The binding RNAs of Prrc2b in brain tissues of Prrc2b-HA knock-in mice [RIP-Seq]
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ABSTRACT:
PROVIDER: PRJNA1017793 | ENA |
REPOSITORIES: ENA
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Project description:We performed HA RIP-seq in cerebral cortexes of WT and Prrc2b-HA knockin mice at P4 to further analyze genes that Prrc2b selectively binds.
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Project description:Two isoforms (Long isoform ~220 KDa, short isoform ~150 KDa) of the human PRRC2B protein (Uniprot accession Q5JSZ5) were recombinantly expressed with an HA-tag in HEK293 cell lines. The goal of the experiment is to identify differential interactors of the short vs long isoforms, in addition to their common interactors. To this aim, 3 sample types were analyzed by IP-MS: HA-tagged long PRRC2B, HA-tagged short PRRC2B, and HA-tagged mCherry (control). Four biological replicates were analyzed from each experimental group.
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Project description:We prepared two types of MEF cells: one is cells with wild-type CtBP2-HA knock-in and the other is Rossmann fold mutant CtBP2-HA knock-in. We isolated exosomes from those cells with the polymer precipitation method and subjected them to proteome analysis.
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Project description:Accumulating evidence suggests that posttranscriptional regulation of gene expression, including regulation of RNA splicing, transport, modification, translation, and degradation, primarily relies on RNA binding proteins (RBPs). However, the functions of many RBPs remain understudied. Here, we characterized the function of a novel RBP, Proline-Rich Coiled-coil 2B (PRRC2B). Through photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and deep sequencing, we identified transcriptome-wide CU- or GA-rich PRRC2B binding sites around the translation initiation codon on a specific cohort of mRNAs in HEK293T cells. These PRRC2B target mRNAs, including oncogenes and cell cycle regulators such as CCND2 (cyclin D2), exhibited decreased translation upon PRRC2B knockdown as revealed by sequencing polysome-associated RNA, resulting in decreased G1/S phase transition and cell proliferation. Antisense oligonucleotides (ASOs) blocking PRRC2B-CCND2 mRNA interaction decreased CCND2 translation, thus inhibited G1/S transition and cell proliferation. Mechanistically, PRRC2B interactome capture analysis revealed RNA-independent interactions with eukaryotic initiation factors eIF4G2, eIF3, and FXR1. The interaction with eIF4G2 is essential for PRRC2B function since unlike wildtype PRRC2B, eIF4G2-interacting defective mutants failed to rescue the translation deficiency caused by PRRC2B knockdown. Taken together, our findings reveal that PRRC2B, by interacting with eIF4G2, is essential for efficient translation of specific proteins required for cell cycle progression and cell proliferation.
2023-04-20 | GSE220058 | GEO
Project description:Accumulating evidence suggests that posttranscriptional regulation of gene expression, including regulation of RNA splicing, transport, modification, translation, and degradation, primarily relies on RNA binding proteins (RBPs). However, the functions of many RBPs remain understudied. Here, we characterized the function of a novel RBP, Proline-Rich Coiled-coil 2B (PRRC2B). Through photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and deep sequencing, we identified transcriptome-wide CU- or GA-rich PRRC2B binding sites around the translation initiation codon on a specific cohort of mRNAs in HEK293T cells. These PRRC2B target mRNAs, including oncogenes and cell cycle regulators such as CCND2 (cyclin D2), exhibited decreased translation upon PRRC2B knockdown as revealed by sequencing polysome-associated RNA, resulting in decreased G1/S phase transition and cell proliferation. Antisense oligonucleotides (ASOs) blocking PRRC2B-CCND2 mRNA interaction decreased CCND2 translation, thus inhibited G1/S transition and cell proliferation. Mechanistically, PRRC2B interactome capture analysis revealed RNA-independent interactions with eukaryotic initiation factors eIF4G2, eIF3, and FXR1. The interaction with eIF4G2 is essential for PRRC2B function since unlike wildtype PRRC2B, eIF4G2-interacting defective mutants failed to rescue the translation deficiency caused by PRRC2B knockdown. Taken together, our findings reveal that PRRC2B, by interacting with eIF4G2, is essential for efficient translation of specific proteins required for cell cycle progression and cell proliferation.
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Project description:It was hypothesized that the human PRRC2B protein (Uniprot accession Q5JSZ5) interacts with its RNA binding targets via methylated arginines. The protein contains at least two RG rich motifs (residues 1061-1080, and 1104-1124), in which the arginines are hypothesized to be methylated. The goal of the experiment was to identify such methylated PRRC2B arginines. To get a wide coverage of the RG rich domains, an immuno-precipitation experiment was performed, followed by digestion with chymotrypsin and LC-MS/MS analysis.
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Project description:PRRC2B is an intrinsically disordered RNA-binding protein that is part of the cell’s translation machinery. Here we show that PRRC2B has two alternatively spliced mRNA transcripts producing major long and minor short isoforms. Mass spectrometry-based interaction studies indicated that both isoforms associate with the 40S ribosomal subunit and translation initiation factors. Importantly, the long isoform also interacted with additional RNA-binding proteins through its unique Arg/Gly-rich region. Among these is LARP1, a regulator of 5' TOP mRNAs under conditions of mTOR inhibition. We discovered that like LARP1, PRRC2B is an important regulator of 5' TOP mRNA levels during starvation, particularly those encoding ribosomal proteins. Overall, our study elucidates a newly discovered function for PRRC2B as an RNA-binding protein that regulates ribosomal biogenesis, establishing it as a global regulator of translation in addition to its function in initiating specific target translation.
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Project description:RIP-Seq of the viral protein EBNA1-HA-FLAG from EBV in HEK293T cells
2019-03-01 | GSE107808 | GEO
Project description:RIP-sequencing in mouse embryonic stem cells. Experiment 1 (Samples 1-18): Base cell line (untagged control cells): GFP_5xT71BS (mES [129S6/SvEvTac], yGlob [GFP_5xT71BS], stable transfection FLAG-Cas9::T2A::Blasticidin). HA-Tnrc6a: endogenously HA-AVI-tagged Tnrc6a. HA-Tnrc6a_Trim71KO: HA-AVI-tagged Tnrc6a, Trim71-/-. Experiment 2 (Samples 19-42): Ctrl: base cell line GFP_5xT71BS. Tnrc6a: endogenously HA-AVI-tagged Tnrc6a. Tnrc6a_Trim71KO: HA-AVI-tagged Tnrc6a, Trim71-/-. Tnrc6a_Ago2KO: HA-AVI-tagged Tnrc6a, Ago2-/-.
2023-06-26 | GSE205966 | GEO
Project description:RIP-sequencing in mouse embryonic stem cells. Experiment 3 (Samples 2-24) & 4 (Samples 25-48): Base cell line (untagged control cells): GFP_5xT71BS (mES [129S6/SvEvTac], yGlob [GFP_5xT71BS], stable transfection FLAG-Cas9::T2A::Blasticidin). HA-Tnrc6a: endogenously HA-AVI-tagged Tnrc6a. HA-Tnrc6a_Trim71KO: HA-AVI-tagged Tnrc6a, Trim71-/-. Experiment 2 (Samples 19-42): Ctrl: base cell line GFP_5xT71BS. Tnrc6a: endogenously HA-AVI-tagged Tnrc6a. Tnrc6a_Trim71KO: HA-AVI-tagged Tnrc6a, Trim71-/-. Tnrc6a_Ago2KO: HA-AVI-tagged Tnrc6a, Ago2-/-.
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