The Length of the Downstream Exon and the Substitution of Specific Sequences Affect Pre-mRNA Splicing in Vitro

Furdon, Paul J.; Kole, Ryszard

doi:10.1128/mcb.8.2.860-866.1988

Cited by 10 publications

(3 citation statements)

References 31 publications

(24 reference statements)

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“…Previous studies by others have shown that the presence within a 3′-terminal exon of a sequence similar to a 5′ splice site can lead to significant reduction in the accumulation of an mRNA (56)(57)(58). Likewise, Furdon and Kole (59) found that polypyrimidine-rich sequences are 'poisonous' to splicing in vitro when placed within the 3′-terminal exon. Negative sequence elements that affect splicing of weak introns in retroviruses have also been identified (26,28).…”

Section: Effects Of Specific Sequences Within the 3′-terminal Exonmentioning

confidence: 99%

Sequence of the Polypyrimidine Tract of the 3'-Terminal 3' Splicing Signal Can Affect Intron-Dependent Pre-mRNA Processing In Vivo

Liu

Mertz

1996

Nucleic Acids Research

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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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Section: Effects Of Specific Sequences Within the 3′-terminal Exonmentioning

confidence: 99%

Sequence of the Polypyrimidine Tract of the 3'-Terminal 3' Splicing Signal Can Affect Intron-Dependent Pre-mRNA Processing In Vivo

Liu

Mertz

1996

Nucleic Acids Research

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“…Although we have shown the positive effect of exon sequences (ERS) in this study, the negative effect of exon sequences should also be considered. In vitro experiments using P-globin pre-mRNA have shown that a sequence containing many U residues inhibits splicing when it is present in the downstream exon (13). Moreover, in vivo selection of the mutants in the hprt gene has been described previously (41).…”

Section: Discussionmentioning

confidence: 99%

Polypurine Sequences within a Downstream Exon Function as a Splicing Enhancer

Tanaka

Watakabe

Shimura

1994

Molecular and Cellular Biology

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We have previously shown that a purine-rich sequence located within exon M2 of the mouse immunoglobulin mu gene functions as a splicing enhancer, as judged by its ability to stimulate splicing of a distant upstream intron. This sequence element has been designated ERS (exon recognition sequence). In this study, we investigated the stimulatory effects of various ERS-like sequences, using the in vitro splicing system with HeLa cell nuclear extracts. Here, we show that purine-rich sequences of several natural exons that have previously been shown to be required for splicing function as a splicing enhancer like the ERS of the immunoglobulin mu gene. Moreover, even synthetic polypurine sequences had stimulatory effects on the upstream splicing. Evaluation of the data obtained from the analyses of both natural and synthetic purine-rich sequences shows that (i) alternating purine sequences can stimulate splicing, while poly(A) or poly(G) sequences cannot, and (ii) the presence of U residues within the polypurine sequence greatly reduces the level of stimulation. Competition experiments strongly suggest that the stimulatory effects of various purine-rich sequences are mediated by the same trans-acting factor(s). We conclude from these results that the purine-rich sequences that we examined in this study also represent examples of ERS. Thus, ERS is considered a general splicing element that is present in various exons and plays an important role in splice site selection.

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“…In certain differentially spliced genes, additional elements located upstream of the branch point (36,58) or downstream of the 5' splice site (7) have been shown to influence recognition of the neighboring exon. In addition, splicing efficiency in a number of systems is influenced by exon sequences (5,6,11,16,18,20,22,23,28,29,36,37,40,51,59,(63)(64)(65)72). Thus, a region encompassing shortly upstream of the branch point to shortly downstream of the 5' splice site usually contains all of the sequences necessary for recognition of an exon.…”

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confidence: 99%

In Vivo Recognition of a Vertebrate Mini-Exon as an Exon-Intron-Exon Unit

Sterner

Berget

1993

Molecular and Cellular Biology

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Very small vertebrate exons are problematic for RNA splicing because of the proximity of their 3' and 5' splice sites. In this study, we investigated the recognition of a constitutive 7-nucleotide mini-exon from the troponin I gene that resides quite close to the adjacent upstream exon. The mini-exon failed to be included in spliced RNA when placed in a heterologous gene unless accompanied by the upstream exon. The requirement for the upstream exon disappeared when the mini-exon was internally expanded, suggesting that the splice sites bordering the mini-exon are compatible with those of other constitutive vertebrate exons and that the small size of the exon impaired inclusion. Mutation of the 5' splice site of the natural upstream exon did not result in either exon skipping or activation of a cryptic 5' splice site, the normal vertebrate phenotypes for such mutants. Instead, a spliced RNA accumulated that still contained the upstream intron. In vitro, the mini-exon failed to assemble into spliceosome complexes unless either internally expanded or accompanied by the upstream exon. Thus, impaired usage of the mini-exon in vivo was accompanied by impaired recognition in vitro, and recognition of the mini-exon was facilitated by the presence of the upstream exon in vivo and in vitro. Cumulatively, the atypical in vivo and in vitro properties of the troponin exons suggest a mechanism for the recognition of this mini-exon in which initial recognition of an exon-intron-exon unit is followed by subsequent recognition of the intron.

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The Length of the Downstream Exon and the Substitution of Specific Sequences Affect Pre-mRNA Splicing in Vitro

Cited by 10 publications

References 31 publications

Sequence of the Polypyrimidine Tract of the 3'-Terminal 3' Splicing Signal Can Affect Intron-Dependent Pre-mRNA Processing In Vivo

Sequence of the Polypyrimidine Tract of the 3'-Terminal 3' Splicing Signal Can Affect Intron-Dependent Pre-mRNA Processing In Vivo

Polypurine Sequences within a Downstream Exon Function as a Splicing Enhancer

In Vivo Recognition of a Vertebrate Mini-Exon as an Exon-Intron-Exon Unit

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