Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro

Krainer, A. R., Maniatis, T., Ruskin, B., Green, M. R. (April 1984) Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell, 36 (4). pp. 993-1005. ISSN 0092-8674 (Print)0092-8674

URL: http://www.ncbi.nlm.nih.gov/pubmed/6323033
DOI: 10.1016/0092-8674(84)90049-7

Abstract

Human beta-globin mRNA precursors (pre-mRNAs) synthesized in vitro from a bacteriophage SP6 promoter/beta-globin gene fusion are accurately and efficiently spliced when added to a HeLa cell nuclear extract. Under optimal conditions, the first intervening sequence (IVS 1) is removed by splicing in up to 90% of the input pre-mRNA. Splicing requires ATP and in its absence the pre-mRNA is neither spliced nor cleaved at splice junctions. Splicing does not require that the pre-mRNA contain a correct 5' or 3' end, a 3' poly A tail, or a 5'-terminal cap structure. However, capping of the pre-mRNA significantly affects the specificity of in vitro processing. In the absence of a cap approximately 30%-40% of the pre-mRNA is accurately spliced, and a number of aberrantly cleaved RNAs are also detected. In contrast, capped pre-mRNAs are spliced more efficiently and produce fewer aberrant RNA species. The specificity of splice-site selection in vitro was tested by analyzing pre-mRNAs that contain beta-thalassemia splicing mutations in IVS 1. Remarkably, these mutations cause the same abnormal splicing events in vitro and in vivo. The ability to synthesize mutant pre-mRNAs and study their splicing in a faithful in vitro system provides a powerful approach to determine the mechanisms of RNA splice-site selection.

Item Type: Paper
Uncontrolled Keywords: Adenosine Triphosphate/metabolism Base Sequence Cations Cell Nucleus/metabolism *Cloning, Molecular DNA Restriction Enzymes Globins/*genetics HeLa Cells/metabolism Humans *Mutation Nucleic Acid Precursors/*genetics Plasmids RNA Precursors RNA, Messenger/*genetics Thalassemia/genetics *Transcription, Genetic
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > pre-mRNA
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > RNA splicing
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > splicing factor
CSHL Authors:
Communities: CSHL labs > Krainer lab
Depositing User: Matt Covey
Date: April 1984
Date Deposited: 11 Mar 2014 20:41
Last Modified: 11 Mar 2014 20:41
Related URLs:
URI: http://repository.cshl.edu/id/eprint/29589

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