The exon junction complex and intron removal prevent re-splicing of mRNA

AbstractAccurate splice site selection is critical for fruitful gene expression. Here, we demonstrate the Drosophila EJC suppresses hundreds of functional cryptic splice sites (SS), even though majority of these bear weak splicing motifs and appear incompetent. Mechanistically, the EJC directly conceals splicing elements through position-specific recruitment, preventing SS definition. We note that intron removal using strong, canonical SS yields AG|GU signatures at exon-exon junctions. Unexpectedly, we discover that scores of these minimal exon junction sequences are in fact EJC-suppressed 5’ and 3’ recursive SS, and that loss of EJC regulation from such transcripts triggers faulty mRNA resplicing. An important corollary is that intronless cDNA expression constructs from aforementioned targets yield high levels of unanticipated, truncated transcripts generated by resplicing. Consequently, we conclude the EJC has ancestral roles to defend transcriptome fidelity by (1) repressing illegitimate splice sites on pre-mRNAs, and (2) preventing inadvertent activation of such sites on spliced segments.

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Usage of Cryptic Splice Sites in Citrullinemia Fibroblasts Suggests Role of Polyadenylation in Splice-Site Selection during Terminal Exon Definition

DNA and Cell Biology ◽

10.1089/dna.1998.17.717 ◽

1998 ◽

Vol 17 (8) ◽

pp. 717-725 ◽

Cited By ~ 3

Author(s):

TING-FEN TSAI ◽

MEI-JANE WU ◽

TSUNG-SHENG SU

Keyword(s):

Splice Site ◽

Site Selection ◽

Splice Sites ◽

Terminal Exon ◽

Splice Site Selection ◽

Exon Definition ◽

Cryptic Splice Sites

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Nuclear pre-mRNA processing in plants: distinct modes of 3'-splice-site selection in plants and animals

Molecular and Cellular Biology ◽

10.1128/mcb.8.5.2042-2051.1988 ◽

1988 ◽

Vol 8 (5) ◽

pp. 2042-2051

Author(s):

K Wiebauer ◽

J J Herrero ◽

W Filipowicz

Keyword(s):

Splice Site ◽

Hela Cells ◽

Site Selection ◽

Human Growth ◽

Mrna Processing ◽

Beta Globin ◽

Splice Sites ◽

Splice Site Selection ◽

Consensus Sequences ◽

Intron 2

The report that human growth hormone pre-mRNA is not processed in transgenic plant tissues (A. Barta, K. Sommergruber, D. Thompson, K. Hartmuth, M.A. Matzke, and A.J.M. Matzke, Plant Mol. Biol. 6:347-357, 1986) has suggested that differences in mRNA splicing processes exist between plants and animals. To gain more information about the specificity of plant pre-mRNA processing, we have compared the splicing of the soybean leghemoglobin pre-mRNA with that of the human beta-globin pre-mRNA in transfected plant (Orychophragmus violaceus and Nicotiana tabacum) protoplasts and mammalian (HeLa) cells. Of the three introns of leghemoglobin pre-mRNA, only intron 2 was correctly and efficiently processed in HeLa cells. The 5' splice sites of the remaining two introns were faithfully recognized, but correct processing of the 3' sites took place only rarely (intron 1) or not at all (intron 3); cryptic 3' splice sites were used instead. While the first intron in human beta-globin pre-mRNA was not spliced in transfected plant protoplasts, intron 2 processing occurred at a low level, indicating that some mammalian introns can be recognized by the plant intron-splicing machinery. However, excision of intron 2 proved to be incorrect, involving the authentic 5' splice site and a cryptic 3' splice site. Our results indicate that the mechanism of 3'-splice-site selection during intron excision differs between plants and animals. This conclusion is supported by analysis of the 3'-splice-site consensus sequences in animal and plant introns which revealed that polypyrimidine tracts, characteristic of animal introns, are not present in plant pre-mRNAs. It is proposed that an elevated AU content of plant introns is important for their processing.

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A novel protein factor is required for use of distal alternative 5' splice sites in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.11.12.5945-5953.1991 ◽

1991 ◽

Vol 11 (12) ◽

pp. 5945-5953

Author(s):

J E Harper ◽

J L Manley

Keyword(s):

Splice Site ◽

Site Selection ◽

Deae Cellulose ◽

Simian Virus 40 ◽

Nuclear Extract ◽

Simian Virus ◽

Splice Sites ◽

Splice Site Selection ◽

Small Nuclear Ribonucleoprotein

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites.

