Saccharomyces cerevisiae
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA129321 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:The vast landscape of RNA-protein interactions at the heart of post-transcriptional regulation remains largely unexplored. Indeed it is likely that, even in yeast, a substantial fraction of the regulatory RNA-binding proteins (RBPs) remain to be discovered. Systematic experimental methods can play a key role in discovering these RBPs - most of the known yeast RBPs lack RNA-binding domains that might enable this activity to be predicted. We describe here a new proteome-wide approach to identify RNA-protein interactions based on in vitro binding of RNA samples to yeast protein microarrays that represent over 80% of the yeast proteome. We used this procedure to screen for novel RBPs and RNA-protein interactions. A complementary mass spectrometry technique also identified proteins that associate with yeast mRNAs. Both the protein microarray and mass spectrometry methods successfully identify previously annotated RBPs, suggesting that other proteins identified in these assays might be novel RBPs. Of 35 putative novel RBPs identified by either or both of these methods, 12, including 75% of the eight most highly-ranked candidates, reproducibly associated with specific cellular RNAs. Surprisingly, most of the 12 newly discovered RBPs were enzymes. Functional characteristics of the RNA targets of some of the novel RBPs suggest coordinated post-transcriptional regulation of subunits of protein complexes and a possible link between mRNA trafficking and vesicle transport. Our results suggest that many more RBPs still remain to be identified and provide a set of candidates for further investigation.
2011-03-10 | GSE22876 | GEO
Project description:The vast landscape of RNA-protein interactions at the heart of post-transcriptional regulation remains largely unexplored. Indeed it is likely that, even in yeast, a substantial fraction of the regulatory RNA-binding proteins (RBPs) remain to be discovered. Systematic experimental methods can play a key role in discovering these RBPs - most of the known yeast RBPs lack RNA-binding domains that might enable this activity to be predicted. We describe here a new proteome-wide approach to identify RNA-protein interactions based on in vitro binding of RNA samples to yeast protein microarrays that represent over 80% of the yeast proteome. We used this procedure to screen for novel RBPs and RNA-protein interactions. A complementary mass spectrometry technique also identified proteins that associate with yeast mRNAs. Both the protein microarray and mass spectrometry methods successfully identify previously annotated RBPs, suggesting that other proteins identified in these assays might be novel RBPs. Of 35 putative novel RBPs identified by either or both of these methods, 12, including 75% of the eight most highly-ranked candidates, reproducibly associated with specific cellular RNAs. Surprisingly, most of the 12 newly discovered RBPs were enzymes. Functional characteristics of the RNA targets of some of the novel RBPs suggest coordinated post-transcriptional regulation of subunits of protein complexes and a possible link between mRNA trafficking and vesicle transport. Our results suggest that many more RBPs still remain to be identified and provide a set of candidates for further investigation. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc.
2011-03-10 | E-GEOD-22876 | biostudies-arrayexpress
Project description:In this study, we systematically identified the RNAs associated with a selective sample of 40 of ~600 yeast RNA-binding proteins (RBPs). To identify RNAs associated with each putative RBP, C-terminal tandem affinity purification (TAP)-tagged proteins, expressed under control of their native promoters, were affinity purified from whole cell extracts of cultures grown to mid-log phase in rich medium [1-3]. Extracts were incubated with immunoglobulin G (IgG) agarose beads, washed, and ribonuclear protein complexes were eluted by tobacco etch virus (TEV) protease treatment (Text S2). We performed two to four independent isolations with each tagged strain. As controls, we performed 13 immunoaffinity purifications (IPs) of untagged strains to identify and exclude potential false-positive RNA targets. We purified total RNA from the whole-cell extracts and TEV-purified fractions, reverse transcribed with an amino-allyl-dUTP/dNTP mix, coupled the purified cDNA to Cy3 and Cy5 dyes, respectively, mixed the two differentially labeled cDNA pools, and then hybridized them to DNA microarrays (Datasets S1-S4). An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Genotype: strain expressing tagged version of RNA-BP Keywords: all_pairs Computed
2010-06-25 | E-GEOD-13135 | biostudies-arrayexpress
