scholarly journals Identification of proteins that interact with exon sequences, splice sites, and the branchpoint sequence during each stage of spliceosome assembly.

1996 ◽  
Vol 16 (7) ◽  
pp. 3317-3326 ◽  
Author(s):  
M D Chiara ◽  
O Gozani ◽  
M Bennett ◽  
P Champion-Arnaud ◽  
L Palandjian ◽  
...  
Keyword(s):  
Splice Site ◽  
Yeast Cells ◽  
Splice Sites ◽  
Spliceosome Assembly ◽  
Systematic Analysis ◽  
Small Nuclear Ribonucleoprotein ◽  
Sequence Elements ◽  
Cross Links ◽  
C Complex ◽  
Branchpoint Sequence

We have carried out a systematic analysis of the proteins that interact with specific intron and exon sequences during each stage of mammalian spliceosome assembly. This was achieved by site-specifically labeling individual nucleotides within the 5' and 3' splice sites, the branchpoint sequence (BPS), or the exons with 32P and identifying UV-cross-linked proteins in the E, A, B, or C spliceosomal complex. Significantly, two members of the SR family of splicing factors, which are known to promote E-complex assembly, cross-link within exon sequences to a region approximately 25 nucleotides upstream from the 5' splice site. At the 5' splice site, cross-linking of the U5 small nuclear ribonucleoprotein particle protein, U5(200), was detected in both the B and C complexes. As observed in yeast cells, U5(200), also cross-links to intron/exon sequences at the 3' splice site in the C complex and may play a role in aligning the 5' and 3' exons for ligation. With label at the branch site, we detected three distinct proteins, designated BPS72,BpS70, and BPS56, which replace one another in the E, A, and C complexes. Another dynamic exchange was detected with pre-mRNA labeled at the AG dinucleotide of the 3' splice site. In this case, a protein, AG100,cross-links in the A complex and is replaced by another protein, AG75, in the C complex. The observation that these proteins are specifically associated with critical pre-mRNA sequence elements in functional complexes at different stages of spliceosome assembly implicates roles for these factors in key recognition events during the splicing pathway.

scholarly journals Short artificial hairpins sequester splicing signals and inhibit yeast pre-mRNA splicing.

1993 ◽  
Vol 13 (11) ◽  
pp. 6841-6848 ◽  
Author(s):  
V Goguel ◽  
Y Wang ◽  
M Rosbash
Keyword(s):  
Splice Site ◽  
Major Effect ◽  
Yeast Saccharomyces Cerevisiae ◽  
Spliceosome Assembly ◽  
In Vivo Experiments ◽  
Small Nuclear Ribonucleoprotein ◽  
Sequence Elements ◽  
The Stability

To examine the stability of yeast (Saccharomyces cerevisiae) pre-mRNA structures, we inserted a series of small sequence elements that generated potential RNA hairpins at the 5' splice site and branch point regions. We analyzed spliceosome assembly and splicing in vitro as well as splicing and nuclear pre-mRNA retention in vivo. Surprisingly, the inhibition of in vivo splicing approximately paralleled that of in vitro splicing. Even a 6-nucleotide hairpin could be shown to inhibit splicing, and a 15-nucleotide hairpin gave rise to almost complete inhibition. The in vitro results indicate that hairpins that sequester the 5' splice site have a major effect on the early steps of spliceosome assembly, including U1 small nuclear ribonucleoprotein binding. The in vivo experiments lead to comparable conclusions as the sequestering hairpins apparently result in the transport of pre-mRNA to the cytoplasm. The observations are compared with previous data from both yeast and mammalian systems and suggest an important effect of pre-mRNA structure on in vivo splicing.

scholarly journals Short artificial hairpins sequester splicing signals and inhibit yeast pre-mRNA splicing

1993 ◽  
Vol 13 (11) ◽  
pp. 6841-6848
Author(s):  
V Goguel ◽  
Y Wang ◽  
M Rosbash
Keyword(s):  
Splice Site ◽  
Major Effect ◽  
Yeast Saccharomyces Cerevisiae ◽  
Spliceosome Assembly ◽  
In Vivo Experiments ◽  
Small Nuclear Ribonucleoprotein ◽  
Sequence Elements ◽  
The Stability

To examine the stability of yeast (Saccharomyces cerevisiae) pre-mRNA structures, we inserted a series of small sequence elements that generated potential RNA hairpins at the 5' splice site and branch point regions. We analyzed spliceosome assembly and splicing in vitro as well as splicing and nuclear pre-mRNA retention in vivo. Surprisingly, the inhibition of in vivo splicing approximately paralleled that of in vitro splicing. Even a 6-nucleotide hairpin could be shown to inhibit splicing, and a 15-nucleotide hairpin gave rise to almost complete inhibition. The in vitro results indicate that hairpins that sequester the 5' splice site have a major effect on the early steps of spliceosome assembly, including U1 small nuclear ribonucleoprotein binding. The in vivo experiments lead to comparable conclusions as the sequestering hairpins apparently result in the transport of pre-mRNA to the cytoplasm. The observations are compared with previous data from both yeast and mammalian systems and suggest an important effect of pre-mRNA structure on in vivo splicing.

