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The binding specificity and regulatory effect of WT and redesigned Puf2p [CLIP-Seq]

ABSTRACT: 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 3âUTRs, 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.Â CLIP-seq was performed in BY4742 S. cerevisiae grown in log phase, and using 2 replicates of TAP-tagged proteins.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Marv Wickens

PROVIDER: E-GEOD-73273 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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The binding specificity and regulatory effect of WT and redesigned Puf2p [RNA-Seq]

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.Â 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

The binding specificity and regulatory effect of WT and redesigned Puf2p [CLIP-Seq]

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

The binding specificity and regulatory effect of WT and redesigned Puf2p [RNA-Seq]

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.

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A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins (CLIP)

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2012-10-25 | E-GEOD-37111 | biostudies-arrayexpress

Global Analysis of Yeast mRNPs

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2013-04-18 | E-GEOD-46142 | biostudies-arrayexpress

Genome-wide Analysis Reveals SR Protein Cooperation and Competition in Regulated Splicing

Project description:SR proteins are well-characterized RNA binding proteins that promote exon inclusion by binding to exonic splicing enhancers (ESEs). However, it has been unclear whether regulatory rules deduced on model genes apply generally to activities of SR proteins in the cell. Here, we report global analyses of two prototypical SR proteins SRSF1 (SF2/ASF) and SRSF2 (SC35) using splicing-sensitive arrays and CLIP-seq on mouse embryo fibroblasts (MEFs). Unexpectedly, we find that these SR proteins promote both inclusion and skipping of exons in vivo, but their binding patterns do not explain such opposite responses. Further analyses reveal that loss of one SR protein is accompanied by coordinated loss or compensatory gain in the interaction of other SR proteins at the affected exons. Therefore, specific effects on regulated splicing by one SR protein actually depend on a complex set of relationships with multiple other SR proteins in mammalian genomes. SRSF1 and SRSF2 CLIP-seq

2013-04-05 | E-GEOD-44591 | biostudies-arrayexpress

Xenopus Piwi protein associated transcripts indicate regulation beyond transposons

Project description:This study examines the population of transcripts associated with the Xenopus Piwi proteins, Xiwi and Xili, from X.laevis and X.tropicalis. RIP-seq, CLIP-seq, piRNA-seq, and mRNA-seq datasets were integrated to determine how the Xenopus Piwi proteins where using piRNAs and binding interactions to associate with transcripts in gonadal cells. We performed several replicates of a Piw CLIP-Seq experiment to isolate RNA fragments as CLIP-tags to discover which transcripts are preferentially bound by the Piwi protein. Then we performed several types of mRNA expression profiling experiments using several forms of mRNA-Seq library construction formats. Finally, we sequenced the piRNAs from the OSS cells

2015-11-10 | E-GEOD-63228 | biostudies-arrayexpress

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Project description:CTCF is a master regulator that plays a role in genome architecture and gene expression. A key aspect of CTCF’s mechanism involves bringing together distant genetic elements for intra- and inter-chromosomal interactions. Evidence from epigenetic processes, such as X-chromosome inactivation (XCI), suggests that CTCF may carry out its functions through interacting RNAs. Using genome-wide approaches to investigate the relationship between CTCF’s RNA interactome and its epigenomic landscape, here we report that CTCF interacts with thousands of transcripts in mouse embryonic stem cells (mESC), many in close proximity to CTCF’s genomic binding sites. Biochemical analysis demonstrates that CTCF is a high-affinity RNA binding protein that contacts RNA directly and specifically. In the XCI model, CTCF binds the active and inactive X-chromosomes allele-specifically. At the X-inactivation center, Tsix RNA binds CTCF and targets CTCF to a region associated with X-chromosome pairing. Our work implicates CTCF-RNA interactions in long-range chromosomal interactions in trans and adds a new layer of complexity to CTCF regulation. The genome-wide datasets reported here will provide a useful resource for further study of CTCF-mediated epigenomic regulation. CTCF RNA interactome was identified by UV-crosslinking and immunoprecipitation followed by high-throughput sequencing (CLIP-seq), and was compared to CTCF's epigenomic landscape as obtained by chromatin immunoprecipitation (ChIP-seq).

2015-01-09 | E-GEOD-58242 | biostudies-arrayexpress

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