In Vitro Splicing of Pre-mRNA in HeLa Extracts
The protein-coding sequences in most messenger RNA precursors (pre-mRNAs) of higher eukaryotes are interrupted by intervening sequences (or introns), which are removed from the primary transcript in a process termed splicing (for review see Moore et al. 1993; Krämer 1996). The reaction occurs in the nucleus either co- or post-transcriptionally. The coding sequences (or exons) are joined and the mature mRNA is exported to the cytoplasm where it is translated into protein. Intron removal is an essential process because introns usually have no or only little protein-coding potential and the failure to remove them from the mRNA can introduce in-frame stop codons resulting in premature termination of translation and thus nonfunctional proteins. Many pre-mRNAs contain more than one intron, which provides a means for the regulation of gene expression by alternative splicing events. The selection of alternative 5’ and 3’ splice sites or the inclusion/exclusion of entire exons can result in an on/off switch of a particular gene or in the translation of different protein isoforms (often with specific functions) from one and the same pre-mRNA, thus augmenting the coding repertoire of the genome.
KeywordsGlycerol Phenol Urea EDTA Chloroform
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