RNAi knock down of mRNA decapping co-activators in Drosophila S2 cells
ABSTRACT: We analyzed mRNA expression profiles in Drosophila melanogaster S2 cells that had been depleted of proteins known as mRNA decapping co-activators. mRNA decapping is catalyzed by DCP2, and DCP2 activity is stimulated by decapping co-activators. This group of proteins includes DCP1, Hedls (also known as Ge-1), LSm16 (also known as EDC3), rck/p54 (also known as DHH1 or Me31B), Pat1, and the heptameric LSm1-7 complex. We used the RNA interference technology to deplete cultured S2 cells of DCP1 (CG11183), Ge-1 (CG6181), Pat1 (CG5208), LSm16 (CG6311), and LSm1 (CG4279). We used Affymetrix oligonucleotide microarrays to analyze two independent samples for each depletion. We included the following controls: mock RNAi treatment and GFP dsRNA treatment (two profiles each). We also profiled AGO1 (CG6671) depleted cells (3 independent samples). AGO1 is a key protein required for miRNA-mediated gene silencing. We had shown previously that silencing by miRNAs involves decapping of target mRNAs.
Project description:To assess the roles of the Dcp2 C-terminal domain and the decapping activators Pat1, Lsm1, and Dhh1 in mRNA decapping, we used RNA-Seq to analyze the expression profiles of yeast cells harboring a truncation of the Dcp2 C-terminal domain, mutations that render Dcp2 catalytically inactive, or deletions of the PAT1, LSM1, and DHH1 genes. Consistent with our recent model for decapping regulation, we found that: i) the Dcp2 C-terminal domain is an effector of both negative and positive regulation and that loss of these control functions causes significant deregulation of mRNA decapping; ii) rather than being global activators of decapping, Pat1, Lsm1, and Dhh1 directly target specific subsets of yeast mRNAs and loss of the functions of each of these factors has substantial indirect consequences for genome-wide mRNA expression; and iii) transcripts targeted by Pat1, Lsm1, and Dhh1 exhibit only partial overlap and, as expected, are targeted to decapping-dependent decay. Overall design: Genome-wide expression profiles of the wild-type strain and each of the mutant strains were generated by RNA-Seq, in triplicate, using Illumina HiSeq4000. The transcripts differentially expressed in different mutant cells were identified by comparisons to the transcripts expressed in wild-type cells.
Project description:miRNAs silence gene expression by repressing translation and/or by promoting mRNA decay. AGO1 is a key protein required for miRNA-mediated gene silencing. In animal cells, mRNA degradation of partially complementary miRNA targets occurs via deadenylation by the CAF1-CCR4-NOT1 deadenylase complex, followed by decapping and subsequent exonucleolytic digestion. To determine how generally miRNAs trigger deadenylation, we compared mRNA expression profiles in D. melanogaster cells depleted of AGO1, CAF1 or NOT1. We show that approximately 45% of AGO1-targets are regulated by both CAF1 and NOT1, indicating deadenylation is a widespread effect of miRNA regulation.<br><br>We employed RNA interference using long double-stranded RNAs to deplete cultured S2 cells of AGO1 (CG6671, 2 independent samples), CAF1 (CG5684, 2 independent samples), or NOT1 (CG1884, 3 independent samples). Further, we used Affymetrix oligonucleotide microarrays to analyze expression profiles in these samples. We included the following controls: mock RNAi treatment and GFP dsRNA treatment (3 and 2 independent samples, respectively).<br>
Project description:To assess the role of the decapping activator Scd6 in mRNA decay, we used RNA-Seq to analyze the expression profile of yeast cells harboring a deletion of the SCD6 gene. Consistent with our recent model for decapping regulation, we found that Scd6 targets a small number of specific mRNAs in yeast cells. Interestingly, degradation of Scd6-targeted transcripts also requires the functions of the decapping activators Pat1, Lsm1, and Dhh1, suggesting that Scd6 functions together with Pat1, Lsm1, and Dhh1 in promoting mRNA decapping. Overall design: Genome-wide expression profiles of the wild-type and scd6∆ strains were generated by RNA-Seq, in triplicate, using Illumina HiSeq4000. Transcripts differentially expressed in the scd6∆ strain were identified by comparing to the wild-type strain.
