In vitro splicing pathways of pre-mRNAs containing multiple intervening sequences?

1987 ◽  
Vol 7 (10) ◽  
pp. 3428-3437
Author(s):  
K M Lang ◽  
R A Spritz
Keyword(s):  
Temporal Order ◽  
Exon Skipping ◽  
Beta Globin ◽  
Splice Site Selection ◽  
Interleukin 3 ◽  
Intervening Sequences ◽  
In Vitro Splicing ◽  
Lag Times ◽  
Alpha Lactalbumin

We analyzed the in vitro splicing pathways of three multi-intervening-sequence (IVS) pre-mRNAs: human beta-globin, which contains two IVSs (K. M. Lang, V. L. van Santen, and R. A. Spritz, EMBO J. 4:1991-1996, 1985); rat alpha-lactalbumin, which contains three IVSs; and murine interleukin-3, which contains four IVSs. We found that there are highly preferred pathways of IVS removal from these multi-IVS pre-mRNAs in vitro. The three IVSs of rat alpha-lactalbumin pre-mRNA were excised sequentially from 5' to 3'; in most molecules, IVS1 was removed first, followed by IVS2 and finally by IVS3. The splicing pathway of interleukin-3 pre-mRNA in vitro was more complex. The four IVSs were excised in a highly preferred temporal order, but the order was not strictly sequential or directional. In most molecules, IVS1 and IVS4 were removed first, either simultaneously or in rapid succession. Subsequently, IVS2 was excised, followed by IVS3. The observed splicing pathways apparently resulted from differences in lag times and maximum excision rates of the different IVSs. We detected no exon skipping during splicing of these transcripts in vitro. These observations have implication for proposed models of splice site selection.

scholarly journals In vitro splicing pathways of pre-mRNAs containing multiple intervening sequences?

1987 ◽  
Vol 7 (10) ◽  
pp. 3428-3437 ◽  
Author(s):  
K M Lang ◽  
R A Spritz
Keyword(s):  
Temporal Order ◽  
Exon Skipping ◽  
Beta Globin ◽  
Splice Site Selection ◽  
Interleukin 3 ◽  
Intervening Sequences ◽  
In Vitro Splicing ◽  
Lag Times ◽  
Alpha Lactalbumin

We analyzed the in vitro splicing pathways of three multi-intervening-sequence (IVS) pre-mRNAs: human beta-globin, which contains two IVSs (K. M. Lang, V. L. van Santen, and R. A. Spritz, EMBO J. 4:1991-1996, 1985); rat alpha-lactalbumin, which contains three IVSs; and murine interleukin-3, which contains four IVSs. We found that there are highly preferred pathways of IVS removal from these multi-IVS pre-mRNAs in vitro. The three IVSs of rat alpha-lactalbumin pre-mRNA were excised sequentially from 5' to 3'; in most molecules, IVS1 was removed first, followed by IVS2 and finally by IVS3. The splicing pathway of interleukin-3 pre-mRNA in vitro was more complex. The four IVSs were excised in a highly preferred temporal order, but the order was not strictly sequential or directional. In most molecules, IVS1 and IVS4 were removed first, either simultaneously or in rapid succession. Subsequently, IVS2 was excised, followed by IVS3. The observed splicing pathways apparently resulted from differences in lag times and maximum excision rates of the different IVSs. We detected no exon skipping during splicing of these transcripts in vitro. These observations have implication for proposed models of splice site selection.

scholarly journals Effects of exon sequences on splicing of model pre-mRNA substrates in vitro.

1996 ◽  
Vol 43 (1) ◽  
pp. 161-173 ◽  
Author(s):  
Z Dominski ◽  
R Kole
Keyword(s):  
Globin Gene ◽  
Beta Globin ◽  
Splice Site Selection ◽  
Sequence Composition ◽  
Recognition Element ◽  
Internal Exon ◽  
Alternatively Spliced ◽  
In Vitro Splicing

We used several related pre-mRNA substrates consisting of two introns and three exons to study effects of exon sequences on in vitro splicing. By varying the sequence of the internal exon and measuring the frequency of its skipping we confirmed that 26-nucleotide exon element naturally existing in beta-globin gene and previously analysed in vivo, has a strong stimulatory effect on splicing. Sequence analysis of this element suggests that it belongs to a family of purine-rich splicing elements found in exons of several alternatively spliced pre-mRNAs. The 26-nucleotide element can efficiently function in enhancing inclusion of internal exons regardless of their size and sequence composition, suggesting that it plays a role of a general exon recognition element. The purine-rich element is dispensable in exons flanked by strong splice sites, which promote efficient inclusion of otherwise poorly recognized exons. A row of six cytidines inserted into the internal exon (GC2 mutation) initially considered to stimulate exon inclusion to a similar extent as the purine-rich element (Dominski & Kole, 1994, J. Biol. Chem. 269, 23590-23596), appears not to affect splice site selection in vitro, and in vivo it is likely to act by stabilizing mRNA that includes the internal exon against rapid cytoplasmic degradation.

scholarly journals Polypurine sequences within a downstream exon function as a splicing enhancer.

1994 ◽  
Vol 14 (2) ◽  
pp. 1347-1354 ◽  
Author(s):  
K Tanaka ◽  
A Watakabe ◽  
Y Shimura
Keyword(s):  
Recognition Sequence ◽  
Splice Site Selection ◽  
Sequence Element ◽  
Nuclear Extracts ◽  
Gene Functions ◽  
Mouse Immunoglobulin ◽  
In Vitro Splicing ◽  
Immunoglobulin Mu ◽  
Splicing Enhancer

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.

