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Douglas L Black - One of the best experts on this subject based on the ideXlab platform.
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Polypyrimidine Tract Binding Protein blocks microRNA-124 biogenesis to enforce its neuronal specific expression.
2018Co-Authors: Kristen W. Yeom, Sika Zheng, Simon Mitchell, Anthony J Linares, Chia-ho Lin, Xiao-jun Wang, Alexander Hoffmann, Douglas L BlackAbstract:MicroRNA-124 is expressed in neurons, where it represses genes inhibitory for neuronal differentiation, including the RNA binding protein PTBP1. PTBP1 maintains non-neuronal splicing patterns of mRNAs that switch to neuronal isoforms upon neuronal differentiation. We find that pri-miR-124-1 is expressed in mouse embryonic stem cells (mESCs) where mature miR-124 is absent. PTBP1 binds to this precursor RNA upstream of the miRNA stem-loop to inhibit mature miR-124 expression in vivo, and DROSHA cleavage of pri-miR-124-1 in vitro. This new function for PTBP1 in repressing miR-124 biogenesis adds an additional regulatory loop to the already intricate interplay between these two molecules. Applying mathematical modeling to examine the dynamics of this regulation, we find that the pool of pri-miR-124 whose maturation is blocked by PTBP1 creates a robust and self-reinforcing transition in gene expression as PTBP1 is depleted during early neuronal differentiation. While interlocking regulatory loops are often modeled between miRNAs and transcriptional regulators, our results indicate that miRNA targeting of posttranscriptional regulators also reinforces developmental decisions. Notably, induction of neuronal differentiation observed upon PTBP1 knockdown likely results from direct de-repression of miR-124, in addition to indirect effects previously described.
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ptbp1 and PTBP2 serve both specific and redundant functions in neuronal pre mrna splicing
Cell Reports, 2016Co-Authors: John K Vuong, Douglas L Black, Chia-ho Lin, Min Zhang, Liang Chen, Sika ZhengAbstract:Families of alternative splicing regulators often contain multiple paralogs presumed to fulfill different functions. Polypyrimidine tract binding proteins PTBP1 and PTBP2 reprogram developmental pre-mRNA splicing in neurons, but how their regulatory networks differ is not understood. To compare their targeting, we generated a knockin allele that conditionally expresses PTBP1. Bred to a PTBP2 knockout, the transgene allowed us to compare the developmental and molecular phenotypes of mice expressing only PTBP1, only PTBP2, or neither protein in the brain. This knockin Ptbp1 rescued a forebrain-specific, but not a pan-neuronal, PTBP2 knockout, demonstrating both redundant and distinct roles for the proteins. Many developmentally regulated exons exhibited different sensitivities to PTBP1 and PTBP2. Nevertheless, the two paralogs displayed similar RNA binding across the transcriptome, indicating that their differential targeting does not derive from their RNA interactions, but from possible different cofactor interactions.
