The binding specificity and regulatory effect of WT and redesigned Puf2p [CLIP-Seq]
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
SUBMITTER: Marv Wickens
PROVIDER: E-GEOD-73273 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:PUF proteins have become a leading scaffold for designing RNA-binding proteins to contact and control RNAs at will. We analyze the effects of that reengineering across the transcriptome in vivo for the first time. We show, by HITS-CLIP and PAR-CLIP, that S. cerevisiae Puf2p, a non-canonical PUF protein, binds more than 1000 mRNA targets. Puf2p binds multiple UAAU elements, unlike canonical PUF proteins. We also perform CLIP-seq on truncations of Puf2p, showing that its prion domain is dispensable for WT binding. We design a modified Puf2p to bind UAAG rather than UAAU, which allows us to align the protein with the binding site. In vivo, the redesigned protein binds UAAG sites. Its altered specificity redistributes the protein away from 3âUTRs, such that the protein tracks with its sites and binds throughout the mRNA. We use RNA-seq to determine that R1 SNE Puf2p represses a novel RNA network. CLIP-seq was performed in BY4742 S. cerevisiae grown in log phase, and using 2 replicates of TAP-tagged proteins. RNA-seq was performed to determine the regulatory effect of WT or mutant Puf2p, using 4 replicates of the control (no Puf2p), 3 of WT Puf2p and 4 of R1 SNE Puf2p.
2015-12-15 | E-GEOD-73227 | biostudies-arrayexpress
Project description:PUF proteins have become a leading scaffold for designing RNA-binding proteins to contact and control RNAs at will. We analyze the effects of that reengineering across the transcriptome in vivo for the first time. We show, by HITS-CLIP and PAR-CLIP, that S. cerevisiae Puf2p, a non-canonical PUF protein, binds more than 1000 mRNA targets. Puf2p binds multiple UAAU elements, unlike canonical PUF proteins. We also perform CLIP-seq on truncations of Puf2p, showing that its prion domain is dispensable for WT binding. We design a modified Puf2p to bind UAAG rather than UAAU, which allows us to align the protein with the binding site. In vivo, the redesigned protein binds UAAG sites. Its altered specificity redistributes the protein away from 3’UTRs, such that the protein tracks with its sites and binds throughout the mRNA. We use RNA-seq to determine that R1 SNE Puf2p represses a novel RNA network.
2015-12-15 | GSE73227 | GEO
Project description:PUF proteins have become a leading scaffold for designing RNA-binding proteins to contact and control RNAs at will. We analyze the effects of that reengineering across the transcriptome in vivo for the first time. We show, by HITS-CLIP and PAR-CLIP, that S. cerevisiae Puf2p, a non-canonical PUF protein, binds more than 1000 mRNA targets. Puf2p binds multiple UAAU elements, unlike canonical PUF proteins. We also perform CLIP-seq on truncations of the Puf2p, showing that its prion domain is dispensable for WT binding. We design a modified Puf2p to bind UAAG rather than UAAU, which allows us to align the protein with the binding site. In vivo, the redesigned protein binds UAAG sites. Its altered specificity redistributes the protein away from 3’UTRs, such that the protein tracks with its sites, binds throughout the mRNA. We use RNA-seq to determine that R1 SNE Puf2p represses a novel RNA network.
2015-12-15 | GSE73273 | GEO
Project description:Crosslinking and immunoprecipitation (CLIP) is increasingly used to map transcriptome-wide binding sites of RNA-binding proteins (RBPs). We developed a method for CLIP data analysis and applied it to compare 254 nm CLIP with PAR-CLIP, which involves crosslinking of photoreactive nucleotides with 365 nm UV light. We found small differences in the accuracy of these methods in identifying binding sites of HuR, a protein that binds low-complexity sequences and Argonaute 2, which has a complex binding specificity. We show that crosslink-induced mutations lead to single-nucleotide resolution for both PAR-CLIP and CLIP. Our results confirm the expectation from original CLIP publications that RNA-binding proteins do not protect sufficiently their sites under the denaturing conditions used during the CLIP procedure, and we show that extensive digestion with sequence-specific ribonucleases strongly biases the set of recovered binding sites. We finally show that this bias can be substantially reduced by milder nuclease digestion conditions. We performed duplicate experiments for each variant of the CLIP protocol (CLIP, PAR-CLIP), each protein (HuR, Ago2), and enzymatic digestion (complete T1 digestion, mild MNase digestion). In addition, we performed a single PAR-CLIP experiment with mild T1 digestion.
