Project description:A variety of small RNAs, including the Dicer-dependent miRNAs and the Dicer-independent Piwi-interacting RNAs, associate with Argonaute family proteins to regulate gene expression in diverse cellular processes. These two species of small RNA have not been found in fungi. Here, by analyzing small RNA associated with the Neurospora Argonaute protein QDE-2, we show that diverse pathways generate miRNA-like small RNAs (milRNAs) and Dicer-independent small interfering RNAs (disiRNAs) in this filamentous fungus. Surprisingly, milRNAs are produced by at least four different mechanisms that use a distinct combination of factors, including Dicers, QDE-2, the exonuclease QIP and an RNAse III domain-containing protein MRPL3. In contrast, disiRNAs originate from loci producing overlapping sense and antisense transcripts, and do not require the known RNAi components for their production. Taken together, these results uncover several pathways for small RNA production in filamentous fungi, shedding light on the diversity and evolutionary origins of eukaryotic small RNAs. One small RNA library was generated using QDE-2 immunoprecipitate from Neurospora crassa.
Project description:A variety of small RNAs, including the Dicer-dependent miRNAs and the Dicer-independent Piwi-interacting RNAs, associate with Argonaute family proteins to regulate gene expression in diverse cellular processes. These two species of small RNA have not been found in fungi. Here, by analyzing small RNA associated with the Neurospora Argonaute protein QDE-2, we show that diverse pathways generate miRNA-like small RNAs (milRNAs) and Dicer-independent small interfering RNAs (disiRNAs) in this filamentous fungus. Surprisingly, milRNAs are produced by at least four different mechanisms that use a distinct combination of factors, including Dicers, QDE-2, the exonuclease QIP and an RNAse III domain-containing protein MRPL3. In contrast, disiRNAs originate from loci producing overlapping sense and antisense transcripts, and do not require the known RNAi components for their production. Taken together, these results uncover several pathways for small RNA production in filamentous fungi, shedding light on the diversity and evolutionary origins of eukaryotic small RNAs.
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.
Project description:In flies, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous selfish genetic elements such as retrotransposons and repetitive sequences. Here, we show that while small interfering RNAs (siRNAs) derive from both the sense and antisense strands of their double-stranded RNA precursors, rasiRNAs arise mainly from the antisense strand. rasiRNA production appear not to require Dicer-1, which makes microRNAs, or Dicer-2, which makes siRNAs, and rasiRNAs lack the 2´,3´ hydroxy termini characteristic of animal siRNA and miRNA. Unlike siRNAs and miRNAs, rasiRNAs function through the Piwi, rather than the Ago, Argonaute protein subfamily. Thus, rasiRNAs define a third RNA silencing pathway distinct from both the miRNA and RNAi pathways. Keywords: gene silencing; post-transcriptional gene regulation; short RNAs; RNAi; rasiRNAs; rasiRNA; microRNAs; microRNA; siRNAs; siRNA
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.
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. small RNA profiling in wild type S. pombe cells and in 12 mutant cells
Project description:DICER has a well-characterized role in the processing of microRNAs (miRNAs) and small interfering RNAs (siRNA) that are important for post-transcriptional gene regulation. Emerging evidence suggests that DICER also has several non-canonical functions beyond miRNA/siRNA biogenesis, for example in transcriptional gene silencing at the chromatin level, as well as in RNA degradation and maintenance of genomic integrity. We have shown that the function of DICER in germ cells is essential for normal spermatogenesis; male mice lacking DICER in postnatal male germ cells are infertile due to severe defects in haploid differentiation. To better understand the function of DICER in male germ cells, we immunoprecipitated DICER from juvenile mouse testes and performed mass spectrometric analysis to identify DICER-interacting proteins.
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:Dicer, also known as endoribonuclease Dicer or helicase with RNase motif, cleaves double-stranded RNA (dsRNA) and pre-microRNA (pre-miRNA) into short mature double-stranded RNA fragments called small interfering RNA and microRNA, respectively. Medullary thymic epithelial cells (mTECs) express close to 90% of the protein-coding genome, besides they play an important role to self-tolerance through the ectopic expression of peripheral tissue antigens (PTAs) in the thymus. MicroRNAs are potent post-transcriptional regulators in developmental switches, lineage commitment and gene expression regulation, but their role in representation of the immunological self in mature mTECs is not fully understood. So we used a Mus musculus mTEC cell line (mTEC 3.10) to knockdown Dicer transcript by means of siRNA transfection and then observe the effect of Dicer knockdown in the transcriptional profile of messenger RNAs (mRNAs) by means of oligo microarray hybridization. The Agilent oligo microarrays were used to determine the large scale mRNA transcriptional profiles of control or Dicer-knockdown 3.10 mTECs.