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De Novo Prediction of PTBP1 Binding and Splicing Targets Reveals Unexpected Features of Its RNA Recognition and Function
2016Co-Authors: A Reum Han, Anthony J Linares, Peter Stoilov, Yu Zhou, Douglas L BlackAbstract:The splicing regulator Polypyrimidine Tract Binding Protein (PTBP1) has four RNA binding domains that each binds a short pyrimidine element, allowing recognition of diverse pyrimidine-rich sequences. This variation makes it difficult to evaluate PTBP1 binding to particular sites based on sequence alone and thus to identify target RNAs. Conversely, transcriptome-wide binding assays such as CLIP identify many in vivo targets, but do not provide a quantitative assessment of binding and are informative only for the cells where the analysis is performed. A general method of predicting PTBP1 binding and possible targets in any cell type is needed. We developed computational models that predict the binding and splicing targets of PTBP1. A Hidden Markov Model (HMM), trained on CLIP-seq data, was used to score probable PTBP1 binding sites. Scores from this model are highly correlated (r=20.9) with experimentally determined dissociation constants. Notably, we find that the protein is not strictly pyrimidine specific, as interspersed Guanosine residues are well tolerated within PTBP1 binding sites. This model identifies many previously unrecognized PTBP1 binding sites, and can score PTBP1 binding acros
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multiple determinants of splicing repression activity in the polypyrimidine tract binding proteins ptbp1 and PTBP2
RNA, 2016Co-Authors: Niroshika Keppetipola, Shalini Sharma, Kyuhyeon Yeom, Adrian L Hernandez, Tessa Bui, Douglas L BlackAbstract:Most human genes generate multiple protein isoforms through alternative pre-mRNA splicing, but the mechanisms controlling alternative splicing choices by RNA binding proteins are not well understood. These proteins can have multiple paralogs expressed in different cell types and exhibiting different splicing activities on target exons. We examined the paralogous polypyrimidine tract binding proteins PTBP1 and PTBP2 to understand how PTBP1 can exhibit greater splicing repression activity on certain exons. Using both an in vivo coexpression assay and an in vitro splicing assay, we show that PTBP1 is more repressive than PTBP2 per unit protein on a target exon. Constructing chimeras of PTBP1 and 2 to determine amino acid features that contribute to their differential activity, we find that multiple segments of PTBP1 increase the repressive activity of PTBP2. Notably, when either RRM1 of PTBP2 or the linker peptide separating RRM2 and RRM3 are replaced with the equivalent PTBP1 sequences, the resulting chimeras are highly active for splicing repression. These segments are distinct from the known region of interaction for the PTBP1 cofactors Raver1 and Matrin3 in RRM2. We find that RRM2 of PTBP1 also increases the repression activity of an otherwise PTBP2 sequence, and that this is potentially explained by stronger binding by Raver1. These results indicate that multiple features over the length of the two proteins affect their ability to repress an exon.
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Li et al. Supplemental File 1: Table 1. Exons validated by RT/PCR.
2016Co-Authors: Sika Zheng, Chia-ho Lin, A Reum Han, Peter Stoilov, Douglas L BlackAbstract:Table S1 for Li et al. A variety of exons showing differences in splicing between wild type and PTBP2 nestin-KO brain at embryonic day 18 were subjected to further analysis by RT/PCR. These included exons identified by MJAY array, RNAseq, and exons previously reported to be PTBP targets. Gene names, exon coordinates, and percent spliced in values (PSI) are listed. Genome coordinates are for mm9
Norman B Hecht - One of the best experts on this subject based on the ideXlab platform.
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NB: Polypyrimidine tract binding protein 2 stabilizes phosphoglycerate kinase 2 mRNA in murine male germ cells by binding to its 3
2016Co-Authors: Norman B HechtAbstract:The mRNA that encodes the testis-specific protein phospho-glycerate kinase (PGK2) is a long-lived mRNA that is transcribed in meiotic and postmeiotic male germ cells. Pgk2 mRNA is present in germ cells for up to 2 wk before its protein product is detected. Using affinity chromatography with the 30-UTR of the Pgk2 mRNA, several proteins, including the RNA-binding protein, polypyrimidine tract binding protein 2 (PTBP2), were identified in mouse testis extracts. Coimmunoprecipitation experiments confirmed that PTBP2 binds to Pgk2 mRNA in the testis and RNA gel shifts demonstrated that PTBP2, but not PTBP1, binds to a specific region of the Pgk2 3 0-UTR. Recombinant PTBP2 increased the stability of reporter con-structs that contained the 30-UTR Pgk2 sequence element in both testis extracts and transfected HeLa cells. We propose that PTBP2 is a trans-acting factor that helps to stabilize Pgk2 mRNA in male mouse germ cells. gametogenesis, gene regulation, posttranscriptional control, RNA-binding proteins, spermatogenesi
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polypyrimidine tract binding protein 2 binds to selective intronic messenger rna and microrna targets in the mouse testis
Biology of Reproduction, 2011Co-Authors: Norman B HechtAbstract:Here we use an in vivo cross-linking and immunoprecipitation procedure to detect RNA targets of the multifunctional RNA-binding protein polypyrimidine tract-binding protein (PTBP) 2 in mouse testis. Eleven known mRNAs, including PTBP2 mRNA, 28 RNAs matching intron sequences, and 12 small RNAs and repeat sequences are identified. The specificity of interaction between PTBP2 and its target RNAs was confirmed using RNA interference with mouse N2A cells. Reduction of PTBP2 levels led to decreases in 7 of 10 of the mRNAs, to the repression of alternative splicing of introns, and to reductions in specific miRNAs.
