Project description:Canonical microRNAs (miRNAs) require two processing steps: the first by the Microprocessor, a complex of DGCR8 and Drosha, and the second by Dicer. dgcr8delta/delta mouse embryonic stem cells (mESCs) have less severe phenotypes than dicer1delta/delta mESCs, suggesting a physiological role for Microprocessor-independent, Dicer-dependent small RNAs. To identify these small RNAs with unusual biogenesis, we performed high-throughput sequencing from wild type, dgcr8delta/delta, and dicer1delta/delta mESCs. Several of the DGCR8-independent, Dicer-dependent RNAs were non-canonical miRNAs. These derived from mirtrons and a newly identified subclass of miRNA precursors, which appears to be the endogenous counterpart of short hairpin RNAs (shRNAs). Our analyses also revealed endogenous siRNAs resulting from Dicer cleavage of long hairpins, the vast majority of which originated from one genomic locus with tandem, inverted short interspersed nuclear elements (SINEs). Our results extend the known diversity of mammalian small-RNA generating pathways and show that mammalian siRNAs exist in tissues outside of oocytes. Small RNAs were sequenced from wt, dgcr8(-), and dicer(-) mouse ES cells and the frequencies of small RNA types compared between the three. This record includes Illumina-platform-generated datasets from all three samples and 454-platform-generated datasets from wt and dgcr8(-) samples. [raw data files are unavailable]
Project description:Canonical microRNAs (miRNAs) require two processing steps: the first by the Microprocessor, a complex of DGCR8 and Drosha, and the second by Dicer. dgcr8delta/delta mouse embryonic stem cells (mESCs) have less severe phenotypes than dicer1delta/delta mESCs, suggesting a physiological role for Microprocessor-independent, Dicer-dependent small RNAs. To identify these small RNAs with unusual biogenesis, we performed high-throughput sequencing from wild type, dgcr8delta/delta, and dicer1delta/delta mESCs. Several of the DGCR8-independent, Dicer-dependent RNAs were non-canonical miRNAs. These derived from mirtrons and a newly identified subclass of miRNA precursors, which appears to be the endogenous counterpart of short hairpin RNAs (shRNAs). Our analyses also revealed endogenous siRNAs resulting from Dicer cleavage of long hairpins, the vast majority of which originated from one genomic locus with tandem, inverted short interspersed nuclear elements (SINEs). Our results extend the known diversity of mammalian small-RNA generating pathways and show that mammalian siRNAs exist in tissues outside of oocytes.
Project description:Non-canonical microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) are distinct subclasses of small RNAs that bypass the DGCR8/Drosha Microprocessor but still require Dicer for their biogenesis. What, if any, role they have in mammals remains unknown. To identify potential roles for these Microprocessor-independent, Dicer-dependent small RNAs, we compared the phenotypes resulting from conditional deletion of dgcr8 versus dicer in post-mitotic neurons. Loss of dicer resulted in an earlier lethality, more severe structural abnormalities, and increased apoptosis relative to dgcr8 loss. Deep sequencing of small RNAs from the hippocampus and cortex of the conditional knockouts and control littermates identified multiple novel non-canonical microRNAs including new mirtrons H/ACA box snoRNA-derived small RNAs, and a previously unidentified mammalian subclass derived from C/D box snoRNAs. These non-canonical miRNAs were expressed at high levels in the brain relative to other tissues. In contrast, we found no evidence for endo-siRNAs in the brain. Taken together, our findings provide evidence for a diverse population of highly expressed non-canonical miRNAs that together play important functional roles in post-mitotic neurons. Examination of small RNA populations in the hippocampus and cortex of 2 conditional knockout and control littermates
Project description:Non-canonical microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) are distinct subclasses of small RNAs that bypass the DGCR8/Drosha Microprocessor but still require Dicer for their biogenesis. What, if any, role they have in mammals remains unknown. To identify potential roles for these Microprocessor-independent, Dicer-dependent small RNAs, we compared the phenotypes resulting from conditional deletion of dgcr8 versus dicer in post-mitotic neurons. Loss of dicer resulted in an earlier lethality, more severe structural abnormalities, and increased apoptosis relative to dgcr8 loss. Deep sequencing of small RNAs from the hippocampus and cortex of the conditional knockouts and control littermates identified multiple novel non-canonical microRNAs including new mirtrons H/ACA box snoRNA-derived small RNAs, and a previously unidentified mammalian subclass derived from C/D box snoRNAs. These non-canonical miRNAs were expressed at high levels in the brain relative to other tissues. In contrast, we found no evidence for endo-siRNAs in the brain. Taken together, our findings provide evidence for a diverse population of highly expressed non-canonical miRNAs that together play important functional roles in post-mitotic neurons.
