Polypyrimidine tract binding proteins PTBP1 and PTBP2 interact with distinct proteins under splicing conditions.

RNA binding proteins play an important role in regulating alternative pre-mRNA splicing and in turn cellular gene expression. Polypyrimidine tract binding proteins, PTBP1 and PTBP2, are paralogous RNA binding proteins that play a critical role in the process of neuronal differentiation and maturation; changes in the concentration of PTBP proteins during neuronal development direct splicing changes in many transcripts that code for proteins critical for neuronal differentiation. How the two related proteins regulate different sets of neuronal exons is unclear. The distinct splicing activities of PTBP1 and PTBP2 can be recapitulated in an in vitro splicing system with the differentially regulated N1 exon of the c-src pre-mRNA. Here, we conducted experiments under these in vitro splicing conditions to identify PTBP1 and PTBP2 interacting partner proteins. Our results highlight that both PTBPs interact with proteins that participate in chromatin remodeling and transcription regulation. Our data reveal that PTBP1 interacts with many proteins involved in mRNA processing including splicing regulation while PTBP2 does not. Our results also highlight enzymes that can serve as potential "writers" and "erasers" in adding chemical modifications to the PTB proteins. Overall, our study highlights important differences in protein-protein interactions between the PTBP proteins under splicing conditions and supports a role for post-translational modifications in dictating their distinct splicing activities.

Identification and Characterization of a Minimal Functional Splicing Regulatory Protein, PTBP1.

Polypyrimidine tract binding protein 1 (PTBP1) is a well-studied RNA binding protein that serves as an important model for understanding molecular mechanisms underlying alternative splicing regulation. PTBP1 has four RNA binding domains (RBDs) connected via linker regions. Additionally, PTBP1 has an N-terminal unstructured region that contains nuclear import and export sequences. Each RBD can bind to pyrimidine rich elements with high affinity to mediate splicing activity. Studies support a variety of models for how PTBP1 can mediate splicing regulation on target exons. Obtaining a detailed atomic view hinges on determining a crystal structure of PTBP1 bound to a target RNA transcript. Here, we created a minimal functional PTBP1 with deletions in both linker 1 and linker 2 regions and assayed for activity on certain regulated exons, including the c-Src N1 exon. We show that for a subset of PTBP1-regulated exons the linker regions are not necessary for splicing repression activity. Gel mobility shift assays reveal the linker deletion mutant binds with 12-fold higher affinity to a target RNA sequence compared to wild-type PTBP1. A minimal PTBP1 that also contains an N-terminal region deletion binds to a target RNA with an affinity higher than that of wild-type PTBP1. Moreover, this minimal protein oligomerizes readily to form a distinct higher-order complex previously shown to be required for mediating splicing repression. This minimal functional PTBP1 protein can serve as a candidate for future structure studies to understand the mechanism of splicing repression for certain regulated exons.

Post-Translational Modifications in Polypyrimidine Tract Binding Proteins PTBP1 and PTBP2.

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.

Multiple determinants of splicing repression activity in the polypyrimidine tract binding proteins, PTBP1 and PTBP2.

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.