Strict 3' splice site sequence requirements for U2 snRNP recruitment after U2AF binding underlie a genetic defect leading to autoimmune disease

Protection experiments with antibodies against small nuclear ribonucleoproteins (snRNPs) have elucidated the location of and requirements for interactions between snRNPs and human beta-globin transcripts during splicing in vitro. U2 snRNP association with the intron branch site continues after branch formation, requires intact U2 RNA, and is affected by some alterations of the 3' splice site sequence. U2 snRNP binding to the branched intermediate and U1 snRNP protection of an extended 5' splice region are detected exclusively in spliceosome fractions, indicating that both snRNPs are spliceosome components. While each snRNP associates specifically with the pre-mRNA, they also appear to interact with each other. The recovery of fragments mapping upstream of the 5' splice site suggests how the excised exon is held in the spliceosome.

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Multiple interactions between the splicing substrate and small nuclear ribonucleoproteins in spliceosomes.

Molecular and Cellular Biology ◽

10.1128/mcb.7.1.281 ◽

1987 ◽

Vol 7 (1) ◽

pp. 281-293 ◽

Cited By ~ 46

Author(s):

B Chabot ◽

J A Steitz

Keyword(s):

Splice Site ◽

Beta Globin ◽

Branch Site ◽

U2 Snrnp ◽

U1 Snrnp ◽

Branch Formation ◽

Splice Site Sequence ◽

Multiple Interactions ◽

Small Nuclear Ribonucleoproteins

Protection experiments with antibodies against small nuclear ribonucleoproteins (snRNPs) have elucidated the location of and requirements for interactions between snRNPs and human beta-globin transcripts during splicing in vitro. U2 snRNP association with the intron branch site continues after branch formation, requires intact U2 RNA, and is affected by some alterations of the 3' splice site sequence. U2 snRNP binding to the branched intermediate and U1 snRNP protection of an extended 5' splice region are detected exclusively in spliceosome fractions, indicating that both snRNPs are spliceosome components. While each snRNP associates specifically with the pre-mRNA, they also appear to interact with each other. The recovery of fragments mapping upstream of the 5' splice site suggests how the excised exon is held in the spliceosome.

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The polypyrimidine tract binding protein binds upstream of neural cell-specific c-src exon N1 to repress the splicing of the intron downstream.

Molecular and Cellular Biology ◽

10.1128/mcb.17.8.4667 ◽

1997 ◽

Vol 17 (8) ◽

pp. 4667-4676 ◽

Cited By ~ 142

Author(s):

R C Chan ◽

D L Black

Keyword(s):

Splice Site ◽

Binding Protein ◽

Cell Extract ◽

Neural Cell ◽

Splicing Regulation ◽

Polypyrimidine Tract ◽

Polypyrimidine Tract Binding ◽

Polypyrimidine Tract Binding Protein ◽

Splice Site Sequence ◽

Hela Extract

The neural cell-specific N1 exon of the c-src pre-mRNA is both negatively regulated in nonneural cells and positively regulated in neurons. We previously identified conserved intronic elements flanking N1 that direct the repression of N1 splicing in a nonneural HeLa cell extract. The upstream repressor elements are located within the polypyrimidine tract of the N1 exon 3' splice site. A short RNA containing this 3' splice site sequence can sequester trans-acting factors in the HeLa extract to allow splicing of N1. We now show that these upstream repressor elements specifically interact with the polypyrimidine tract binding protein (PTB). Mutations in the polypyrimidine tract reduce both PTB binding and the ability of the competitor RNA to derepress splicing. Moreover, purified PTB protein restores the repression of N1 splicing in an extract derepressed by a competitor RNA. In this system, the PTB protein is acting across the N1 exon to regulate the splicing of N1 to the downstream exon 4. This mechanism is in contrast to other cases of splicing regulation by PTB, in which the protein represses the splice site to which it binds.

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Vitiligo pathogenesis: autoimmune disease, genetic defect, excessive reactive oxygen species, calcium imbalance, or what else?

