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PMID: 10022858 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Selection and characterization of pre-mRNA splicing enhancers: identification of novel SR protein-specific enhancer sequences.

Molecular and cellular biology ·Vol. 19 ·No. 3 ·1999-03-00 ·Pages 1705-19

Schaal TD, Maniatis T

Abstract

Splicing enhancers are RNA sequences required for accurate splice site recognition and the control of alternative splicing. In this study, we used an in vitro selection procedure to identify and characterize novel RNA sequences capable of functioning as pre-mRNA splicing enhancers. Randomized 18-nucleotide RNA sequences were inserted downstream from a Drosophila doublesex pre-mRNA enhancer-dependent splicing substrate. Functional splicing enhancers were then selected by multiple rounds of in vitro splicing in nuclear extracts, reverse transcription, and selective PCR amplification of the spliced products. Characterization of the selected splicing enhancers revealed a highly heterogeneous population of sequences, but we identified six classes of recurring degenerate sequence motifs five to seven nucleotides in length including novel splicing enhancer sequence motifs. Analysis of selected splicing enhancer elements and other enhancers in S100 complementation assays led to the identification of individual enhancers capable of being activated by specific serine/arginine (SR)-rich splicing factors (SC35, 9G8, and SF2/ASF). In addition, a potent splicing enhancer sequence isolated in the selection specifically binds a 20-kDa SR protein. This enhancer sequence has a high level of sequence homology with a recently identified RNA-protein adduct that can be immunoprecipitated with an SRp20-specific antibody. We conclude that distinct classes of selected enhancers are activated by specific SR proteins, but there is considerable sequence degeneracy within each class. The results presented here, in conjunction with previous studies, reveal a remarkably broad spectrum of RNA sequences capable of binding specific SR proteins and/or functioning as SR-specific splicing enhancers.

MeSH Terms
Alternative Splicing Animals Drosophila/genetics Enhancer Elements, Genetic Exons Genes, Insect Nuclear Proteins/genetics,metabolism Nucleocytoplasmic Transport Proteins Purines Pyrimidines RNA Precursors RNA-Binding Proteins/genetics,metabolism Ribonucleoproteins Serine-Arginine Splicing Factors
Chemicals
Nuclear Proteins Nucleocytoplasmic Transport Proteins Purines Pyrimidines RNA Precursors RNA-Binding Proteins Ribonucleoproteins SRSF3 protein, human Serine-Arginine Splicing Factors
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Schaal T D
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Maniatis T
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.19.3.1705
Published
1999-03-00
Pages
1705-19
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC83964
Subset
IM
Grants
NIGMS NIH HHS · R01 GM042231 · United States
NIGMS NIH HHS · GM42231 · United States
External Links
PubMed source DOI: 10.1128/MCB.19.3.1705 PMC Full Text View journal details
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