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: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: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: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:This SuperSeries is composed of the following subset Series: GSE31288: The impact of RNAi on the Saccharomyces cerevisiae transcriptome GSE31290: Small RNAs in S. cerevisiae reconstituted with RNAi Refer to individual Series
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:Most eukaryotic genes express mRNAs with alternative polyadenylation sites at their 3’ ends. Here we show that polyadenylated 3’ termini in three yeast species (S. cerevisiae, K. lactis, D. hansenii) are remarkably heterogeneous. Instead of a few discrete 3’ ends, the average yeast gene has an “end zone”, a >200 bp window with >60 distinct poly(A) sites, the most utilized of which represents only 20% of the mRNA molecules. The pattern of polyadenylation within this zone varies across species, with D. hansenii possessing a higher focus on a single dominant point closer to the ORF terminus. Some polyadenylation occurs within mRNA coding regions with a strong bias towards the promoter. The polyadenylation pattern is determined by a highly degenerate sequence over a broad region and by a local sequence that relies on A residues after the cleavage point. Many dominant poly(A) sites are predicted to adopt a common secondary structure that may be recognized by the cleavage/polyadenylation machinery. We suggest that the end zone reflects a region permissive for polyadenylation, within which cleavage occurs preferentially at the A-rich sequence. In S. cerevisiae strains, D. hansenii genes adopt the S. cerevisiae polyadenylation profile, indicating that the polyadenylation pattern is mediated primarily by species-specific factors. Four sequencing lanes containing direct RNA sequence from S. cerevisiae (strains JGY2000 and two replicates of AB1380), K. lactis strain CLIB209, D. hansenii strain NCYC2572, and S. cerevisiae strains JYAC06 and JYAC07, each harboring D. hansenii sequences on a YAC. AB1380 is the non-YAC-containing S. cerevisiae parental strain for JYAC06 and JYAC07.
Project description:The RNA interference (RNAi) pathway is found in most eukaryotic lineages but curiously is absent in others, including that of Saccharomyces cerevisiae. Here, we show that reconstituting RNAi in S. cerevisiae causes loss of a beneficial dsRNA virus, known as killer virus. Incompatibility between RNAi and killer viruses extends to other fungal species, in that RNAi is absent in all species known to possess dsRNA killer viruses, whereas killer viruses are absent in closely related species that retained RNAi. Thus, the advantage imparted by acquiring and retaining killer viruses explains the persistence of RNAi-deficient species during fungal evolution. Employ high-throughput sequencing of endogenous small RNAs from Saccharomyces cerevisiae wild-type and RNAi-reconstituted strains.
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:RNAi, a gene-silencing pathway triggered by double-stranded RNA, is conserved in diverse eukaryotic species but has been lost in the model budding yeast, Saccharomyces cerevisiae. We report that RNAi is present in other budding-yeast species, including Saccharomyces castellii and Candida albicans. These species use noncanonical Dicer proteins to generate siRNAs, which mostly correspond to transposable elements and Y´ subtelomeric repeats. In S. castellii, RNAi mutants are viable but have excess Y´ mRNA levels. In S. cerevisiae, introducing Dicer and Argonaute of S. castellii restores RNAi, and the reconstituted pathway silences endogenous retrotransposons. These results identify a novel class of Dicer proteins, bring the tool of RNAi to the study of budding yeasts, and bring the tools of budding yeast to the study of RNAi. Employ high-throughput sequencing of endogenous small RNAs from the budding yeasts Saccharomyces castellii, Kluyveromyces polysporus, Candida albicans, Saccharomyces cerevisiae, and Saccharomyces bayanus.