Exon definition may facilitate splice site selection in RNAs with multiple exons

1990 ◽  
Vol 10 (1) ◽  
pp. 84-94 ◽  
Author(s):  
B L Robberson ◽  
G J Cote ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Exon Skipping ◽  
Splice Sites ◽  
Splice Site Selection ◽  
Spliceosome Assembly ◽  
Exon Definition ◽  
Exon 2 ◽  
Correct Orientation

Interactions at the 3' end of the intron initiate spliceosome assembly and splice site selection in vertebrate pre-mRNAs. Multiple factors, including U1 small nuclear ribonucleoproteins (snRNPs), are involved in initial recognition at the 3' end of the intron. Experiments were designed to test the possibility that U1 snRNP interaction at the 3' end of the intron during early assembly functions to recognize and define the downstream exon and its resident 5' splice site. Splicing precursor RNAs constructed to have elongated second exons lacking 5' splice sites were deficient in spliceosome assembly and splicing activity in vitro. Similar substrates including a 5' splice site at the end of exon 2 assembled and spliced normally as long as the second exon was less than 300 nucleotides long. U2 snRNPs were required for protection of the 5' splice site terminating exon 2, suggesting direct communication during early assembly between factors binding the 3' and 5' splice sites bordering an exon. We suggest that exons are recognized and defined as units during early assembly by binding of factors to the 3' end of the intron, followed by a search for a downstream 5' splice site. In this view, only the presence of both a 3' and a 5' splice site in the correct orientation and within 300 nucleotides of one another will stable exon complexes be formed. Concerted recognition of exons may help explain the 300-nucleotide-length maximum of vertebrate internal exons, the mechanism whereby the splicing machinery ignores cryptic sites within introns, the mechanism whereby exon skipping is normally avoided, and the phenotypes of 5' splice site mutations that inhibit splicing of neighboring introns.

scholarly journals Nonconsensus branch-site sequences in the in vitro splicing of transcripts of mutant rabbit beta-globin genes.

1985 ◽  
Vol 82 (24) ◽  
pp. 8349-8353 ◽  
Author(s):  
R. A. Padgett ◽  
M. M. Konarska ◽  
M. Aebi ◽  
H. Hornig ◽  
C. Weissmann ◽  
...  
Keyword(s):  
Globin Genes ◽  
Beta Globin ◽  
Branch Site ◽  
In Vitro Splicing

scholarly journals The length of the downstream exon and the substitution of specific sequences affect pre-mRNA splicing in vitro.

1988 ◽  
Vol 8 (2) ◽  
pp. 860-866 ◽  
Author(s):  
P J Furdon ◽  
R Kole
Keyword(s):  
Splice Site ◽  
Mrna Splicing ◽  
Beta Globin ◽  
Specific Sequence ◽  
In Vitro Splicing ◽  
Intron 2 ◽  
Specific Sequences

We have shown previously that truncation of the human beta-globin pre-mRNA in the second exon, 14 nucleotides downstream from the 3' splice site, leads to inhibition of splicing but not cleavage at the 5' splice site. We now show that several nonglobin sequences substituted at this site can restore splicing and that the efficiency of splicing depends on the length of the second (downstream) exon and not a specific sequence. Deletions in the first exon have no effect on the efficiency of in vitro splicing. Surprisingly, an intron fragment from the 5' region of the human or rabbit beta-globin intron 2, when placed 14 nucleotides downstream from the 3' splice site, inhibited all the steps in splicing beginning with cleavage at the 5' splice site. This result suggests that the intron 2 fragment carries a "poison" sequence that can inhibit the splicing of an upstream intron.

Polypurine sequences within a downstream exon function as a splicing enhancer

1994 ◽  
Vol 14 (2) ◽  
pp. 1347-1354
Author(s):  
K Tanaka ◽  
A Watakabe ◽  
Y Shimura
Keyword(s):  
Recognition Sequence ◽  
Splice Site Selection ◽  
Sequence Element ◽  
Nuclear Extracts ◽  
Gene Functions ◽  
Mouse Immunoglobulin ◽  
In Vitro Splicing ◽  
Immunoglobulin Mu ◽  
Splicing Enhancer

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.

scholarly journals Cooperation of pre-mRNA sequence elements in splice site selection

1992 ◽  
Vol 12 (5) ◽  
pp. 2108-2114
Author(s):  
Z Dominski ◽  
R Kole
Keyword(s):  
Splice Site ◽  
Hela Cells ◽  
Site Selection ◽  
Exon Skipping ◽  
Polypyrimidine Tract ◽  
Splice Site Selection ◽  
Branch Point Sequence ◽  
Sequence Elements

We have recently demonstrated that short internal exons in pre-mRNA transcripts with three exons and two introns are ignored by splicing machinery in vitro and in vivo, resulting in exon skipping. Exon skipping is reversed when the pyrimidine content of the polypyrimidine tract in the upstream intron is increased (Z. Dominski and R. Kole, Mol. Cell. Biol. 11:6075-6083, 1991). Here we show that skipping of the short internal exon can be partially reversed by mutations which modify the upstream branch point sequence of the 5' splice site at the end of the exon to their respective consensus sequences. When the modified elements are combined with one another in the same pre-mRNA, exon skipping is fully reversed. Full reversion of exon skipping is also observed when these elements are combined individually with the upstream polypyrimidine tract strengthened by three purine-to-pyrimidine mutations. The observed patterns of splice site selection are similar in vitro (in nuclear extracts from HeLa cells) and in vivo (in transfected HeLa cells). We also show that the length of the downstream intron plays a role in splice site selection. Our data indicate that the interplay between the sequence elements in pre-mRNA controls the outcome of each splicing event, providing the means for very subtle regulation of alternative splicing.

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