ABSTRACT: We use small RNA sequencing to look for spreading of secondary and tertiary siRNAs along transcripts targeted by ectopic hairpin-derived primary siRNAs. We find that signals within the 3' UTR inhibit siRNA spreading. sequencing of 20-30nt sRNAs from Argonaute immunoprecipitation
Project description:We tested whether we could increase the population of Rdp1-independent primary siRNAs in fission yeast by overexpressing the Dcr1 ribonuclease. We found that in addition to generation of centromeric siRNAs, which was expected, Dcr1 overexpression also resulted in generation of genome-wide primary siRNAs, mapping to many open reading frames. sequencing of 20-30nt sRNAs from total RNA
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:We analyzed the small RNAs bound to the fission yeast RITS and ARC complexes, and to Ago1 in wild-type and ARC mutant cells, to gain insight into the molecular role of ARC. We found that ARC contains longer heterochromatic siRNAs than RITS, and that ARC subunit Arb1 is required for loading small RNAs onto Ago1. Sequencing of 18-28 nt small RNAs from Tas3, Arb1 and Ago1 purifications.
Project description:We test the effects of overexpressing RNAi proteins Dcr1, Rdp1, or Ago1 in wildtype, dcr1∆, ago1∆, or rdp1∆ cells. We find that overexpression does not generally affect H3K9me2 at euchromatic loci, but there are varying effects in H3K9me2 on centromeric heterochromatin. Examination of genome-wide enrichment in H3K9 dimethylation in fission yeast haploid cells
Project description:Purpose: Identification of miRNA-mRNA interactions in human cells. Method: We used the CLASH technique with PTH-tagged AGO1 protein as a bait. Results: In 6 experiments we identified more than 18,000 miRNA-mRNA interactions in human HEK293 cells, corresponding to targets of 399 miRNAs. Conclusions: The binding of most miRNAs includes the 5' seed region, but around 60% of seed interactions are noncanonical, containing bulged or mismatched nucleotides. Moreover, seed interactions are generally accompanied by specific, non-seed basepairing. 18% of miRNA-mRNA interactions involve the miRNA 3' end, with little evidence for 5' contacts, and some of these were functionally validated. Analyses of miRNA:mRNA basepairing showed that miRNA species systematically differ in their target RNA interactions, and strongly overrepresented motifs were found in the interaction sites of several miRNAs. We speculate that these affect the response of RISC to miRNA-target binding. 6 samples (E1-E6) were prepared with comparable but not identical protocols, described in more detail in Helwak et al. Cell 2013. Samples E7-E10 were used for the determination of the background of the method resulting from RNA-RNA interactions formed after cell lysis.
Project description:microRNAs (miRNAs) are a class of small silencing RNAs that have regulatory roles in gene expression. miRNAs interact with Argonaute (AGO) proteins to form effector complexes that can cleave target mRNAs or repress their translation. Rice encodes four AGO1 homologs (AGO1a, AGO1b, AGO1c, and AGO1d). We used an RNAi approach to knock down the four AGO1s. The RNAi lines displayed pleiotropic developmental phenotypes and had increased accumulation of miRNA targets, suggesting the involvement of AGO1s in the miRNA pathway. Three of the AGO1s (AGO1a, AGO1b, and AGO1c complexes) were purified and further characterized. We showed that the three AGO1s all have a strong preference for binding small RNAs (sRNAs) with 5’ U and have Slicer activity. We catalogued the sRNAs in each AGO1 complex by deep sequencing, and found all three AGO1s predominantly bound known miRNAs. Most of the miRNAs were evenly distributed in the three AGO1 complexes, suggesting a redundant role for the AGO1s in the function of these miRNAs. Intriguingly, we also found a subset of miRNAs were specifically incorporated into or excluded from one of the AGO1s, suggesting that there is also functional specialization among the rice AGO1s. Four samples, total extract and three AGO1 complexes were analyzed
Project description:This SuperSeries is composed of the following subset Series: GSE18248: Sequencing of rice degradome GSE18250: Profiling of small RNA populations in rice total extract and purified AGO1 complexes Refer to individual Series
Project description:We provide evidence of the generation of Rdp1-mediated secondary siRNAs in vivo in fission yeast. Secondly, we show that the presence of Ago1-associated siRNAs does not guarantee robust silencing. We obtained sequences from gel purified small RNAs of wt and rdp1delta fission yeast cells and FLAG-ago1 associated small RNAs.
Project description:Double-stranded RNA-binding proteins are key elements in the intracellular localization of mRNA and its local translation. Staufen is a double-stranded RNA binding protein involved in the localised translation of specific mRNAs during Drosophila early development and neuronal cell fate. The human homologue Staufen1 forms RNA-containing complexes that include proteins involved in translation and motor proteins to allow their movement within the cell, but the mechanism underlying translation repression in these complexes is poorly understood. Here we show that human Staufen1-containing complexes contain essential elements of the gene silencing apparatus, like Ago1-3 proteins, and we describe a set of miRNAs specifically associated to complexes containing human Staufen1. Among these, miR124 stands out as particularly relevant because it appears enriched in human Staufen1 complexes and is over-expressed upon differentiation of human neuroblastoma cells in vitro. In agreement with these findings, we show that expression of human Staufen1 is essential for proper dendritic arborisation during neuroblastoma cell differentiation, yet it is not necessary for maintenance of the differentiated state, and suggest potential human Staufen1 mRNA targets involved in this process. Three or four biological replicates per condition were independently hybridized to GeneChip Human Genome U133 Plus 2.0