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Competing Upstream 5′ Splice Sites Enhance the Rate of Proximal Splicing

Molecular and Cellular Biology ◽

10.1128/mcb.01071-09 ◽

2010 ◽

Vol 30 (8) ◽

pp. 1878-1886 ◽

Cited By ~ 24

Author(s):

Martin J. Hicks ◽

William F. Mueller ◽

Peter J. Shepard ◽

Klemens J. Hertel

Keyword(s):

Splice Site ◽

Site Selection ◽

Regulatory Element ◽

Regulatory Elements ◽

Splice Sites ◽

Base Pairing ◽

Splice Site Selection ◽

U1 Snrna ◽

Pairing Potential

ABSTRACT Alternative 5′ splice site selection is one of the major pathways resulting in mRNA diversification. Regulation of this type of alternative splicing depends on the presence of regulatory elements that activate or repress the use of competing splice sites, usually leading to the preferential use of the proximal splice site. However, the mechanisms involved in proximal splice site selection and the thermodynamic advantage realized by proximal splice sites are not well understood. Here, we have carried out a systematic analysis of alternative 5′ splice site usage using in vitro splicing assays. We show that observed rates of splicing correlate well with their U1 snRNA base pairing potential. Weak U1 snRNA interactions with the 5′ splice site were significantly rescued by the proximity of the downstream exon, demonstrating that the intron definition mode of splice site recognition is highly efficient. In the context of competing splice sites, the proximity to the downstream 3′ splice site was more influential in dictating splice site selection than the actual 5′ splice site/U1 snRNA base pairing potential. Surprisingly, the kinetic analysis also demonstrated that an upstream competing 5′ splice site enhances the rate of proximal splicing. These results reveal the discovery of a new splicing regulatory element, an upstream 5′ splice site functioning as a splicing enhancer.

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Factors influencing alternative splice site utilization in vivo.

Molecular and Cellular Biology ◽

10.1128/mcb.7.2.738 ◽

1987 ◽

Vol 7 (2) ◽

pp. 738-748 ◽

Cited By ~ 45

Author(s):

X Y Fu ◽

J L Manley

Keyword(s):

Splice Site ◽

Site Selection ◽

Simian Virus 40 ◽

Cell Types ◽

Simian Virus ◽

Splice Sites ◽

Splice Site Selection ◽

Culture Cells ◽

293 Cells

To study factors that influence the choice of alternative pre-mRNA splicing pathways, we introduced plasmids expressing either wild-type or mutated simian virus 40 (SV40) early regions into tissue culture cells and then measured the quantities of small-t and large-T RNAs produced. One important element controlling splice site selection was found to be the size of the intron removed in the production of small-t mRNA; expansion of this intron (from 66 to 77 or more nucleotides) resulted in a substantial increase in the amount of small-t mRNA produced relative to large-T mRNA. This suggests that in the normal course of SV40 early pre-mRNA processing, large-T splicing is at a competitive advantage relative to small-t splicing because of the small size of the latter intron. Several additional features of the pre-mRNA that can influence splice site selection were also identified by analyzing the effects of mutations containing splice site duplications. These include the strengths of competing 5' splice sites and the relative positions of splice sites in the pre-mRNA. Finally, we showed that the ratio of small-t to large-T mRNA was 10 to 15-fold greater in human 293 cells than in HeLa cells or other mammalian cell types. These results suggest the existence of cell-specific trans-acting factors that can dramatically alter the pattern of splice site selection in a pre-mRNA.

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Comparison of in vitro and in vivo splice site selection in kappa-immunoglobulin precursor mRNA.

