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. 1996 May 1;24(9):1765–1773. doi: 10.1093/nar/24.9.1765

Sequence of the polypyrimidine tract of the 3'-terminal 3' splicing signal can affect intron-dependent pre-mRNA processing in vivo.

X Liu 1, J E Mertz 1
PMCID: PMC145838  PMID: 8649998

Abstract

Most pre-mRNAs require an intron for efficient processing in higher eukaryotes. However, not all introns can provide this function. For example, transcripts synthesized from a variant of the human beta-globin gene lacking its second intervening sequence (IVS2), yet retaining its first intervening sequence (IVS1), exhibit multiple defects in mRNA biogenesis. To investigate why, we transfected into monkey cells plasmids containing the human beta-globin gene and variants of it altered in (i) IVS1, (ii) the 3'-terminal exon, and (iii) the polyadenylation signal. The beta-globin RNAs accumulated in these cells were analyzed by quantitative S1 nuclease mapping for nuclear accumulation, intron excision, polyadenylation and cytoplasmic accumulation. We found that the 3' splicing signal of IVS1, with multiple purines interrupting its polypyrimidine tract, could efficiently function as an internal 3' splicing signal; however, it could not efficiently function as the 3'-terminal 3' splicing signal for any of these steps in intron-dependent mRNA biogenesis unless (i) its polypyrimidine tract was made uninterrupted in pyrimidines, or (ii) specific sequences were deleted from the 3'-terminal exon. We conclude that whether an intron can provide the function necessary for efficient processing of intron-dependent pre-mRNA is dependent upon the ability of its 3' splicing signal to define the 3'-terminal exon. On the practical side, this finding means one needs to consider both the sequence and location of the intron to be included in an intron-dependent gene to obtain efficient expression in vivo.

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Selected References

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