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Coordinate repression of a trio of neuron-specific splicing events by the splicing regulator PTB.
RNA. 1999 Jan; 5(1):117-30.RNA

Abstract

In this study, we demonstrate the ability of the polypyrimidine tract binding protein PTB to function as a coordinator of splicing regulation for a trio of neuron-specific exons that are subject to developmental splicing changes in the rat cerebellum. Three neuron-specific exons that show positive regulation are derived from the GABA(A) receptor gamma2 subunit 24 nucleotide exon, clathrin light chain B exon EN, and N-methyl-D-aspartate receptor NR1 subunit exon 5 pre-mRNAs. The functional activity of splicing repressor signals located in the 3' splice site regions adjacent to the neural exons is shown using an alternative splicing switch assay, in which these short RNA sequences function in trans to switch splicing to the neural pathway in HeLa splicing reactions. Parallel UV crosslinking/competition assays demonstrate selective binding of PTB in comparison to substantially lower binding at adjacent, nonneural 3' splice sites. Substantially lower PTB binding and splicing switch activity is also observed for the 3' splice site of NMDA exon 21, which is subject to negative regulation in cerebellum tissue in the same time frame. In splicing active neural extracts, the balance of control shifts to positive regulation, and this shift correlates with a PTB status that is predominantly the neural form. In this context, the addition of recombinant PTB is sufficient to switch splicing to the nonneural pathway. The neural extracts also reveal specific binding of the CUG triplet repeat binding protein to a subset of regulatory 3' splice site regions. These interactions may interfere with PTB function or modulate splicing levels in a substrate-specific manner within neural tissue. Together these results strengthen the evidence that PTB is a splicing regulator with multiple targets and demonstrate its ability to discriminate among neural and nonneural substrates. Thus, a variety of mechanisms that counterbalance the splicing repressor function of PTB in neural tissue are capable of mediating developmental splicing control. Altered expression of PTB isoforms during cerebellar development, as documented by Western blot analysis, is proposed to be a contributing mechanism.

Authors+Show Affiliations

Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9917071

Citation

Zhang, L, et al. "Coordinate Repression of a Trio of Neuron-specific Splicing Events By the Splicing Regulator PTB." RNA (New York, N.Y.), vol. 5, no. 1, 1999, pp. 117-30.
Zhang L, Liu W, Grabowski PJ. Coordinate repression of a trio of neuron-specific splicing events by the splicing regulator PTB. RNA. 1999;5(1):117-30.
Zhang, L., Liu, W., & Grabowski, P. J. (1999). Coordinate repression of a trio of neuron-specific splicing events by the splicing regulator PTB. RNA (New York, N.Y.), 5(1), 117-30.
Zhang L, Liu W, Grabowski PJ. Coordinate Repression of a Trio of Neuron-specific Splicing Events By the Splicing Regulator PTB. RNA. 1999;5(1):117-30. PubMed PMID: 9917071.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Coordinate repression of a trio of neuron-specific splicing events by the splicing regulator PTB. AU - Zhang,L, AU - Liu,W, AU - Grabowski,P J, PY - 1999/1/23/pubmed PY - 1999/1/23/medline PY - 1999/1/23/entrez SP - 117 EP - 30 JF - RNA (New York, N.Y.) JO - RNA VL - 5 IS - 1 N2 - In this study, we demonstrate the ability of the polypyrimidine tract binding protein PTB to function as a coordinator of splicing regulation for a trio of neuron-specific exons that are subject to developmental splicing changes in the rat cerebellum. Three neuron-specific exons that show positive regulation are derived from the GABA(A) receptor gamma2 subunit 24 nucleotide exon, clathrin light chain B exon EN, and N-methyl-D-aspartate receptor NR1 subunit exon 5 pre-mRNAs. The functional activity of splicing repressor signals located in the 3' splice site regions adjacent to the neural exons is shown using an alternative splicing switch assay, in which these short RNA sequences function in trans to switch splicing to the neural pathway in HeLa splicing reactions. Parallel UV crosslinking/competition assays demonstrate selective binding of PTB in comparison to substantially lower binding at adjacent, nonneural 3' splice sites. Substantially lower PTB binding and splicing switch activity is also observed for the 3' splice site of NMDA exon 21, which is subject to negative regulation in cerebellum tissue in the same time frame. In splicing active neural extracts, the balance of control shifts to positive regulation, and this shift correlates with a PTB status that is predominantly the neural form. In this context, the addition of recombinant PTB is sufficient to switch splicing to the nonneural pathway. The neural extracts also reveal specific binding of the CUG triplet repeat binding protein to a subset of regulatory 3' splice site regions. These interactions may interfere with PTB function or modulate splicing levels in a substrate-specific manner within neural tissue. Together these results strengthen the evidence that PTB is a splicing regulator with multiple targets and demonstrate its ability to discriminate among neural and nonneural substrates. Thus, a variety of mechanisms that counterbalance the splicing repressor function of PTB in neural tissue are capable of mediating developmental splicing control. Altered expression of PTB isoforms during cerebellar development, as documented by Western blot analysis, is proposed to be a contributing mechanism. SN - 1355-8382 UR - https://www.unboundmedicine.com/medline/citation/9917071/Coordinate_repression_of_a_trio_of_neuron_specific_splicing_events_by_the_splicing_regulator_PTB_ L2 - http://www.rnajournal.org/cgi/pmidlookup?view=long&pmid=9917071 DB - PRIME DP - Unbound Medicine ER -