Project description:Canonical small interfering RNAs (siRNAs) are generated by the cleavage of double-stranded RNA (dsRNA) by the ribonuclease Dicer. siRNAs are found in plants, animals, and some fungi where they bind to Argonautes to direct RNA silencing. In this study, we characterized the canonical Dicer-dependent siRNAs of C. elegans. We identified thousands of endogenous loci, representing dozens of unique elements, that give rise to low to moderate levels of siRNAs, called 23H-RNAs. These loci include repetitive elements, alleged coding genes, pseudogenes, non-coding RNAs, and unannotated features, many of which adopt hairpin structures. Using protein-small RNA co-immunoprecipitation, we identified the Argonautes that associate with 23H-RNAs and using mRNA-sequencing we explored their roles in gene regulation.
Project description:Dgcr8 and Dicer are both important components of the microRNA biogenesis pathway while Dicer is also implicated in biogenesis of other types of small RNAs such as siRNAs and mirtrons. Here we performed microarray analysis of WT, Dgcr8 and Dicer knockout ES cells to identify mRNAs differentially regulated upon loss of Dgcr8 and Dicer.
Project description:RNA interference (RNAi) is a phylogenetically widespread gene silencing process triggered by doublestranded RNA (dsRNA). In plants and C. elegans, two distinct populations of small RNAs have been proposed to participate in RNAi : "Primary siRNAs" (derived from Dicer nuclease-mediated cleavage of the original trigger) and "Secondary siRNAs" (additional small RNAs whose synthesis requires an RNA-directed RNA polymerase [RdRP]). Analyzing small RNAs associated with ongoing RNAi in C. elegans, we found secondary siRNAs to comprise the vast majority. The bulk of secondary siRNAs exhibited structure and sequence indicative of a biosynthetic mode where each molecule derives from an independent de novo initiation by RdRP. Analysis of endogenous small RNAs indicated that a fraction derive from a biosynthetic mechanism that is similar to that of secondary siRNAs formed during RNAi, suggesting that small antisense transcripts derived from cellular mRNAs by RdRP activity may have key roles in cellular regulation. Keywords: C. elegans small RNA sequences from wild type animals fed on sel-1 dsRNA producing bacteria
Project description:Plants and invertebrates protect themselves from viruses through RNA interference (RNAi), yet it remains unknown whether this defense mechanism exists in mammals. Antiviral RNAi involves the processing of viral long double-stranded (ds) RNA molecules into small interfering RNAs (siRNAs) by the ribonuclease (RNAse) III Dicer. These siRNAs are incorporated into effector complex(es) containing members of the Argonaute (Ago) protein family and guide silencing of complementary target viral RNAs. Here, we detect the accumulation of phased Dicer-dependent virus-derived siRNA (viRNAs) and demonstrate their loading into Ago2 after infection of mouse embryonic stem (ES) cells with Encephalomyocarditis virus (EMCV). We further show that the production of these viRNAs is drastically reduced, yet not completely abolished, if ES cells are first induced to differentiate before infection. Finally, we reveal that the mammalian virus Nodamura virus (NoV) encodes for a protein that counteracts such antiviral RNAi in ES cells supporting the existence of an effective RNAi-based immunity in mammals.
Project description:Plants and invertebrates protect themselves from viruses through RNA interference (RNAi), yet it remains unknown whether this defense mechanism exists in mammals. Antiviral RNAi involves the processing of viral long double-stranded (ds) RNA molecules into small interfering RNAs (siRNAs) by the ribonuclease (RNAse) III Dicer. These siRNAs are incorporated into effector complex(es) containing members of the Argonaute (Ago) protein family and guide silencing of complementary target viral RNAs. Here, we detect the accumulation of phased Dicer-dependent virus-derived siRNA (viRNAs) and demonstrate their loading into Ago2 after infection of mouse embryonic stem (ES) cells with Encephalomyocarditis virus (EMCV). We further show that the production of these viRNAs is drastically reduced, yet not completely abolished, if ES cells are first induced to differentiate before infection. Finally, we reveal that the mammalian virus Nodamura virus (NoV) encodes for a protein that counteracts such antiviral RNAi in ES cells supporting the existence of an effective RNAi-based immunity in mammals. Infection of wild-type or mutant mouse ES cells and analysis of small RNAs from total extracts or immunoprecipitated components of the RNAi pathway
Project description:The assembly of fission yeast pericentromeric heterochromatin and generation of small interfering RNAs (siRNAs) from noncoding centromeric transcripts are mutually dependent processes. How this interdependent positive feedback loop is first triggered is a fundamental unanswered question. Here we show that two distinct Argonaute (Ago1)-dependent pathways mediate small RNA generation. RNA-dependent RNA polymerase complex (RDRC) and Dicer act on specific noncoding RNAs to generate siRNAs by a mechanism that requires the slicer activity of Ago1 but is independent of pre-existing heterochromatin. In the absence of RDRC or Dicer, a distinct class of small RNAs, called primal small RNAs (priRNAs), associate with Ago1. priRNAs are degradation products of abundant transcripts, which bind to Ago1 and target antisense transcripts that result from bidirectional transcription of DNA repeats. Our results suggest that a transcriptome surveillance mechanism based on the random association of RNA degradation products with Argonaute triggers siRNA amplification and heterochromatin assembly within DNA repeats.