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

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.

scholarly journals Synergistic assembly of human pre-spliceosomes across introns and exons

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Joerg E Braun ◽  
Larry J Friedman ◽  
Jeff Gelles ◽  
Melissa J Moore
Keyword(s):  
Splice Site ◽  
Additive Effects ◽  
Splice Sites ◽  
Dynamic Interactions ◽  
Spliceosome Assembly ◽  
Branch Site ◽  
Nuclear Extracts ◽  
Human Genes

Most human genes contain multiple introns, necessitating mechanisms to effectively define exons and ensure their proper connection by spliceosomes. Human spliceosome assembly involves both cross-intron and cross-exon interactions, but how these work together is unclear. We examined in human nuclear extracts dynamic interactions of single pre-mRNA molecules with individual fluorescently tagged spliceosomal subcomplexes to investigate how cross-intron and cross-exon processes jointly promote pre-spliceosome assembly. U1 subcomplex bound to the 5' splice site of an intron acts jointly with U1 bound to the 5' splice site of the next intron to dramatically increase the rate and efficiency by which U2 subcomplex is recruited to the branch site/3' splice site of the upstream intron. The flanking 5' splice sites have greater than additive effects implying distinct mechanisms facilitating U2 recruitment. This synergy of 5' splice sites across introns and exons is likely important in promoting correct and efficient splicing of multi-intron pre-mRNAs.

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.

Direct interactions between pre-mRNA and six U2 small nuclear ribonucleoproteins during spliceosome assembly

1994 ◽  
Vol 14 (5) ◽  
pp. 2994-3005
Author(s):  
D Staknis ◽  
R Reed
Keyword(s):  
Splice Site ◽  
Specific Protein ◽  
Cross Link ◽  
Protection Factor ◽  
Intimate Contact ◽  
Branch Site ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
Cross Links ◽  
Protein Components

Highly purified mammalian spliceosomal complex B contains more than 30 specific protein components. We have carried out UV cross-linking studies to determine which of these components directly contacts pre-mRNA in purified prespliceosomal and spliceosomal complexes. We show that heterogeneous nuclear ribonucleoproteins cross-link in the nonspecific complex H but not in the B complex. U2AF65, which binds to the 3' splice site, is the only splicing factor that cross-links in purified prespliceosomal complex E. U2AF65 and the U1 small nuclear ribonucleoprotein particle (snRNP) are subsequently destabilized, and a set of six spliceosome-associated proteins (SAPs) cross-links to the pre-mRNA in the prespliceosomal complex A. These proteins require the 3' splice site for binding and cross-link to an RNA containing only the branch site and 3' splice site. Significantly, all six of these SAPs are specifically associated with U2 snRNP. These proteins and a U5 snRNP component cross-link in the fully assembled B complex. Previous work detected an ATP-dependent, U2 snRNP-associated factor that protects a 30- to 40-nucleotide region surrounding the branchpoint sequence from RNase digestion. Our data indicate that the six U2 snRNP-associated SAPs correspond to this branchpoint protection factor. Four of the snRNP proteins that are in intimate contact with the pre-mRNA are conserved between Saccharomyces cerevisiae and humans, consistent with the possibility that these factors play key roles in mediating snRNA-pre-mRNA interactions during the splicing reaction.

Recognition of mutant and cryptic 5' splice sites by the U1 small nuclear ribonucleoprotein in vitro

1987 ◽  
Vol 7 (2) ◽  
pp. 698-707
Author(s):  
B Chabot ◽  
J A Steitz
Keyword(s):  
Splice Site ◽  
Beta Globin ◽  
Splice Sites ◽  
Protection Assay ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoproteins

We examined the ability of U1 small nuclear ribonucleoproteins (U1 snRNPs) to recognize mutant and cryptic 5' splice sites on beta-globin pre-mRNA substrates using an RNase T1 protection assay. When U1 snRNPs were prebound to anti-(U1)RNP antibodies, we detected binding to mutant but not to cryptic 5' splice sites on several substrates. By contrast, in a splicing extract at 0 degree C, neither the mutated nor cryptic 5' splice sites of a human beta-globin transcript were selected as protected fragments with the same antibodies. However, after incubation of the transcript in the extract to yield splicing intermediates, fragments that included a cryptic 5' splice site were detected. The results of our analyses suggest that U1 snRNPs are involved in recognizing cryptic 5' splice sites but that interactions with other splicing components are required to stabilize the association.

scholarly journals The Pre-mRNA 5′ Cap Determines Whether U6 Small Nuclear RNA Succeeds U1 Small Nuclear Ribonucleoprotein Particle at 5′ Splice Sites

