PUF partner interactions at a conserved interface shape the RNA binding landscape and cell fate
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA976659 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR24747767_1.fastq.gz | Fastqsanger.gz | |||
| SRR24747767_2.fastq.gz | Fastqsanger.gz | |||
| SRR24747768_1.fastq.gz | Fastqsanger.gz | |||
| SRR24747768_2.fastq.gz | Fastqsanger.gz | |||
| SRR24747769_1.fastq.gz | Fastqsanger.gz |
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Project description:Protein-RNA regulatory networks underpin much of biology. C. elegans FBF-2, a PUF RNA-binding protein, binds over 1000 RNAs to govern stem cells and differentiation. FBF-2 interacts with multiple protein partners via a key tyrosine, Y479. Here we investigate the in vivo significance of partnerships using a Y479A mutant. Y479A occupancy increases or decreases at specific sites across the transcriptome, varying with the RNA, and shifts from 3’UTRs to other regions. Germline development also changes in a very specific fashion: Y479A abolishes one FBF-2 function – the sperm to oocyte cell fate switch. Y479A changes to regulation of one mRNA, gld-1, are critical to this fate change, though other network changes are also important. FBF-2 switches from repression to activation of gld-1 RNA, likely by distinct FBF-2 partnerships. The role of partnerships in governing RNA regulatory networks will extend broadly, including for human health and disease, as protein partnerships pervade RNA control.
2023-12-22 | GSE233561 | GEO
Project description:Members of the PUF (Pumilio and FBF) and CPEB (cytoplasmic polyadenylation element-binding) protein families collaborate to regulate mRNA expression throughout eukaryotes. Here, we focus on the physical interactions between members of these two families, concentrating on Caenorhabditis elegans FBF-2 and CPB-1. To localize the site of interaction on FBF-2, we identified conserved amino acids within C. elegans PUF proteins. Deletion of an extended loop containing several conserved residues abolished binding to CPB-1. We analyzed alanine substitutions at 13 individual amino acids in FBF-2, each identified via its conservation. Multiple single point mutations disrupted binding to CPB-1 but not to RNA. Position Tyr-479 was particularly critical as multiple substitutions to other amino acids at this position did not restore binding. The complex of FBF-2 and CPB-1 repressed translation of an mRNA containing an FBF binding element. Repression required both proteins and was disrupted by FBF-2 alleles that failed to bind CPB-1 or RNA. The equivalent loop in human PUM2 is required for binding to human CPEB3 in vitro, although the primary sequences of the human and C. elegans PUF proteins have diverged in that region. Our findings define a key region in PUF/CPEB interactions and imply a conserved platform through which PUF proteins interact with their protein partners.
| S-EPMC3365739 | biostudies-literature
Project description:The molecular mechanisms of aging are unsolved and fascinating fundamental biological questions. Caenorhabditis elegans is an ideal model organism for investigating aging. PUF-8, a PUF (Pumilio and FBF) protein in C. elegans, is crucial for germline development through binding to the 3’ untranslated regions (3’ UTR) in the mRNA of target genes. Recently, PUF-8 was reported to alter mitochondrial dynamics and mitophagy by regulating MFF, a mitochondrial fission factor, and subsequently regulate longevity. Here, we determined the crystal structure of the PUF domain of PUF-8 with an RNA substrate. Mutagenesis experiments were performed to alter PUF-8 recognition of its target mRNAs. We generate these mutations in C. elegans, those mutations reduced the fertility and extended the lifespan. We deep sequenced total mRNAs from wild-type and puf-8 mutant worms and conducted in vitro RNA pull-down experiments. Six PUF-8 regulated genes were identified, in which their mRNA 3’ UTRs contain at least one PUF-binding element (PBE). One of the six genes, pqm-1, is crucial for lipid storage and aging process. Knockdown of pqm-1 could revert the lifespan extension of puf-8(-) animals. Therefore, PUF-8 may regulate the lifespan of C. elegans via modulating pqm-1-related pathways
2021-05-01 | GSE165061 | GEO
Project description:The PUF family of RNA binding proteins has a conserved role in maintaining stem cell self-renewal. FBF is a C. elegans PUF that is required to maintain germline stem cells (GSCs). To understand how FBF controls GSCs, we sought to identify is target mRNAs. Briefly, we immunoprecipitated FBF-mRNA complexes from worm extracts and then used microarrays to identify the FBF-associated mRNAs. To focus on germline targets of FBF, we used a FBF-GFP transgene under the control of a germline promoter and we used an anti-GFP antibody to purify FBF-GFP from worm extracts. In parallel, we also processed a strain expressing TUBULIN-GFP in the germline to control for mRNAs that non-specifically co-purify with GFP. We found that FBF associates with >1,000 unique mRNAs and likely controls a broad network of key cellular and developmental regulators.