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polypyrimidine tract binding protein 2 stabilizes phosphoglycerate kinase 2 mrna in murine male germ cells by binding to its 3 utr
Biology of Reproduction, 2007Co-Authors: Mingang Xu, Norman B HechtAbstract:Abstract The mRNA that encodes the testis-specific protein phosphoglycerate kinase (PGK2) is a long-lived mRNA that is transcribed in meiotic and postmeiotic male germ cells. Pgk2 mRNA is present in germ cells for up to 2 wk before its protein product is detected. Using affinity chromatography with the 3′-UTR of the Pgk2 mRNA, several proteins, including the RNA-binding protein, polypyrimidine tract binding protein 2 (PTBP2), were identified in mouse testis extracts. Coimmunoprecipitation experiments confirmed that PTBP2 binds to Pgk2 mRNA in the testis and RNA gel shifts demonstrated that PTBP2, but not PTBP1, binds to a specific region of the Pgk2 3′-UTR. Recombinant PTBP2 increased the stability of reporter constructs that contained the 3′-UTR Pgk2 sequence element in both testis extracts and transfected HeLa cells. We propose that PTBP2 is a trans-acting factor that helps to stabilize Pgk2 mRNA in male mouse germ cells.
Bert L Semler - One of the best experts on this subject based on the ideXlab platform.
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the linker domain of poly rc binding protein 2 is a major determinant in poliovirus cap independent translation
Virology, 2008Co-Authors: Polen Sean, Joseph H C Nguyen, Bert L SemlerAbstract:Poliovirus, a member of the enterovirus genus in the family Picornaviridae, is the causative agent of poliomyelitis. Translation of the viral genome is mediated through an internal ribosomal entry site (IRES) encoded within the 5' noncoding region (5' NCR). IRES elements are highly structured RNA sequences that facilitate the recruitment of ribosomes for translation. Previous studies have shown that binding of a cellular protein, poly(rC) binding protein 2 (PCBP2), to a major stem-loop structure in the genomic 5' NCR is necessary for the translation of picornaviruses containing type I IRES elements, including poliovirus, coxsackievirus, and human rhinovirus. PCBP1, an isoform that shares approximately 90% amino acid identity to PCBP2, cannot efficiently stimulate poliovirus IRES-mediated translation, most likely due to its reduced binding affinity to stem-loop IV within the poliovirus IRES. The primary differences between PCBP1 and PCBP2 are found in the so-called linker domain between the second and third K-homology (KH) domains of these proteins. We hypothesize that the linker region of PCBP2 augments binding to poliovirus stem-loop IV RNA. To test this hypothesis, we generated six PCBP1/PCBP2 chimeric proteins. The recombinant PCBP1/PCBP2 chimeric proteins were able to interact with poliovirus stem-loop I RNA and participate in protein-protein interactions. We demonstrated that the PCBP1/PCBP2 chimeric proteins with the PCBP2 linker, but not with the PCBP1 linker, were able to interact with poliovirus stem-loop IV RNA, and could subsequently stimulate poliovirus IRES-mediated translation. In addition, using a monoclonal anti-PCBP2 antibody (directed against the PCBP2 linker domain) in mobility shift assays, we showed that the PCBP2 linker domain modulates binding to poliovirus stem-loop IV RNA via a mechanism that is not inhibited by the antibody.
