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3' isoform specific RNA IP (3' isRIP) for PUF3 RBP in budding yeast

ABSTRACT: 3' isoform specific RNA IP (3' isRIP) for PUF3 RBP in budding yeast

PROVIDER: PRJEB5011 | ENA |

REPOSITORIES: ENA

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Dataset's files

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			Action	DRS
	ERR375918_1.fastq.gz	Fastqsanger.gz
	ERR375918_2.fastq.gz	Fastqsanger.gz
	ERR375919_1.fastq.gz	Fastqsanger.gz
	ERR375919_2.fastq.gz	Fastqsanger.gz
	ERR375920_1.fastq.gz	Fastqsanger.gz

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3' isoform specific RNA IP (3' isRIP-seq) for PUF3 RBP in budding yeast

Project description:Exponentially growing cells (OD 0.5-0.8) from the TAP-tagged strain were harvested by ultracentrifugation followed by of washing and flash freezing in liquid nitrogen and breakage in FastPrep24 (ZYmoresearch S6005). The lysate was subjected to IP with Dyna M280 sheep anti rabbit IgG beads for 2h at 4C. An aliquot was taken before the IP as an input control. The IP of the RNA-protein complex was subjected to cleavage by TEV protease to cleave the TAP-tag off the protein. The bound RNA was then precipitated in phenol/chloroform/isoamyl-alcohol and sequenced using 3' T-filling protocol as described by Wilkening et al 2013 to obtain accurate quantification of 3' isoforms. We used the R Bioconductor package DESeq2 (Anders and Huber 2010) to call for differential log change between the Input and the RNA-IP sample and took all those isoforms as differentially regulated which had more than 4-fold change at a FDR of 10% as reported by the package.

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Rpb4 and Puf3

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Project description:To specifically explore such mRNA localization events, we immunopurified the entire Nuclear Pore Complex scaffold using Nup59 as a bait and systematically analyzed all co-isolating mRNAs using Agilent microarrays in the budding yeast Saccharomyces cerevisiae. A mock IP was performed as a control experiment, using a yeast strain in which Nup59 was untagged. Two replicates of the Nup59 IP and of the mock IP were performed. The mRNAs with a significant enrichment (p<0.05 in the bayesian test of the LIMMA package) in the mock IP were removed from the further analyses of the Nup59-IP.

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Project description:Puf3 is a RNA-binding protein, a member of the conserved Puf-protein family. Combining different functional genomics data, we have analyzed the role of Puf3 in post-transcriptional gene regulation in S. pombe. We present data on Puf3 interacting proteins and regulatory mRNA targets.

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Mass spectrometry data for Leishmania major proteins obtained by immunoprecipitation with anti-PUF1, anti-PUF3 or pre-immunization antibodies.

Project description:This project was aimed to the characterization of proteins interacting with the RNA binding proteins PUF1 and PUF3 of Leishmania major (Friedlin strain). For this purpose an immunoprecipitation (IP) strategy was followed. Firstly, protein extracts from L. major promastigotes were incubated with specific antibodies, raised in rabbits, against recombinant proteins PUF1 or PUF3. In parallel, a control IP using a pre-immune serum was performed. After affinity purification of the antibody-protein complexes, proteins were analyzed by Reverse phase- Liquid Chomatography/Mass spectrometry.

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Project description:Pbp1 (polyA-binding protein - binding protein 1) is a stress granule marker and polyglutamine expansions in its mammalian ortholog ataxin-2 have been linked to neurodegenerative conditions. Pbp1 was recently shown to form intracellular assemblies that function in the negative regulation of TORC1 signaling under respiratory conditions. Furthermore, it was observed that loss of Pbp1 leads to mitochondrial dysfunction. Here, we show that loss of Pbp1 leads to a specific decrease in mitochondrial proteins whose encoding mRNAs are targets of the RNA-binding protein Puf3, suggesting a functional relationship between Pbp1 and Puf3. We found that Pbp1 stabilizes and promotes the translation of Puf3-target mRNAs in respiratory conditions, such as those involved in the assembly of cytochrome c oxidase. We further show that Pbp1 and Puf3 associate through their respective low complexity domains, which is required for target mRNA stabilization and translation. Our findings reveal a key role for Pbp1-containing assemblies in enabling the translation of mRNAs critical for mitochondrial biogenesis and respiration under metabolically challenging conditions. They may further explain prior associations of Pbp1/ataxin-2 with stress granule biology and RNA metabolism.

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Project description:Cells expressing Tap-Tagged PUF3 were used for selection on IgG beads, then released using TEV protease. Samples were input and bound fraction without rRNA removal, and unbound fraction and input after rRNA removal with oligonucleotides and RNase H.

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