Publications by authors named "Robert J Ontiveros"

2 Publications

  • Page 1 of 1

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

Biochemistry 2020 12 7;59(50):4766-4774. Epub 2020 Dec 7.

Department of Chemistry and Biochemistry, California State University Fullerton, Fullerton, California 92831, United States.

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.
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http://dx.doi.org/10.1021/acs.biochem.0c00664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7821650PMC
December 2020

Reciprocal regulation of hnRNP C and CELF2 through translation and transcription tunes splicing activity in T cells.

Nucleic Acids Res 2020 06;48(10):5710-5719

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

RNA binding proteins (RBPs) frequently regulate the expression of other RBPs in mammalian cells. Such cross-regulation has been proposed to be important to control networks of coordinated gene expression; however, much remains to be understood about how such networks of cross-regulation are established and what the functional consequence is of coordinated or reciprocal expression of RBPs. Here we demonstrate that the RBPs CELF2 and hnRNP C regulate the expression of each other, such that depletion of one results in reduced expression of the other. Specifically, we show that loss of hnRNP C reduces the transcription of CELF2 mRNA, while loss of CELF2 results in decreased efficiency of hnRNP C translation. We further demonstrate that this reciprocal regulation serves to fine tune the splicing patterns of many downstream target genes. Together, this work reveals new activities of hnRNP C and CELF2, provides insight into a previously unrecognized gene regulatory network, and demonstrates how cross-regulation of RBPs functions to shape the cellular transcriptome.
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http://dx.doi.org/10.1093/nar/gkaa295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261192PMC
June 2020