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multimerization of poly rc binding protein 2 is required for translation initiation mediated by a viral ires
RNA, 2004Co-Authors: Kristin M Bedard, Brandon L Walter, Bert L SemlerAbstract:The cellular protein, poly(rC) binding protein 2 (PCBP2), is known to function in picornavirus cap-independent translation. We have further examined the RNA binding properties and protein–protein interactions of PCBP2 necessary for translation. We have studied its putative multimerization properties utilizing the yeast two-hybrid assay and in vitro biochemical methods, including glutathione S-transferase (GST) pull-down assays and gel filtration. Through genetic analysis, the multimerization domain has been localized to the second K-homologous (KH) RNA binding domain of the protein between amino acids 125 and 158. To examine the function of multimerization in poliovirus translation, we utilized the truncated protein, ΔKH1-PCBP2, which is capable of multimer formation, but does not bind poliovirus stem–loop IV RNA (an interaction required for translation). Utilizing RNA binding and in vitro translation assays, this protein was shown to act as a dominant negative, suggesting that PCBP2 multimerization functions in poliovirus translation and RNA binding. Additionally, PCBP2 containing a deletion in the multimerization domain (ΔKH2-PCBP2) was not able to bind poliovirus stem–loop IV RNA and could not rescue translation in extracts that were depleted of endogenous PCBP2. Results from these experiments suggest that the multimerization of PCBP2 is required for efficient RNA binding and cap-independent translation of poliovirus RNA. By examining the functional interactions of the cellular protein PCBP2, we have discovered a novel determinant in the mechanism of picornavirus cap-independent translation.
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requirement of poly rc binding protein 2 for translation of poliovirus rna
Journal of Virology, 1997Co-Authors: Lawrence B Blyn, Bert L Semler, Jonathan S Towner, Ellie EhrenfeldAbstract:Poly(rC) binding protein 2 (PCBP2) is one of several cellular proteins that interact specifically with a major stem-loop domain in the poliovirus internal ribosome entry site. HeLa cell extracts subjected to stem-loop IV RNA affinity chromatography were depleted of all detectable PCBP2. Such extracts were unable to efficiently translate poliovirus RNA, although extracts recovered from control columns of matrix unlinked to RNA retained full translation activity. Both translation and production of infectious progeny virus were restored in the PCBP2-depleted extracts by addition of recombinant PCBP2, but not by PCBP1, which is a closely related member of the protein family. The data show that PCBP2 is an essential factor, which is required for efficient translation of poliovirus RNA in HeLa cells.
Niroshika Keppetipola - One of the best experts on this subject based on the ideXlab platform.
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post translational modifications in polypyrimidine tract binding proteins ptbp1 and PTBP2
Biochemistry, 2018Co-Authors: Jeffrey Pina, Janice Reynaga, Anthony Truong, Niroshika KeppetipolaAbstract:RNA binding proteins play an important role in regulating alternative pre-mRNA splicing and in turn cellular gene expression. Many of these RNA binding proteins occur as gene families with members sharing a high degree of primary structure identity and domain organization yet have tissue-specific expression patterns and regulate different sets of target exons. How highly similar members in a gene family can exert different splicing outcomes is not well understood. We conducted mass spectrometry analysis of post-translational phosphorylation and acetylation modifications for two paralogs of the polypyrimidine tract binding protein family, PTBP1 and PTBP2, to discover modifications that occur in splicing reaction mixtures and to identify discrete modifications that may direct their different splicing activities. We find that PTBP1 and PTBP2 have many distinct phosphate modifications located in the unstructured N-terminal, linker 1, and linker 2 regions. We find that the two proteins have many overlapping acetate modifications in the RNA recognition motifs (RRMs) with a few distinct sites in PTBP1 RRM2 and RRM3. Our data also reveal that lysine residues in the nuclear localization sequence of PTBP2 are acetylated. Collectively, our results highlight important differences in post-translational modifications between the paralogs and suggest a role for them in the differential splicing activity of PTBP1 and PTBP2.