2011-05-15 | E-GEOD-28859 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE3968: Puf proteins, RNA co-immunopurification GSE3969: Puf3delta vs. wild type comparison Abstract: Genes encoding RNA-binding proteins are diverse and abundant in eukaryotic genomes. Although some have been shown to have roles in post-transcriptional regulation of the expression of specific genes, few of these proteins have been studied systematically. We have used an affinity tag to isolate each of the five members of the Puf family of RNA-binding proteins in Saccharomyces cerevisiae and DNA microarrays to comprehensively identify the associated mRNAs. Distinct groups of 40-220 different mRNAs with striking common themes in the functions and subcellular localization of the proteins they encode are associated with each of the five Puf proteins: Puf3p binds nearly exclusively to cytoplasmic mRNAs that encode mitochondrial proteins; Puf1p and Puf2p interact preferentially with mRNAs encoding membrane-associated proteins; Puf4p preferentially binds mRNAs encoding nucleolar ribosomal RNA-processing factors; and Puf5p is associated with mRNAs encoding chromatin modifiers and components of the spindle pole body. We identified distinct sequence motifs in the 3'-untranslated regions of the mRNAs bound by Puf3p, Puf4p, and Puf5p. Three-hybrid assays confirmed the role of these motifs in specific RNA-protein interactions in vivo. The results suggest that combinatorial tagging of transcripts by specific RNA-binding proteins may be a general mechanism for coordinated control of the localization, translation, and decay of mRNAs and thus an integral part of the global gene expression program. Refer to individual Series
2006-03-07 | E-GEOD-4393 | biostudies-arrayexpress
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
2013-04-18 | E-GEOD-46142 | biostudies-arrayexpress
Project description:PUF family proteins are among the best characterized regulatory RNA-binding proteins in non-mammalian species, but relatively little is known about mRNA targets or functions of mammalian PUF proteins. In this study, we used ribonomic analysis to identify and analyze mRNAs associated with ribonucleoproteins containing an endogenous human PUF protein, Pum1. Pum1 associated mRNAs were highly enriched for genes encoding proteins that function in transcriptional regulation and cell cycle/proliferation, results consistent with the post-transcriptional RNA regulon model and the proposed ancestral functions of PUF proteins in stem cell biology. Analysis of 3’UTR sequences of Pum1 associated mRNAs revealed a core Pum1 consensus sequence, UGUAHAUA. Pum1 knockdown demonstrated that Pum1 enhances decay of associated mRNAs, and re-localization of Pum1 to stress granules suggested that Pum1 functions in repression of translation. This study is the first in vivo genome-wide mRNA target identification of a mammalian PUF protein and provides direct evidence that human PUF proteins regulate stability of associated mRNAs. Comparison of Pum1 associated mRNAs to mRNA targets of PUF proteins from S. cerevisiae and Drosophila demonstrates how a well conserved RNA-binding domain and cognate binding sequence have been evolutionarily rewired to regulate the collective expression of different sets of functionally related genes. Keywords: RIP-chip
2008-05-01 | GSE11301 | GEO
Project description:Dicer is a deeply conserved endoribonuclease with key functions in small RNA biogenesis. Here we employed PAR-CLIP/iPAR-CLIP to identify direct Dicer binding sites in the transcriptomes of human cells and human. We found hundreds of novel miRNAs and non-canonical Dicer substrates with high sensitivity. Small RNA production depended on structure of the binding site and is globally biased towards the 5' arm of hairpins. Unexpectedly, in both species Dicer bound numerous hairpins inside mRNAs without observable small RNA production. Our data revealed ~100 mRNAs of protein coding genes to be targeted in both human and worm. These mRNAs significantly overlapped with the RNAi pathway. We also, unexpectedly, found that mitochondrial transcripts are Dicer targets in both species. We demonstrate functional consequences of Dicer binding by perturbation analysis. Taken together,we provide the first genome-wide catalog of direct Dicer targets. Our results suggest widespread function outside of miRNA biogenesis. PAR-CLIP basically as described previously (Hafner et al. 2010).
