Project description:MicroRNAs (miRNAs) and small-interfering RNAs (siRNAs) negatively regulate their targets by 1) repressing translation, 2) endonucleolytic RNA cleavage, or 3) DNA methylation resulting in transcriptional silencing. P-body/decapping components are likely required for translational repression, but are not known to function in other posttranscriptional regulatory pathways or to affect smRNA levels. Here, we show that the P-body/decapping protein DCP5 is required for miRNA-mediated translational repression but not cleavage, and to regulate the transcription of specific miRNAs. We find that this protein also affects the abundance of tRNA-derived smRNAs. Significantly, DCP5 is required for the transcriptional silencing and DNA methylation of numerous transposable/repetitive elements and imprinted genes, indicating that it is a novel component of the RNA-directed DNA methylation pathway. Our results demonstrate that DCP5 and likely the P-body itself are required for multiple smRNA-mediated silencing pathways and provide the first evidence for the spatial separation of translational inhibition and cleavage by miRNAs. small RNA (smRNA) expression comparison between wildtype (Col-0) and dcp5 mutant plants in Arabidopsis
Project description:MicroRNAs (miRNAs) and small-interfering RNAs (siRNAs) negatively regulate their targets by 1) repressing translation, 2) endonucleolytic RNA cleavage, or 3) DNA methylation resulting in transcriptional silencing. P-body/decapping components are likely required for translational repression, but are not known to function in other posttranscriptional regulatory pathways or to affect smRNA levels. Here, we show that the P-body/decapping protein DCP5 is required for miRNA-mediated translational repression but not cleavage, and to regulate the transcription of specific miRNAs. We find that this protein also affects the abundance of tRNA-derived smRNAs. Significantly, DCP5 is required for the transcriptional silencing and DNA methylation of numerous transposable/repetitive elements and imprinted genes, indicating that it is a novel component of the RNA-directed DNA methylation pathway. Our results demonstrate that DCP5 and likely the P-body itself are required for multiple smRNA-mediated silencing pathways and provide the first evidence for the spatial separation of translational inhibition and cleavage by miRNAs. total RNA expression comparison with between wildtype (Col-0) and dcp5 mutant plants in Arabidopsis
Project description:MicroRNAs (miRNAs) and small-interfering RNAs (siRNAs) negatively regulate their targets by 1) repressing translation, 2) endonucleolytic RNA cleavage, or 3) DNA methylation resulting in transcriptional silencing. P-body/decapping components are likely required for translational repression, but are not known to function in other posttranscriptional regulatory pathways or to affect smRNA levels. Here, we show that the P-body/decapping protein DCP5 is required for miRNA-mediated translational repression but not cleavage, and to regulate the transcription of specific miRNAs. We find that this protein also affects the abundance of tRNA-derived smRNAs. Significantly, DCP5 is required for the transcriptional silencing and DNA methylation of numerous transposable/repetitive elements and imprinted genes, indicating that it is a novel component of the RNA-directed DNA methylation pathway. Our results demonstrate that DCP5 and likely the P-body itself are required for multiple smRNA-mediated silencing pathways and provide the first evidence for the spatial separation of translational inhibition and cleavage by miRNAs.
Project description:MicroRNAs (miRNAs) and small-interfering RNAs (siRNAs) negatively regulate their targets by 1) repressing translation, 2) endonucleolytic RNA cleavage, or 3) DNA methylation resulting in transcriptional silencing. P-body/decapping components are likely required for translational repression, but are not known to function in other posttranscriptional regulatory pathways or to affect smRNA levels. Here, we show that the P-body/decapping protein DCP5 is required for miRNA-mediated translational repression but not cleavage, and to regulate the transcription of specific miRNAs. We find that this protein also affects the abundance of tRNA-derived smRNAs. Significantly, DCP5 is required for the transcriptional silencing and DNA methylation of numerous transposable/repetitive elements and imprinted genes, indicating that it is a novel component of the RNA-directed DNA methylation pathway. Our results demonstrate that DCP5 and likely the P-body itself are required for multiple smRNA-mediated silencing pathways and provide the first evidence for the spatial separation of translational inhibition and cleavage by miRNAs.
Project description:Background: RNA silencing pathways play critical roles in gene regulation, virus infection, and transposon control. RNA interference (RNAi) is mediated by small interfering RNAs (siRNAs), which are liberated from double stranded (ds) RNA precursors by Dicer and direct the RNA-induced silencing complex (RISC) to target transcripts. Recent efforts have uncovered important principles governing small RNA (smRNA) sorting into RISC, yet mechanisms defining substrate selection by Dicer proteins remain uncharacterized. Methodology: To better characterize Dicer-2 substrates in Drosophila, we examined the antiviral RNAi response, which generates virus-derived siRNAs from viral RNA. Using high-throughput sequencing, we found that diverse viruses were uniquely targeted; substrates included dsRNA replication intermediates and intramolecular RNA stem loops. smRNA distribution patterns from viral and synthetic dsRNA precursors were highly reproducible, and machine learning techniques identified characteristics of precursor molecules and smRNA duplexes important in determining relative smRNA abundance. Significance: To our knowledge, this study provides the first description of the rules governing Dicer-2 substrate selection, which has important implications for exogenous RNA silencing technologies and the development of smRNA-based antiviral therapeutics.
Project description:Arabidopsis DCP5, a homolog of human RNA-associated protein 55, is a nessary component of eukaryotic processing bodies (P-bodies). knockdown mutant of dcp5-1 showed compromised RNA decapping activity and reduced P-body size. Here we profiled Arabidopsis transcriptome of roots, shoots, and inflorensences in Col-0 and DCP5-1 mutant using strand-specific RNA-sequencing. Our analysis identified a large number of DCP5-regulatd transcripts in Arabidopsis.
Project description:Background: RNA silencing pathways play critical roles in gene regulation, virus infection, and transposon control. RNA interference (RNAi) is mediated by small interfering RNAs (siRNAs), which are liberated from double stranded (ds) RNA precursors by Dicer and direct the RNA-induced silencing complex (RISC) to target transcripts. Recent efforts have uncovered important principles governing small RNA (smRNA) sorting into RISC, yet mechanisms defining substrate selection by Dicer proteins remain uncharacterized. Methodology: To better characterize Dicer-2 substrates in Drosophila, we examined the antiviral RNAi response, which generates virus-derived siRNAs from viral RNA. Using high-throughput sequencing, we found that diverse viruses were uniquely targeted; substrates included dsRNA replication intermediates and intramolecular RNA stem loops. smRNA distribution patterns from viral and synthetic dsRNA precursors were highly reproducible, and machine learning techniques identified characteristics of precursor molecules and smRNA duplexes important in determining relative smRNA abundance. Significance: To our knowledge, this study provides the first description of the rules governing Dicer-2 substrate selection, which has important implications for exogenous RNA silencing technologies and the development of smRNA-based antiviral therapeutics. virus-derived siRNA (vsiRNA) expression comparison between control and 4 different virus-infected cells in control as well as 5 different RNAi pathway protein knock-downs in Drosophila dl1 cells