2010-01-27 | GSE19922 | GEO
Project description:We determined the genomic landscape of FBF-1 and FBF-2 binding in germline stem cells using iCLIP, a method that allows identification of protein-RNA interactions at high resolution. We first developed reagents to explore the genomic binding landscapes of full-length FBF-1 and FBF-2 in vivo and then used our iCLIP data to test the precision of several commonly used methods for CLIP peak calling. Based on this iCLIP data, we discovered that FBF-1 and FBF-2 have similar global protein-RNA interaction profiles and that they both target conserved cell cycle regulators and lincRNAs. We found that FBF-1 and FBF-2 regulate their targets through canonical as well as unexpected motif sequences. We elucidated the first in vivo crosslink site analysis for a PUF protein from which we precisely determined FBF-1 and FBF-2 binding sites. Taken together, our data provide an updated model of PUF binding in stem cells. Our study also provides new insight on the control of gene expression in stem cells by RNA binding proteins.
2016-05-11 | GSE76136 | GEO
Project description:RNA-coimmunopurifications with TAP-tagged Puf proteins from Saccharomyces cereviseae. Untagged strain (BY4741) served as a control. Cells were grown to midlog phase and harvested by centrifugation. TAP-tagged Puf proteins were affinity purified from cell-free extracts with IgG sepharose and eluted with TEV protease. RNA was isolated from extract (=input)and from purified protein samples by phenol-chloroform extraction. RNA samples 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 DNA microarrays over night at 65 degrees. For a detailed procedure see http://microarray-pubs.stanford.edu/yeast_puf and also Gerber AP et al. PLoS Biology, 2004. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set
2006-01-04 | GSE3968 | GEO
Project description:Gene expression profiling was performed to compare RNA abundances in mycelium of Puf knockout strains compared to that in mycelium of wild-type N. crassa
2015-03-16 | GSE50995 | GEO
Project description:RNA-coimmunopurifications with TAP-tagged Puf proteins from Saccharomyces cereviseae. Untagged strain (BY4741) served as a control. Cells were grown to midlog phase and harvested by centrifugation. TAP-tagged Puf proteins were affinity purified from cell-free extracts with IgG sepharose and eluted with TEV protease. RNA was isolated from extract (=input)and from purified protein samples by phenol-chloroform extraction. RNA samples 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 DNA microarrays over night at 65 degrees. For a detailed procedure see http://microarray-pubs.stanford.edu/yeast_puf and also Gerber AP et al. PLoS Biology, 2004. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Computed
2006-01-03 | E-GEOD-3968 | biostudies-arrayexpress
Project description:Eukaryotic gene expression is controlled by a number of RNA-binding proteins (RBP), such as the proteins from the Puf (Pumilio and FBF) superfamily (PufSF). These proteins bind to RNA via multiple Puf repeat domains, each of which specifically recognizes a single RNA base. Recently, three diversified PufSF proteins have been described in model organisms, each of which are responsible for the maturation of ribosomal RNA or the translational regulation of mRNAs, however, less is known about the role of these proteins across eukaryotic diversity.Here, we investigated the distribution and function of Puf superfamily RBPs in the tree of eukaryotes. We determined that the following PufSF proteins are universally conserved across eukaryotes and can be broadly classified into three groups: (i) Nop9 orthologues, which participate in the nucleolar processing of immature 18S rRNA; (ii) ‘classical’ Pufs which control the translation of mRNA and; (iii) PUM3 orthologues, which are involved in the maturation of 7S rRNA. In nearly all eukaryotes, the rRNA maturation proteins, Nop9 and PUM3, are retained as a single copy, while mRNA effectors (‘classical’ Pufs) underwent multiple lineage-specific expansions. We propose that the variation in number of ‘classical’ Pufs relates to the size of the transcriptome and thus the potential mRNA targets. We further distinguished full set of PufSF proteins in divergent metamonad Giardia intestinalis and initiated their cellular and biochemical characterization
2020-06-16 | PXD019608 | Pride