Sequences within the last intron function in RNA 3'-end formation in cultured cells.
In cultured cells, little if any mRNA accumulates from an intronless version of the human gene for triosephosphate isomerase (TPI), a gene that normally contains six introns. By deleting introns either individually or in combinations, it was demonstrated by Northern (RNA) blot hybridization that while the deletion of a greater number of introns generally results in a lower level of product mRNA, not all introns contribute equally to mRNA formation. For example, intron 1 appeared to be dispensable, at least when the remaining introns are present, but deletion of the last intron, intron 6, reduced the level of product mRNA to 51% of normal. To determine how intron 6 contributes to mRNA formation, partial deletions of intron 6 were constructed and analyzed. Deletion of the lariat and acceptor splice sites or the donor, lariat, and acceptor splice sites, each of which precluded removal of the intron 6 sequences that remained, reduced the level of product mRNA to < 1 or 27% of normal, respectively. As measured by RNase mapping and cDNA sequencing, the decrease in mRNA abundance that was attributable to the complete and partial intron 6 deletions was accompanied by an increase in the abundance of pre-mRNA that lacked a mature 3' end, i.e., that was neither cleaved nor polyadenylated. We infer from these and other data that sequences within the final intron facilitate proper 3'-end formation, possibly through an association with the components of a productive spliceosome.
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Department of Human Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263.
Cheng
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Maquat
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D000483:Alleles; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D006801:Humans; D007438:Introns; D007739:L Cells; D008545:Melanoma; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011061:Poly A / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012322:RNA Precursors / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D015183:Restriction Mapping; D017384:Sequence Deletion; D014162:Transfection; D014305:Triose-Phosphate Isomerase / Q000235:genetics*; D014407:Tumor Cells, Cultured
D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D014305:Triose-Phosphate Isomerase