Mapping of dsRNA in yeast using reconstituted RNAi pathway
Ontology highlight
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:To examine whether the mutations in NLS1/2 affect the interaction with mRNAs, we determined the in vitro and in vivo interaction of WT Xrn1 and Xrn1 Delta NLS1/2 mutant with cellular mRNAs.
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: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.
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.
Project description:To investigate the role of cytoplasmic helicases in the decay of Xrn1-sensitive lncRNAs, we performed RNA-Seq in WT, ecm32-delta, ski2-delta, slh1-delta, dbp1-delta and dhh1-delta yeast cells.
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: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:mRNA level is controlled by factors that mediate both mRNA synthesis and decay, including the 5’ to 3’ exonuclease Xrn1 - a major mRNA synthesis and decay factor. Here we show that nucleocytoplasmic shuttling of several mRNA decay factors plays a key role in determining both mRNA synthesis and decay. Shuttling is regulated by RNA-controlled binding of the karyopherin Kap120 to two nuclear localization sequences (NLSs) in Xrn1, location of one of which is conserved from yeast to human. The decaying RNA binds and masks NLS1, establishing a link between mRNA decay and Xrn1 shuttling. Preventing Xrn1 import, either by deleting KAP120 or mutating the two Xrn1 NLSs, compromise transcription and, unexpectedly, also the cytoplasmic decay, uncovering a cytoplasmic decay pathway that initiates in the nucleus. Most mRNAs are degraded by both the “classical” and the novel pathways, the ratio between them represents a full spectrum. Importantly, Xrn1 shuttling is required for proper adaptation to environmental changes, in particular to ever changing environmental fluctuations.
Project description:Antisense (as)lncRNAs are extensively degraded by the nuclear exosome and the cytoplasmic exoribonuclease Xrn1 in the budding yeast Saccharomyces cerevisiae, lacking RNA interference (RNAi). Whether the ribonuclease III Dicer affects aslncRNAs in close RNAi-capable relatives remains unknown. Using genome-wide RNA profiling, here we show that aslncRNAs are primarily targeted by the exosome and Xrn1 in the RNAi-capable budding yeast Naumovozyma castellii, Dicer only affecting Xrn1-sensitive lncRNAs (XUTs) levels in Xrn1-deficient cells. The dcr1 and xrn1 mutants display synergic growth defects, indicating that Dicer becomes critical in the absence of Xrn1. Small RNA sequencing showed that Dicer processes aslncRNAs into small RNAs, with a preference for asXUTs. Consistently, Dicer localizes into the cytoplasm. Finally, we observed an expansion of the exosome-sensitive antisense transcriptome in N. castellii compared to S. cerevisiae, suggesting that the presence of cytoplasmic RNAi has reinforced the nuclear RNA surveillance machinery to temper aslncRNAs expression. Our data provide fundamental insights into aslncRNAs metabolism and open perspectives into the possible evolutionary contribution of RNAi in shaping the aslncRNAs transcriptome.