show that in Caenorhabditis elegans, the small RNA 3'-2'-O-methyltransferase henn-1/HEN1 is required for sustained fertility across generations. In the absence of henn-1, animals become progressively less fertile, becoming sterile after ∼30 generations at 25°C.
View Article and Find Full Text PDFpiRNAs are known to silence transposable elements, but not all piRNAs match transposon sequences. Recent studies from Shen et al. (2018) and Zhang et al.
View Article and Find Full Text PDFCaenorhabditis elegans contains 25 Argonautes, of which, ALG-1 and ALG-2 are known to primarily interact with miRNAs. ALG-5 belongs to the AGO subfamily of Argonautes that includes ALG-1 and ALG-2, but its role in small RNA pathways is unknown. We analyzed by high-throughput sequencing the small RNAs associated with ALG-5, ALG-1 and ALG-2, as well as changes in mRNA expression in alg-5, alg-1 and alg-2 mutants.
View Article and Find Full Text PDFReversible changes in gene expression independent of the genetic code can be transmitted from one generation to the next via poorly understood mechanisms. In worms, a histone-modifying enzyme is necessary to keep small RNA-guided transgenerational gene silencing in check.
View Article and Find Full Text PDFGermline-expressed endogenous small interfering RNAs (endo-siRNAs) transmit multigenerational epigenetic information to ensure fertility in subsequent generations. In Caenorhabditis elegans, nuclear RNAi ensures robust inheritance of endo-siRNAs and deposition of repressive H3K9me3 marks at target loci. How target silencing is maintained in subsequent generations is poorly understood.
View Article and Find Full Text PDFpiRNAs silence foreign genes, such as transposons, to preserve genome integrity, but they also target endogenous mRNAs by mechanisms that are poorly understood. Caenorhabditis elegans piRNAs interact with both transposon and nontransposon mRNAs to initiate sustained silencing via the RNAi pathway. To assess the dysregulation of gene silencing caused by lack of piRNAs, we restored RNA silencing in RNAi-defective animals in the presence or absence of piRNAs.
View Article and Find Full Text PDFMore than 2,000 C. elegans genes are targeted for RNA silencing by the mutator complex, a specialized small interfering RNA (siRNA) amplification module which is nucleated by the Q/N-rich protein MUT-16. The mutator complex localizes to Mutator foci adjacent to P granules at the nuclear periphery in germ cells.
View Article and Find Full Text PDFLittle is known about where microRNAs (miRNAs) regulate their targets within the cell. In this issue, Li et al. identify a new player in the plant miRNA pathway that implicates the endoplasmic reticulum in miRNA-mediated gene silencing.
View Article and Find Full Text PDFThe nematode Caenorhabditis elegans contains each of the broad classes of eukaryotic small RNAs, including microRNAs (miRNAs), endogenous small-interfering RNAs (endo-siRNAs), and piwi-interacting RNAs (piRNAs). To better understand the evolution of these regulatory RNAs, we deep-sequenced small RNAs from C. elegans and three closely related nematodes: C.
View Article and Find Full Text PDFIn RNA-directed silencing pathways, ternary complexes result from small RNA-guided ARGONAUTE (AGO) associating with target transcripts. Target transcripts are often silenced through direct cleavage (slicing), destabilization through slicer-independent turnover mechanisms, and translational repression. Here, wild-type and active-site defective forms of several Arabidopsis thaliana AGO proteins involved in posttranscriptional silencing were used to examine several AGO functions, including small RNA binding, interaction with target RNA, slicing or destabilization of target RNA, secondary small interfering RNA formation, and antiviral activity.
View Article and Find Full Text PDFRNA silencing can be initiated by endogenous or exogenously delivered siRNAs. In Caenorhabditis elegans, RNA silencing guided by primary siRNAs is inefficient and therefore requires an siRNA amplification step involving RNA-dependent RNA polymerases (RdRPs). Many factors involved in RNA silencing localize to protein- and RNA-rich nuclear pore-associated P granules in the germline, where they are thought to surveil mRNAs as they exit the nucleus.
View Article and Find Full Text PDFBackground: In nematodes, plants, and fungi, RNAi is remarkably potent and persistent due to the amplification of initial silencing signals by RNA-dependent RNA polymerases (RdRPs). In Caenorhabditis elegans (C. elegans), the interaction between the RNA-induced silencing complex (RISC) loaded with primary small interfering RNAs (siRNAs) and the target messenger RNA (mRNA) leads to the recruitment of RdRPs and synthesis of secondary siRNAs using the target mRNA as the template.
View Article and Find Full Text PDFSmall RNAs--including piRNAs, miRNAs, and endogenous siRNAs--bind Argonaute proteins to form RNA silencing complexes that target coding genes, transposons, and aberrant RNAs. To assess the requirements for endogenous siRNA formation and activity in Caenorhabditis elegans, we developed a GFP-based sensor for the endogenous siRNA 22G siR-1, one of a set of abundant siRNAs processed from a precursor RNA mapping to the X chromosome, the X-cluster. Silencing of the sensor is also dependent on the partially complementary, unlinked 26G siR-O7 siRNA.
View Article and Find Full Text PDFEndogenous small interfering RNAs (siRNAs) are a class of naturally occuring regulatory RNAs found in fungi, plants, and animals. Some endogenous siRNAs are required to silence transposons or function in chromosome segregation; however, the specific roles of most endogenous siRNAs are unclear. The helicase gene eri-6/7 was identified in the nematode Caenorhabditis elegans by the enhanced response to exogenous double-stranded RNAs (dsRNAs) of the null mutant.
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