Project description:In this study, we systematically identified the RNAs associated with a selective sample of 40 of ~600 yeast RNA-binding proteins (RBPs). To identify RNAs associated with each putative RBP, C-terminal tandem affinity purification (TAP)-tagged proteins, expressed under control of their native promoters, were affinity purified from whole cell extracts of cultures grown to mid-log phase in rich medium [1-3]. Extracts were incubated with immunoglobulin G (IgG) agarose beads, washed, and ribonuclear protein complexes were eluted by tobacco etch virus (TEV) protease treatment (Text S2). We performed two to four independent isolations with each tagged strain. As controls, we performed 13 immunoaffinity purifications (IPs) of untagged strains to identify and exclude potential false-positive RNA targets. We purified total RNA from the whole-cell extracts and TEV-purified fractions, reverse transcribed with an amino-allyl-dUTP/dNTP mix, coupled the purified cDNA to Cy3 and Cy5 dyes, respectively, mixed the two differentially labeled cDNA pools, and then hybridized them to DNA microarrays (Datasets S1-S4). An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Genotype: strain expressing tagged version of RNA-BP Keywords: all_pairs
2008-10-28 | GSE13135 | GEO
Project description:Protein binding is essential to the transport, decay and regulation of almost all RNA molecules. However, the structural preference of protein binding on RNAs and their cellelar functions and dynamics upon changing environmental condictions are poorly understood. Here, we integrated various high-throughput data and introduced a computational framework to describe the global interactions between RNA binding proteins (RBPs) and structured RNAs in yeast at single-nucleotide resolution. We found that on average, in terms of percent total lengths, ~15% of mRNA untranslated regions (UTRs), ~37% of canonical ncRNAs and ~11% of long ncRNA (lncRNAs) are bound by proteins. The RBP binding sites, in general, tend to occur at single-stranded loops, with evolutionarily conserved signatures, and often facilitate a specific RNA structure conformation in vivo. We found that four nucleotide modifications of tRNA are significantly associated with RBP binding. We also identified various structural motifs bound by RBPs in the UTRs of mRNAs, associated with localization, degradation and stress responces. Moreover, we identified >200 novel lncRNAs bound by RBPs, and about half of them contain conserved secondary structures. We present the first ensemble pattern of RBP binding sites in the structured noncoding regions of a eukaryotic genome, emphasizing their structural context and cellular functions.
2013-07-15 | GSE48888 | GEO
Project description:Protein binding is essential to the transport, decay and regulation of almost all RNA molecules. However, the structural preference of protein binding on RNAs and their cellelar functions and dynamics upon changing environmental condictions are poorly understood. Here, we integrated various high-throughput data and introduced a computational framework to describe the global interactions between RNA binding proteins (RBPs) and structured RNAs in yeast at single-nucleotide resolution. We found that on average, in terms of percent total lengths, ~15% of mRNA untranslated regions (UTRs), ~37% of canonical ncRNAs and ~11% of long ncRNA (lncRNAs) are bound by proteins. The RBP binding sites, in general, tend to occur at single-stranded loops, with evolutionarily conserved signatures, and often facilitate a specific RNA structure conformation in vivo. We found that four nucleotide modifications of tRNA are significantly associated with RBP binding. We also identified various structural motifs bound by RBPs in the UTRs of mRNAs, associated with localization, degradation and stress responces. Moreover, we identified >200 novel lncRNAs bound by RBPs, and about half of them contain conserved secondary structures. We present the first ensemble pattern of RBP binding sites in the structured noncoding regions of a eukaryotic genome, emphasizing their structural context and cellular functions. Duplicate gPAR-CLIP libraries were sequenced from yeast strains for each of three conditions: log-phase growth, growth after 2 hour glucose starvation, and growth after 2 hour nitrogen starvation. polyA RNAs were isolated for all conditions. Total RNA were isolated from log phase growth conditions. Sucrose gradient fractionation was performed: some RNAs were isolated from the "light" fraction (lighter than 40S ribosome) and some from the "heavy" fraction. gPAR-CLIP libraries were used to determine regions of RNA bound by proteins.
2013-07-15 | E-GEOD-48888 | biostudies-arrayexpress
Project description:To identify RNAs specifically associated with potential RBPs, yeast cells expressing TAP-tagged RBP or the wild-type strain BY4741 (mock control) were grown to mid-log phase in rich medium and harvested by centrifugation. RNA affinity isolations were essentially performed as described (Gerber et al. 2004 PLoS Biol.; see protocol). In brief, TAP-tagged protein were captured from cell extracts with IgG coupled agarose beads (Sigma) and released by incubation with a site-specific protease (AcTEV, Sigma). from the extract (input) and from the affinity isolates was purified with the RNeasy Mini/ Micro Kit (Qiagen). RNAs were reverse transcribed using a mixture of oligo-dT and random nonamer oligos in the presence of amino-allyl dUTP/dNTP mixture. cDNAs were fluorescently labeled and hybridized on yeast oligo microarrays over night at 42 degrees in formamide-based hybridization buffer. Antigenic peptide used in IP: TAP-tag An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.
2010-12-06 | E-GEOD-21864 | biostudies-arrayexpress