Experimental Dermatology ◽

10.1111/j.1600-0625.2007.00666_1.x ◽

2008 ◽

Vol 17 (2) ◽

pp. 139-140 ◽

Cited By ~ 48

Author(s):

Lübeck Ralf Paus ◽

K. U. Schallreuter ◽

P. Bahadoran ◽

M. Picardo ◽

A. Slominski ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Autoimmune Disease ◽

Genetic Defect ◽

Reactive Oxygen ◽

Oxygen Species

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Multiple Roles for SR Proteins in trans Splicing

Molecular and Cellular Biology ◽

10.1128/mcb.22.15.5337-5346.2002 ◽

2002 ◽

Vol 22 (15) ◽

pp. 5337-5346 ◽

Cited By ~ 15

Author(s):

Suzanne Furuyama ◽

James P. Bruzik

Keyword(s):

Splice Site ◽

Rna Binding ◽

Sr Proteins ◽

Multiple Roles ◽

Separate Experiment ◽

Splice Sites ◽

U2 Snrnp ◽

In Trans ◽

Two Stages

ABSTRACT The trans-splicing reaction involves the association of 5′; and 3′; splice sites contained on separate transcripts. The mechanism by which these splice sites are juxtaposed during trans-spliceosome assembly and the role of SR proteins at each stage in this process have not been determined. Utilizing a system that allows for the separation of the RNA binding and RS domains of SR proteins, we have found that SR proteins are required for at least two stages of the trans-splicing reaction. They are important both prior to and subsequent to the addition of U2 snRNP to the 3′; acceptor. In addition, we have demonstrated a role for RS domain phosphorylation in both of these activities. Dephosphorylation of the RS domain led to a block in U2 snRNP binding to the substrate. In a separate experiment, RS domain phosphorylation was also determined to be necessary for trans splicing to proceed on a substrate that had U2 snRNP already bound. This newly identified role for phosphorylated SR proteins post-U2-snRNP addition coincides with the recruitment of the 5′; splice site contained on the SL RNP, suggesting a role for SR proteins in splice site communication in trans splicing.

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Outcome of donor splice site mutations accounting for congenital afibrinogenemia reflects order of intron removal in the fibrinogen alpha gene (FGA)

Blood ◽

10.1182/blood-2002-03-0853 ◽

2003 ◽

Vol 101 (5) ◽

pp. 1851-1856 ◽

Cited By ~ 30

Author(s):

Catia Attanasio ◽

Armelle David ◽

Marguerite Neerman-Arbez

Keyword(s):

Splice Site ◽

Genetic Defect ◽

Autosomal Recessive Disorder ◽

Mendelian Inheritance ◽

Donor Splice ◽

Intron 1 ◽

Congenital Afibrinogenemia ◽

Alpha Gene ◽

Splice Mutations ◽

Intron 4

Congenital afibrinogenemia (Mendelian Inheritance in Man #202400) is a rare, autosomal recessive disorder characterized by the complete absence of circulating fibrinogen. Our recent studies on the molecular basis of the disease showed that the most common genetic defect is a donor splice mutation in fibrinogen alpha gene (FGA)intron 4, IVS4+1G>T. Two other FGA donor splice mutations, in intron 1 (IVS1+3A>G) and intron 3 (IVS3+1_+4delGTAA), were identified in afibrinogenemia patients. Because it was impossible to directly study the effect of these mutations on mRNA splicing in patient hepatocytes, we used a transfected cell approach, which previously allowed us to show that the common IVS4 mutation causes afibrinogenemia due to the activation of multiple cryptic donor splice sites. In this study, analysis of the IVS3delGTAA mutation showed exon 3 skipping in 99% of transcripts and exons 2 and 3 skipping in 1% of transcripts. The different outcomes of these donor splice mutations appear to follow the model proposed in a study of fibrillar collagen genes, where donor splice mutations occurring in a rapidly spliced intron with respect to upstream introns lead in most cases to exon skipping, while mutations in later-spliced introns lead to intron inclusion or cryptic splice-site utilization. Indeed, we found that inFGA intron 3 was preferentially spliced first, followed by intron 2, intron 4, and intron 1.

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Multiple Splicing Defects in an Intronic False Exon

Molecular and Cellular Biology ◽

10.1128/mcb.20.17.6414-6425.2000 ◽

2000 ◽

Vol 20 (17) ◽

pp. 6414-6425 ◽

Cited By ~ 113

Author(s):

Hanzhen Sun ◽

Lawrence A. Chasin

Keyword(s):