2014-11-20 | E-GEOD-55324 | biostudies-arrayexpress
Project description:To exert regulatory function, miRNAs guide Argonaute (AGO) proteins to partially complementary sites on target RNAs. Crosslinking and immunoprecipitation (“re state-of-the-art to map AGO binding sites, but assigning the targeting miRNA to these sites relies on bioinformatics predictions and is therefore indirect. To directly and unambiguously identify miRNA:target site interactions, we modified our CLIP methodology in C. elegans to experimentally ligate miRNAs to their target sites. Unexpectedly, ligation reactions also occurred in absence of the exogenous ligase. Our in vivo dataset and re-analysis of published mammalian AGO-CLIP data for miRNA-chimeras yielded >17,000 miRNA:target site interactions. Analysis of interactions and extensive experimental validation of chimera-discovered targets of viral miRNAs suggest that our strategy identifies canonical, non-canonical, and non-conserved miRNA interactions. Our data suggest that ~80% of miRNA:targets have perfect or partial seed complementarity. In summary, analysis of miRNA:target chimeras enables the systematic, context-specific, in vivo discovery of miRNA interactions. In vivo PAR-CLIP basically as described previously (Jungkamp et al. 2011) using GFP-tagged ALG-1 expressing worms in L3 stage. Worm lysate was treated with RNase T1. Following immunoprecipitation and a second RNase T1 digest, it was proceeded as described in Hafner et al. 2010. For the modified iPAR-CLIP ligation samples and its control samples immuno-purified complexes were treated with PNK phospathase minus, subjected to ligation with T4 RNA ligase/no ligase added and subsequently phosphorylated with PNK. Protein purification and RNA library preparation essentially as described in Hafner et al., but with the selection of longer RNA products.
2014-05-23 | E-GEOD-56180 | biostudies-arrayexpress
Project description:PUF family proteins are among the best characterized regulatory RNA-binding proteins in non-mammalian species, but relatively little is known about mRNA targets or functions of mammalian PUF proteins. In this study, we used ribonomic analysis to identify and analyze mRNAs associated with ribonucleoproteins containing an endogenous human PUF protein, Pum1. Pum1 associated mRNAs were highly enriched for genes encoding proteins that function in transcriptional regulation and cell cycle/proliferation, results consistent with the post-transcriptional RNA regulon model and the proposed ancestral functions of PUF proteins in stem cell biology. Analysis of 3’UTR sequences of Pum1 associated mRNAs revealed a core Pum1 consensus sequence, UGUAHAUA. Pum1 knockdown demonstrated that Pum1 enhances decay of associated mRNAs, and re-localization of Pum1 to stress granules suggested that Pum1 functions in repression of translation. This study is the first in vivo genome-wide mRNA target identification of a mammalian PUF protein and provides direct evidence that human PUF proteins regulate stability of associated mRNAs. Comparison of Pum1 associated mRNAs to mRNA targets of PUF proteins from S. cerevisiae and Drosophila demonstrates how a well conserved RNA-binding domain and cognate binding sequence have been evolutionarily rewired to regulate the collective expression of different sets of functionally related genes. Pum1 IP, negative IP, and total IP samples were analyzed for each of 3 biological replicates. Each IP or total RNA sample was run on a separate array versus a common reference sample
2008-05-01 | E-GEOD-11301 | biostudies-arrayexpress