Mapping of dsRNA in yeast using reconstituted RNAi pathway
ABSTRACT: Small RNA produced by Dicer (Dcr1) are used to map dsRNA in wild-type strain and a xrn1-delta mutant of S. cerevisiae, inactivated for the cytoplasmic 5'-3' RNA decay pathway. Small RNA sequencing in wild-type and xrn1-delta strains of S. cerevisiae, with or without reconstituted RNAi pathway.
Project description:Long non-coding RNAs (lncRNAs) have been shown to regulate gene expression, chromatin domains and chromosome stability in eukaryotic cells. Recent observations have reported the existence of telomere associated long ncRNAs (TERRA, telomeric repeat containing RNA) in mammalian and yeast cells but their function(s) remain(s) poorly characterized. Here, we report the existence in S. cerevisiae of several sense and antisense Cryptic Unstable Transcripts (CUTs) and Xrn1-sensitive Unstable Transcripts (XUT) initiating within the subtelomeric repeated region Y’. We show that the Y’ ncRNAs, subTERRA, are distinct from TERRA and are mainly destabilized by the general cytoplasmic and nuclear 5’- and 3’- RNA decays in a sense-dependent manner. subTERRA transcription is mainly sustained by RNAPII and subTERRA accumulate preferentially during the G1/S transition and in C-terminal rap1 mutants independently of Rap1p function in silencing. The accumulation of subTERRA in RNA decay mutants coincides with telomere misregulation: shortening of telomere length, loss of telomeric clustering in mitotic cells, indicating that subTERRA might compete with factors involved in telomere elongation, tethering and/or clustering. We propose that subtelomeric RNAs expression links telomere maintenance with RNA degradation pathways. Exmination of two yeast mutants for RNA decay.
Project description:Comparison of WT, xrn1 delta and upf1 delta strains were used in a tiling array to yield genomic regions regulated by these proteins The supplementary CHP files record either the signal in log2 space or the p-values in linear space, per TAS output. The CHP files are further divided between UPF1 delta vs. WT and XRN1 delta vs. WT. Overall design: Four biological replicates of WT, upf1 delta, and xrn1 delta from the BY4741 background were grown independently and analyzied by Affymetrix yeast tiling microarray analysis.
Project description:Small RNA produced by Dicer (Dcr1) are used to map dsRNA in wild-type strain and a xrn1-delta mutant of S. cerevisiae, inactivated for the cytoplasmic 5'-3' RNA decay pathway. Overall design: Small RNA sequencing in wild-type and xrn1-delta strains of S. cerevisiae, with or without reconstituted RNAi pathway.
Project description:Purpose: identify sites in endogenous mRNAs that are cut by KSHV SOX; Method: parallel analysis of RNA ends (PARE, following Zhai et al., 2014); Results: SOX cuts at discrete locations in mRNAs human Xrn1 was knocked down in HEK293T cells by shRNAs or siRNAs to stabilize degradation fragments with free 5' ends; GFP-SOX or GFP were transfected for ~24 hrs; total RNA samples were collected and subjected to PARE protocol (Zhai et al., 2014)
Project description:Antisense long non-coding (aslnc)RNAs represent a substantial part of eukaryotic transcriptomes that are, in yeast, controlled by the Xrn1 exonuclease. Nonsense-Mediated Decay (NMD) destabilizes the Xrn1-sensitive aslncRNAs (XUT), but what determines their sensitivity remains unclear. We report that 3’ single-stranded (3’-ss) extension mediates XUTs degradation by NMD, assisted by the Mtr4 and Dbp2 helicases. Single-gene investigation, genome-wide RNA analyses and double-stranded (ds)RNA mapping revealed that 3'-ss extensions discriminate the NMD-targeted XUTs from stable lncRNAs. Ribosome profiling showed that XUT are translated locking them for NMD activity. Interestingly, mutants of the Mtr4 and Dbp2 helicases accumulated XUTs, suggesting that dsRNA unwinding is a critical step for degradation. Indeed, expression of anti-complementary transcripts protects cryptic intergenic lncRNAs from NMD. Our results indicate that aslncRNAs form dsRNA that are only translated and targeted to NMD if dissociated by Mtr4 and Dbp2. We propose that NMD buffers genome expression by discarding pervasive regulatory transcripts. Overall design: Strand-specific transcriptome analysis of biological replicates (1) of WT and xrn1-delta cells of the S288C, W303 and SK1 (n & 2n) genetic background of S. cerevisiae; (2) of WT, dcp2-7 and upf1-delta cells; (3) of WT, xrn1-delta and dcp2-7 cells upon treatment of total RNA with Terminator 5'-Phosphate-Dependent Exonuclease. This record also contains CAGE-Seq analysis in wild-type and decapping-deficient cells of the budding yeast S. cerevisiae.
Project description:We report flg22 regulate the accumulation of AGO1-bound small RNA in arabidopsis. We find that a number of miRNAs are up- or down-regulated by flg22, a well-studied PAMP. Examination of AGO1-bound small RNAs with or without flg22 treatment.
Project description:We performed ChIP-Seq for hallmark TFs (Ets1, Runx1), histone modification marks (H3K4me1, H3K4me2, H3K4me3, H3K27me3, H3K36me3), total RNA Pol II, short RNA-Seq as well as nucleosome mapping mainly in murine Rag2 -/- thymocytes. We also performed ChIP-Seq for E47 as well as nucleosome mapping, gene expression microarray analysis in CD4+ CD8+ DP thymocytes. Overall, we find a key role for the transcription factor Ets1, contributing towards alpha beta T cell lineage commitment via differential transactivation of stage-specific genes orchestrated by dynamic, co-association -mediated chromatin remodeling, as well as transcription dependent generation of a specialized chromatin structure at the TCR beta locus. Genome-wide analysis via ChIP-Seq for Ets1, Runx1, total RNA Pol II binding, H3K4me1, H3K4me2, H3K4me3, H3K27me3, H3K36me3, short RNA-Seq, Mnase-Seq in murine Rag2 -/- thymocytes, ChIP-Seq for E47, Mnase-Seq and gene expression microarray analysis in DP thymocytes This Series represents ShortRNA-Seq data.
Project description:In plants, the known microRNAs (miRNAs) are produced as ~21 nucleotide (nt) duplexes from their precursors by Dicer like 1 (DCL1). They are incorporated into Argonaute 1 (AGO1) protein to regulate target gene expression primarily through mRNA cleavage. We report here the discovery of a new class of miRNAs in the model monocot rice (Oryza sativa). These are 24 nt in length and require another member of the Dicer family, DCL3, for their biogenesis. The 24 nt long miRNAs (lmiRNAs) are loaded into AGO4 clade proteins according to hierarchical rules, depending on the upstream biogenesis machinery and the 5’ terminal nucleotide. We demonstrated that lmiRNAs direct DNA methylation at loci from which they are produced as well as in trans at their target genes and play roles in gene regulation. Considered together, our findings define a novel miRNA pathway that mediates DNA methylation. Small RNAs were prepared from Rice total extract in wide type, dcl1, dcl3, rdr2 dbsRNA mutant and AGO4a, AGO4b, and AGO16 complexes, ligated to a 3' adaptor and a 5' acceptor sequentially, and then RT-PCR amplified. PCR products were reamplified using a pair of solexa cloning primers and then provided for sequencing. For technical details, see Wu, L., Zhang, Q., Zhou, H., Ni, F., Wu, X., and Qi, Y. (2009). Rice microRNA effector complexes and targets. The Plant Cell, 21: 3421-3435.
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