Molecular and Cellular Biology ◽

10.1128/mcb.8.6.2610 ◽

1988 ◽

Vol 8 (6) ◽

pp. 2610-2619 ◽

Cited By ~ 7

Author(s):

D E Lowery ◽

B G Van Ness

Keyword(s):

Splice Site ◽

Site Selection ◽

Donor Site ◽

Splice Donor Site ◽

Splice Sites ◽

Splice Donor ◽

Splice Site Selection ◽

Nuclear Extracts

The processing of a number of kappa-immunoglobulin primary mRNA (pre-mRNA) constructs has been examined both in vitro and in vivo. When a kappa-immunoglobulin pre-mRNA containing multiple J segment splice sites is processed in vitro, the splice sites are used with equal frequency. The presence of signal exon, S-V intron, or variable (V) region has no effect on splice site selection in vitro. Nuclear extracts prepared from a lymphoid cell line do not restore correct splice site selection. Splice site selection in vitro can be altered by changing the position or sequence of J splice donor sites. These results differ from the processing of similar pre-mRNAs expressed in vivo by transient transfection. The 5'-most J splice donor site was exclusively selected in vivo, even in nonlymphoid cells, and even in transcripts where in vitro splicing favored a 3' J splice site. The in vitro results are consistent with a model proposing that splice site selection is influenced by splice site strength and proximity; however, our in vivo results demonstrate a number of discrepancies with such a model and suggest that splice site selection may be coupled to transcription or a higher-order nuclear structure.

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

Molecular and Cellular Biology ◽

10.1128/mcb.10.1.84-94.1990 ◽

1990 ◽

Vol 10 (1) ◽

pp. 84-94 ◽

Cited By ~ 1

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.

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Factors influencing alternative splice site utilization in vivo

Molecular and Cellular Biology ◽

10.1128/mcb.7.2.738-748.1987 ◽

1987 ◽

Vol 7 (2) ◽

pp. 738-748

Author(s):

X Y Fu ◽

J L Manley

Keyword(s):

Splice Site ◽

Site Selection ◽

Simian Virus 40 ◽

Cell Types ◽

Simian Virus ◽

Splice Sites ◽

Splice Site Selection ◽

Culture Cells ◽

293 Cells

To study factors that influence the choice of alternative pre-mRNA splicing pathways, we introduced plasmids expressing either wild-type or mutated simian virus 40 (SV40) early regions into tissue culture cells and then measured the quantities of small-t and large-T RNAs produced. One important element controlling splice site selection was found to be the size of the intron removed in the production of small-t mRNA; expansion of this intron (from 66 to 77 or more nucleotides) resulted in a substantial increase in the amount of small-t mRNA produced relative to large-T mRNA. This suggests that in the normal course of SV40 early pre-mRNA processing, large-T splicing is at a competitive advantage relative to small-t splicing because of the small size of the latter intron. Several additional features of the pre-mRNA that can influence splice site selection were also identified by analyzing the effects of mutations containing splice site duplications. These include the strengths of competing 5' splice sites and the relative positions of splice sites in the pre-mRNA. Finally, we showed that the ratio of small-t to large-T mRNA was 10 to 15-fold greater in human 293 cells than in HeLa cells or other mammalian cell types. These results suggest the existence of cell-specific trans-acting factors that can dramatically alter the pattern of splice site selection in a pre-mRNA.

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Role of the 3′ Splice Site in U12-Dependent Intron Splicing

Molecular and Cellular Biology ◽

10.1128/mcb.21.6.1942-1952.2001 ◽

2001 ◽

Vol 21 (6) ◽

pp. 1942-1952 ◽

Cited By ~ 18

Author(s):

Rosemary C. Dietrich ◽

Marian J. Peris ◽

Andrew S. Seyboldt ◽

Richard A. Padgett

Keyword(s):

Splice Site ◽

Site Selection ◽

Intron Splicing ◽

Splice Sites ◽

Splice Site Selection ◽

Branch Site ◽

Site Function

ABSTRACT U12-dependent introns containing alterations of the 3′ splice site AC dinucleotide or alterations in the spacing between the branch site and the 3′ splice site were examined for their effects on splice site selection in vivo and in vitro. Using an intron with a 5′ splice site AU dinucleotide, any nucleotide could serve as the 3′-terminal nucleotide, although a C residue was most active, while a U residue was least active. The penultimate A residue, by contrast, was essential for 3′ splice site function. A branch site-to-3′ splice site spacing of less than 10 or more than 20 nucleotides strongly activated alternative 3′ splice sites. A strong preference for a spacing of about 12 nucleotides was observed. The combined in vivo and in vitro results suggest that the branch site is recognized in the absence of an active 3′ splice site but that formation of the prespliceosomal complex A requires an active 3′ splice site. Furthermore, the U12-type spliceosome appears to be unable to scan for a distal 3′ splice site.

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