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Regulation of alternative 3' splice site selection by constitutive splicing factors.
RNA. 1995 May; 1(3):234-45.RNA

Abstract

We have devised an in vitro splicing assay in which the mutually exclusive exons 2 and 3 of alpha-tropomyosin act as competing 3' splice sites for joining to exon 1. Splicing in normal HeLa cell nuclear extracts results in almost exclusive joining of exons 1 and 3. Splicing in decreased nuclear extract concentrations and decreased ionic strength results in increased 1-2 splicing. We have used this assay to determine the role of three constitutive pre-mRNA splicing factors on alternative 3' splice site selection. Polypyrimidine tract binding protein (PTB) was found to inhibit the splicing of introns containing a strong binding site for this factor. However, the inhibitory effect of PTB could be partially reversed if pre-mRNAs were preincubated with U2 auxiliary factor (U2AF) prior to splicing in PTB-supplemented extracts. For alpha-tropomyosin, regulation of splicing by PTB and U2AF primarily affected the joining of exons 1-3 with no dramatic increases in 1-2 splicing being detected. Preincubation of pre-mRNAs with SR proteins led to small increases in 1-2 splicing. However, if pre-mRNAs were preincubated with SR proteins followed by splicing in PTB-supplemented extracts, there was a nearly complete reversal of the normal 1-2 to 1-3 splicing ratios. Thus, multiple pairwise, and sometimes antagonizing, interactions between constitutive pre-mRNA splicing factors and the pre-mRNA can regulate 3' splice site selection.

Links

Publisher Full Text
ncbi.nlm.nih.gov
rnajournal.org
PMC Free PDF

Authors+Show Affiliations

Lin CH
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.
Patton JG
No affiliation info available

MeSH

Base SequenceCell NucleusDNA-Binding ProteinsExonsGene Expression RegulationHeLa CellsHumansMolecular Sequence DataNuclear ProteinsPhosphoproteinsPolypyrimidine Tract-Binding ProteinRNA SplicingRNA-Binding ProteinsRibonucleoproteinsSerine-Arginine Splicing FactorsSplicing Factor U2AFSubcellular FractionsTropomyosin

Pub Type(s)

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

Language

eng

PubMed ID

7489496

Citation

Lin, C H., and J G. Patton. "Regulation of Alternative 3' Splice Site Selection By Constitutive Splicing Factors." RNA (New York, N.Y.), vol. 1, no. 3, 1995, pp. 234-45.
Lin CH, Patton JG. Regulation of alternative 3' splice site selection by constitutive splicing factors. RNA. 1995;1(3):234-45.
Lin, C. H., & Patton, J. G. (1995). Regulation of alternative 3' splice site selection by constitutive splicing factors. RNA (New York, N.Y.), 1(3), 234-45.
Lin CH, Patton JG. Regulation of Alternative 3' Splice Site Selection By Constitutive Splicing Factors. RNA. 1995;1(3):234-45. PubMed PMID: 7489496.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Regulation of alternative 3' splice site selection by constitutive splicing factors. AU - Lin,C H, AU - Patton,J G, PY - 1995/5/1/pubmed PY - 1995/5/1/medline PY - 1995/5/1/entrez SP - 234 EP - 45 JF - RNA (New York, N.Y.) JO - RNA VL - 1 IS - 3 N2 - We have devised an in vitro splicing assay in which the mutually exclusive exons 2 and 3 of alpha-tropomyosin act as competing 3' splice sites for joining to exon 1. Splicing in normal HeLa cell nuclear extracts results in almost exclusive joining of exons 1 and 3. Splicing in decreased nuclear extract concentrations and decreased ionic strength results in increased 1-2 splicing. We have used this assay to determine the role of three constitutive pre-mRNA splicing factors on alternative 3' splice site selection. Polypyrimidine tract binding protein (PTB) was found to inhibit the splicing of introns containing a strong binding site for this factor. However, the inhibitory effect of PTB could be partially reversed if pre-mRNAs were preincubated with U2 auxiliary factor (U2AF) prior to splicing in PTB-supplemented extracts. For alpha-tropomyosin, regulation of splicing by PTB and U2AF primarily affected the joining of exons 1-3 with no dramatic increases in 1-2 splicing being detected. Preincubation of pre-mRNAs with SR proteins led to small increases in 1-2 splicing. However, if pre-mRNAs were preincubated with SR proteins followed by splicing in PTB-supplemented extracts, there was a nearly complete reversal of the normal 1-2 to 1-3 splicing ratios. Thus, multiple pairwise, and sometimes antagonizing, interactions between constitutive pre-mRNA splicing factors and the pre-mRNA can regulate 3' splice site selection. SN - 1355-8382 UR - https://www.unboundmedicine.com/medline/citation/7489496/Regulation_of_alternative_3'_splice_site_selection_by_constitutive_splicing_factors_ L2 - http://www.rnajournal.org/cgi/pmidlookup?view=long&pmid=7489496 DB - PRIME DP - Unbound Medicine ER -