Project description:RNA silencing pathways are conserved gene regulation mechanisms that lead to degradation, translational repression or transcriptional silencing of target-transcripts selected based on complementarity with small RNA molecules. The fraction of the genome regulated by these pathways has not been established. Argonaute proteins play fundamental roles in RNA silencing. To identify their physiological targets at the genomic level, we examined expression profiles in Drosophila cells depleted of 4 Argonaute paralogs (i.e. AGO1, AGO2, PIWI or Aubergine). We also profiled cells depleted of the microRNA (miRNA)-processing enzyme Drosha.
Project description:This study involves the role of yeast mRNA decay factors in transcription. The experiment included here are the ChIP-exo results of three decay factors: Xrn1, Dcp2 & Lsm1. Four experiments were made: Xrn1, Dcp2, Lsm1 and control (no-TAP tag), in two replicates.
Project description:Proteins regulate gene expression by controlling mRNA biogenesis, localization, translation and decay. Identifying the composition, diversity and function of mRNPs (mRNA protein complexes) is essential to understanding these processes. In a global survey of S. cerevisiae mRNA binding proteins we identified 120 proteins that cross-link to mRNA, including 66 new mRNA binding proteins. These include kinases, RNA modification enzymes, metabolic enzymes, and tRNA and rRNA metabolism factors. These proteins show dynamic subcellular localization during stress, including assembly into stress granules and P-bodies (Processing-bodies). CLIP (cross-linking and immunoprecipitation) analyses of the P-body components Pat1, Lsm1, Dhh1 and Sbp1 identified sites of interaction on specific mRNAs revealing positional binding preferences and co-assembly preferences. Taken together, this work defines the major yeast mRNP proteins, reveals widespread changes in their subcellular location during stress, and begins to define assembly rules for P-body mRNPs. CLIP-seq analysis of Dhh1, Lsm1, Pat1 and Sbp1
Project description:RNA silencing pathways are conserved gene regulation mechanisms that lead to degradation, translational repression or transcriptional silencing of target-transcripts selected based on complementarity with small RNA molecules. Using firefly luciferase reporters, we previously were able show that the RNA binding protein GW182 plays a role in microRNA-mediated gene silencing (Rehwinkel, J. et al., RNA J. 2005). In this study, we provide further evidence for the role GW182 in the miRNA pathway by determining targets regulated by this protein at the genomic level. We examined expression profiles in Drosophila cells depleted of GW182 and compared these profiles to data obtained from cells depleted of AGO1, a key effector of miRNA-mediated gene silencing.
Project description:Identification of sites of replication initiation by copy number analysis, comparing samples before and after entry into S phase. Experiments were performed in the following strains and conditions for mitotic cells: cdc25-22 (temperature-sensitive mutation arrests cells at G2/M for synchrony, otherwise wild type), nitrogen-rich conditions; wild type, haploid and diploid, nitrogen-depletion conditions. Experiments were performed in the following strains and conditions for meiotic cells: pat1-114 diploid (temperature-sensitive mutation induces a synchronous meiosis, otherwise wild type), nitrogen-depletion and nitrogen-rich conditions; pat1-114 diploid with altered Cdc45 levels, nitrogen-depletion conditions. Comparison of samples before and after S phase entry; dye-swap experiments were performed and averaged.
Project description:we determined the contribution of the decapping enzyme Dcp2 to maternal mRNA clearance in zebrafish embryos by overexpressing a dominant-negative form of Dcp2. Overall design: zebrafish embryonic mRNA profile at 6 hpf in mock-inejcted or Dcp2-DN expressing embryos. Experiments are performed as triplicates.
Project description:The general pathways of eukaryotic mRNA decay occur via deadenylation followed by 3’ to 5’ degradation or decapping, although some endonuclease sites have been identified in metazoan mRNAs. To determine the role of endonucleases in mRNA degradation in Saccharomyces cerevisiae, we mapped 5’ monophosphate ends on mRNAs in wild-type and dcp2∆ xrn1∆ yeast cells, wherein mRNA endonuclease cleavage products are stabilized. This led to three important observations. First, only few mRNAs that undergo low level endonucleotyic cleavage were observed suggesting that endonucleases are not a major contributor to yeast mRNA decay. Second, independent of known decapping enzymes, we observed low levels of 5’ monophosphates on some mRNAs suggesting that an unknown mechanism can generate 5' exposed ends, although for all substrates tested Dcp2 was the primary decapping enzyme. Finally, we identified debranched lariat intermediates from intron-containing genes, demonstrating a significant discard pathway for mRNAs during the second step of pre-mRNA splicing, which is a potential new step to regulate gene expression. 5' monophosphorylated ends of poly(A) RNA from wild-type and dcp2D xrn1D strains were identified in duplicates and triplicates, respectively.