1998 ◽  
Vol 18 (12) ◽  
pp. 7510-7520 ◽  
Author(s):  
Laura O’Mullane ◽  
Ian C. Eperon
Keyword(s):  
Splice Site ◽  
Major Effect ◽  
Small Nuclear Rna ◽  
Splice Sites ◽  
U6 Snrna ◽  
Small Nuclear Ribonucleoprotein ◽  
U1 Snrna ◽  
U1 Snrnp ◽  
Nuclear Rna ◽  
Nuclear Ribonucleoprotein

ABSTRACT Efficient splicing of the 5′-most intron of pre-mRNA requires a 5′ m7G(5′)ppp(5′)N cap, which has been implicated in U1 snRNP binding to 5′ splice sites. We demonstrate that the cap alters the kinetic profile of U1 snRNP binding, but its major effect is on U6 snRNA binding. With two alternative wild-type splice sites in an adenovirus pre-mRNA, the cap selectively alters U1 snRNA binding at the site to which cap-independent U1 snRNP binding is stronger and that is used predominantly in splicing; with two consensus sites, the cap acts on both, even though one is substantially preferred for splicing. However, the most striking quantitative effect of the 5′ cap is neither on U1 snRNP binding nor on the assembly of large complexes but on the replacement of U1 snRNP by U6 snRNA at the 5′ splice site. Inhibition of splicing by a cap analogue is correlated with the loss of U6 interactions at the 5′ splice site and not with any loss of U1 snRNP binding.

scholarly journals HNRNPA1 promotes recognition of splice site decoys by U2AF2 in vivo

2017 ◽  
Author(s):  
Jonathan M. Howard ◽  
Hai Lin ◽  
Garam Kim ◽  
Jolene M Draper ◽  
Maximilian Haeussler ◽  
...  
Keyword(s):  
Splice Site ◽  
Binding Sites ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Regulatory Elements ◽  
Splice Sites ◽  
Spliceosome Assembly ◽  
Over Expression

AbstractAlternative pre-mRNA splicing plays a major role in expanding the transcript output of human genes. This process is regulated, in part, by the interplay of trans-acting RNA binding proteins (RBPs) with myriad cis-regulatory elements scattered throughout pre-mRNAs. These molecular recognition events are critical for defining the protein coding sequences (exons) within pre-mRNAs and directing spliceosome assembly on non-coding regions (introns). One of the earliest events in this process is recognition of the 3’ splice site by U2 small nuclear RNA auxiliary factor 2 (U2AF2). Splicing regulators, such as the heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), influence spliceosome assembly both in vitro and in vivo, but their mechanisms of action remain poorly described on a global scale. HNRNPA1 also promotes proof reading of 3’ss sequences though a direct interaction with the U2AF heterodimer. To determine how HNRNPA1 regulates U2AF-RNA interactions in vivo, we analyzed U2AF2 RNA binding specificity using individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) in control- and HNRNPA1 over-expression cells. We observed changes in the distribution of U2AF2 crosslinking sites relative to the 3’ splice sites of alternative cassette exons but not constitutive exons upon HNRNPA1 over-expression. A subset of these events shows a concomitant increase of U2AF2 crosslinking at distal intronic regions, suggesting a shift of U2AF2 to “decoy” binding sites. Of the many non-canonical U2AF2 binding sites, Alu-derived RNA sequences represented one of the most abundant classes of HNRNPA1-dependent decoys. Splicing reporter assays demonstrated that mutation of U2AF2 decoy sites inhibited HNRNPA1-dependent exon skipping in vivo. We propose that HNRNPA1 regulates exon definition by modulating the interaction of U2AF2 with decoy or bona fide 3’ splice sites.

The natural 5' splice site of simian virus 40 large T antigen can be improved by increasing the base complementarity to U1 RNA

1987 ◽  
Vol 7 (8) ◽  
pp. 3018-3020
Author(s):  
Y Zhuang ◽  
H Leung ◽  
A M Weiner
Keyword(s):  
Splice Site ◽  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Splice Sites ◽  
Small Nuclear Ribonucleoprotein ◽  
Antigen 5

The use of alternative 5' splice sites in the simian virus 40 early-transcription unit controls the ratio of large T to small t antigen during viral infection. To study the regulation of these alternative 5' splice sites, we made two mutants which improve the match of the large-T-antigen 5' splice site to the 5' splice site consensus sequence. Whether these mutants were assayed in vitro or in vivo, we found that the efficiency of large-T splicing is increased by improving the match of the large-T-antigen 5' splice site to the consensus. We conclude that the match of a 5' splice site is an important determinant of 5' splice site utilization and that the simian virus 40 large-T-antigen 5' splice site is almost certainly recognized by the U1 small nuclear RNA component of the U1 small nuclear ribonucleoprotein particle.

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