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multiple determinants of splicing repression activity in the polypyrimidine tract binding proteins ptbp1 and PTBP2
RNA, 2016Co-Authors: Niroshika Keppetipola, Shalini Sharma, Kyuhyeon Yeom, Adrian L Hernandez, Tessa Bui, Douglas L BlackAbstract:Most human genes generate multiple protein isoforms through alternative pre-mRNA splicing, but the mechanisms controlling alternative splicing choices by RNA binding proteins are not well understood. These proteins can have multiple paralogs expressed in different cell types and exhibiting different splicing activities on target exons. We examined the paralogous polypyrimidine tract binding proteins PTBP1 and PTBP2 to understand how PTBP1 can exhibit greater splicing repression activity on certain exons. Using both an in vivo coexpression assay and an in vitro splicing assay, we show that PTBP1 is more repressive than PTBP2 per unit protein on a target exon. Constructing chimeras of PTBP1 and 2 to determine amino acid features that contribute to their differential activity, we find that multiple segments of PTBP1 increase the repressive activity of PTBP2. Notably, when either RRM1 of PTBP2 or the linker peptide separating RRM2 and RRM3 are replaced with the equivalent PTBP1 sequences, the resulting chimeras are highly active for splicing repression. These segments are distinct from the known region of interaction for the PTBP1 cofactors Raver1 and Matrin3 in RRM2. We find that RRM2 of PTBP1 also increases the repression activity of an otherwise PTBP2 sequence, and that this is potentially explained by stronger binding by Raver1. These results indicate that multiple features over the length of the two proteins affect their ability to repress an exon.
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neuronal regulation of pre mrna splicing by polypyrimidine tract binding proteins ptbp1 and PTBP2
Critical Reviews in Biochemistry and Molecular Biology, 2012Co-Authors: Niroshika Keppetipola, Shalini Sharma, Douglas L BlackAbstract:Alternative splicing patterns are regulated by RNA binding proteins that assemble onto each pre-mRNA to form a complex RNP structure. The polypyrimidine tract binding protein, PTB, has served as an informative model for understanding how RNA binding proteins affect spliceosome assembly and how changes in the expression of these proteins can control complex programs of splicing in tissues. In this review, we describe the mechanisms of splicing regulation by PTB and its function, along with its paralog PTBP2, in neuronal development.
Sika Zheng - One of the best experts on this subject based on the ideXlab platform.
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axonogenesis is coordinated by neuron specific alternative splicing programming and splicing regulator PTBP2
Neuron, 2019Co-Authors: Min Zhang, Liang Chen, Volkan Ergin, Lin Lin, Cheryl Stork, Sika ZhengAbstract:Summary How a neuron acquires an axon is a fundamental question. Piecemeal identification of many axonogenesis-related genes has been done, but coordinated regulation is unknown. Through unbiased transcriptome profiling of immature primary cortical neurons during early axon formation, we discovered an association between axonogenesis and neuron-specific alternative splicing. Known axonogenesis genes exhibit little expression alternation but widespread splicing changes. Axonogenesis-associated splicing is governed by RNA binding protein PTBP2, which is enriched in neurons and peaks around axonogenesis in the brain. Cortical depletion of PTBP2 prematurely induces axonogenesis-associated splicing, causes imbalanced expression of axonogenesis-associated isoforms, and specifically affects axon formation in vitro and in vivo. PTBP2-controlled axonogenesis-associated Shtn1 splicing determines SHTN1’s capacity to regulate actin interaction, polymerization, and axon growth. Precocious Shtn1 isoform switch contributes to disorganized axon formation of PTBP2−/− neurons. We conclude that PTBP2-orchestrated alternative splicing programming is required for robust generation of a single axon in mammals.