Splice Site ◽

Negative Influence ◽

Splice Sites ◽

Polypyrimidine Tract ◽

Exon Definition ◽

Exon 2 ◽

Vast Number ◽

Consensus Sequences ◽

Splice Site Sequence ◽

Necessary And Sufficient

ABSTRACT Splice site consensus sequences alone are insufficient to dictate the recognition of real constitutive splice sites within the typically large transcripts of higher eukaryotes, and large numbers of pseudoexons flanked by pseudosplice sites with good matches to the consensus sequences can be easily designated. In an attempt to identify elements that prevent pseudoexon splicing, we have systematically altered known splicing signals, as well as immediately adjacent flanking sequences, of an arbitrarily chosen pseudoexon from intron 1 of the human hprt gene. The substitution of a 5′ splice site that perfectly matches the 5′ consensus combined with mutation to match the CAG/G sequence of the 3′ consensus failed to get this model pseudoexon included as the central exon in a dhfr minigene context. Provision of a real 3′ splice site and a consensus 5′ splice site and removal of an upstream inhibitory sequence were necessary and sufficient to confer splicing on the pseudoexon. This activated context also supported the splicing of a second pseudoexon sequence containing no apparent enhancer. Thus, both the 5′ splice site sequence and the polypyrimidine tract of the pseudoexon are defective despite their good agreement with the consensus. On the other hand, the pseudoexon body did not exert a negative influence on splicing. The introduction into the pseudoexon of a sequence selected for binding to ASF/SF2 or its replacement with β-globin exon 2 only partially reversed the effect of the upstream negative element and the defective polypyrimidine tract. These results support the idea that exon-bridging enhancers are not a prerequisite for constitutive exon definition and suggest that intrinsically defective splice sites and negative elements play important roles in distinguishing the real splicing signal from the vast number of false splicing signals.

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

Molecular and Cellular Biology ◽

10.1128/mcb.14.5.2994-3005.1994 ◽

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.

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Sequence requirements in different steps of the pre-mRNA splicing reaction: analysis by the RNA modification-exclusion technique

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4942-4947.1990 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4942-4947

Author(s):

K M Lang ◽

W Keller

Keyword(s):

Complex Formation ◽

Splice Site ◽

Point Mutations ◽

Mrna Splicing ◽

Rna Modification ◽

Modified Nucleotides ◽

Complete Set ◽

Stepwise Assembly ◽

Sequence Requirements ◽

Single Reaction

The stepwise assembly of splicing complexes and the subsequent splicing reaction were analyzed by the RNA modification-exclusion technique, which generates the equivalent of a complete set of point mutations in a single reaction. We found that although the sequences surrounding the 5' splice site, the branch point, and the 3' splice site, including the 3' AG, were required for presplicing complex formation, modified nucleotides at these positions were not completely excluded. The same sequences were required for splicing complex formation; however, modified nucleotides in these sequences were excluded to a much greater extent.

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Nuclear proteins that bind the pre-mRNA 3' splice site sequence r(UUAG/G) and the human telomeric DNA sequence d(TTAGGG)n

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.4301-4310.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 4301-4310 ◽

Cited By ~ 3

Author(s):

F Ishikawa ◽

M J Matunis ◽

G Dreyfuss ◽

T R Cech

Keyword(s):

Splice Site ◽

Peptide Sequencing ◽

Nuclear Proteins ◽

Beta Globin ◽

Telomeric Dna ◽

Two Dimensional Gel Electrophoresis ◽

Mrna Metabolism ◽

Dna Repeat ◽

Splice Site Sequence ◽

Heterogeneous Nuclear Ribonucleoproteins

HeLa cell nuclear proteins that bind to single-stranded d(TTAGGG)n, the human telomeric DNA repeat, were identified and purified by a gel retardation assay. Immunological data and peptide sequencing experiments indicated that the purified proteins were identical or closely related to the heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, A2-B1, D, and E and to nucleolin. These proteins bound to RNA oligonucleotides having r(UUAGGG) repeats more tightly than to DNA of the same sequence. The binding was sequence specific, as point mutation of any of the first 4 bases [r(UUAG)] abolished it. The fraction containing D and E hnRNPs was shown to bind specifically to a synthetic oligoribonucleotide having the 3' splice site sequence of the human beta-globin intervening sequence 1, which includes the sequence UUAGG. Proteins in this fraction were further identified by two-dimensional gel electrophoresis as D01, D02, D1*, and E0; intriguingly, these members of the hnRNP D and E groups are nuclear proteins that are not stably associated with hnRNP complexes. These studies establish the binding specificities of these D and E hnRNPs. Furthermore, they suggest the possibility that these hnRNPs could perhaps bind to chromosome telomeres, in addition to having a role in pre-mRNA metabolism.

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