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Polypyrimidine Tract Binding Protein blocks microRNA-124 biogenesis to enforce its neuronal specific expression.
2018Co-Authors: Kristen W. Yeom, Sika Zheng, Simon Mitchell, Anthony J Linares, Chia-ho Lin, Xiao-jun Wang, Alexander Hoffmann, Douglas L BlackAbstract:MicroRNA-124 is expressed in neurons, where it represses genes inhibitory for neuronal differentiation, including the RNA binding protein PTBP1. PTBP1 maintains non-neuronal splicing patterns of mRNAs that switch to neuronal isoforms upon neuronal differentiation. We find that pri-miR-124-1 is expressed in mouse embryonic stem cells (mESCs) where mature miR-124 is absent. PTBP1 binds to this precursor RNA upstream of the miRNA stem-loop to inhibit mature miR-124 expression in vivo, and DROSHA cleavage of pri-miR-124-1 in vitro. This new function for PTBP1 in repressing miR-124 biogenesis adds an additional regulatory loop to the already intricate interplay between these two molecules. Applying mathematical modeling to examine the dynamics of this regulation, we find that the pool of pri-miR-124 whose maturation is blocked by PTBP1 creates a robust and self-reinforcing transition in gene expression as PTBP1 is depleted during early neuronal differentiation. While interlocking regulatory loops are often modeled between miRNAs and transcriptional regulators, our results indicate that miRNA targeting of posttranscriptional regulators also reinforces developmental decisions. Notably, induction of neuronal differentiation observed upon PTBP1 knockdown likely results from direct de-repression of miR-124, in addition to indirect effects previously described.
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ptbp1 and PTBP2 serve both specific and redundant functions in neuronal pre mrna splicing
Cell Reports, 2016Co-Authors: John K Vuong, Douglas L Black, Chia-ho Lin, Min Zhang, Liang Chen, Sika ZhengAbstract:Families of alternative splicing regulators often contain multiple paralogs presumed to fulfill different functions. Polypyrimidine tract binding proteins PTBP1 and PTBP2 reprogram developmental pre-mRNA splicing in neurons, but how their regulatory networks differ is not understood. To compare their targeting, we generated a knockin allele that conditionally expresses PTBP1. Bred to a PTBP2 knockout, the transgene allowed us to compare the developmental and molecular phenotypes of mice expressing only PTBP1, only PTBP2, or neither protein in the brain. This knockin Ptbp1 rescued a forebrain-specific, but not a pan-neuronal, PTBP2 knockout, demonstrating both redundant and distinct roles for the proteins. Many developmentally regulated exons exhibited different sensitivities to PTBP1 and PTBP2. Nevertheless, the two paralogs displayed similar RNA binding across the transcriptome, indicating that their differential targeting does not derive from their RNA interactions, but from possible different cofactor interactions.
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Li et al. Supplemental File 1: Table 1. Exons validated by RT/PCR.
2016Co-Authors: Sika Zheng, Chia-ho Lin, A Reum Han, Peter Stoilov, Douglas L BlackAbstract:Table S1 for Li et al. A variety of exons showing differences in splicing between wild type and PTBP2 nestin-KO brain at embryonic day 18 were subjected to further analysis by RT/PCR. These included exons identified by MJAY array, RNAseq, and exons previously reported to be PTBP targets. Gene names, exon coordinates, and percent spliced in values (PSI) are listed. Genome coordinates are for mm9
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Li et al. Supplemental File 2: Table 2. Splicing Changes Identified by Exon Junction Microarray.
2016Co-Authors: Sika Zheng, Chia-ho Lin, A Reum Han, Peter Stoilov, Douglas L BlackAbstract:RNA from WT and PTBP2-NestinKO E18 pups was analyzed on Affymetrix MJAY arrays (see Methods). Splicing events yielding Sepscores greater than 0.345 are listed