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At least 235 records · Page 13Linked to original sources
PubMed1995

Splicing of two weak 3' splice sites from the adenovirus major late region is enhanced in nuclear extracts from adenovirus infected cells.

The adenovirus major late transcription unit (MLTU) is an example of a complex alternatively spliced gene, in which more than 15 different 3' splice sites can be joined to a common 5' splice site. Maturation of the full repertoire of possible mRNAs requires late viral protein synthesis and occurs only at late stages of the infectious cycle (16-24 hpi). We are trying to decipher the mechanisms regulating alternative 3' splice site choice during the infectious cycle. Therefore, we examined the splicing activity of several 3' splice sites from the MLTU in vitro in nuclear extracts prepared from adenovirus infected cells (Ad NE) and from uninfected cells. The results suggest that pre-mRNAs with "weak" 3' splice sites (short, atypical polypyrimidine tracts) are activated and pre-mRNAs with long, prototypical polypyrimidine tracts are repressed in Ad NE. In fact, our data show a reciprocal correlation between the strength of a polypyrimidine tract, defined by its affinity for U2AF65K in vitro, and the efficiency of splicing in Ad NE. We are currently investigating the possible mechanisms responsible for this observed shift in 3' splice site choice during an adenovirus infection.

Adenoviridae Infections↗
PubMed1998

SPLICE SITE SELECTION IN PLANT PRE-mRNA SPLICING.

The purpose of this review is to highlight the unique and common features of splice site selection in plants compared with the better understood yeast and vertebrate systems. A key question in plant splicing is the role of AU sequences and how and at what stage they are involved in spliceosome assembly. Clearly, intronic U- or AU-rich and exonic GC- and AG-rich elements can influence splice site selection and splicing efficiency and are likely to bind proteins. It is becoming clear that splicing of a particular intron depends on a fine balance in the "strength" of the multiple intron signals involved in splice site selection. Individual introns contain varying strengths of signals and what is critical to splicing of one intron may be of less importance to the splicing of another. Thus, small changes to signals may severely disrupt splicing or have little or no effect depending on the overall sequence context of a specific intron/exon organization.

Journal Article↗
PubMed2000

Three novel types of splicing aberrations in the tuberous sclerosis TSC2 gene caused by mutations apart from splice consensus sequences.

Disease causing aberrations in both tuberous sclerosis predisposing genes, TSC1 and TSC2, comprise nearly every type of alteration with a predominance of small truncating mutations distributed over both genes. We performed an RNA based screening of the entire coding regions of both TSC genes applying the protein truncation test (PTT) and identified a high proportion of unusual splicing abnormalities affecting the TSC2 gene. Two cases exhibited different splice acceptor mutations in intron 9 (IVS9-15G-->A and IVS9-3C-->G) both accompanied by exon 10 skipping and simultaneous usage of a cryptic splice acceptor in exon 10. Another splice acceptor mutation (IVS38-18A-->G) destroyed the putative polypyrimidine structure in intron 38 and resulted in simultaneous intron retention and usage of a downstream cryptic splice acceptor in exon 39. Another patient bore a C-->T transition in intron 8 (IVS8+281C-->T) activating a splice donor site and resulting in the inclusion of a newly recognised exon in the mRNA followed by a premature stop. These splice variants deduced from experimental results are additionally supported by RNA secondary structure analysis based on free energy minimisation. Three of the reported splicing anomalies are due to sequence changes remote from exon/intron boundaries, described for the first time in TSC. These findings highlight the significance of investigating intronic changes and their consequences on the mRNA level as disease causing mutations in TSC.

Base Sequence↗
PubMed1992

The 35-kDa mammalian splicing factor SC35 mediates specific interactions between U1 and U2 small nuclear ribonucleoprotein particles at the 3' splice site.

The splicing factor SC35 is required for the first step of the splicing reaction and for the assembly of the earliest ATP-dependent complex detected by native gel electrophoresis (A complex). Here we investigate the role of SC35 in mediating specific interactions between U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) and the 5' and 3' splice sites of pre-mRNA. We show that U1 snRNP interacts specifically with both the 5' and 3' splice sites in the presence of ATP and that SC35 is required for these ATP-dependent interactions. Significantly, the SC35-dependent interaction between U1 snRNP and the 3' splice site requires U2 snRNP but not the 5' splice site. We also show that SC35 is required for the ATP-dependent interaction between U2 snRNP and the branch-point sequence. We conclude that SC35 may play an important role in mediating specific interactions between splicing components bound to the 5' and 3' splice sites.

Adenosine Triphosphate↗
PubMed1998

A pyrimidine-rich exonic splicing suppressor binds multiple RNA splicing factors and inhibits spliceosome assembly.

The bovine papillomavirus type 1 (BPV-1) exonic splicing suppressor (ESS) is juxtaposed immediately downstream of BPV-1 splicing enhancer 1 and negatively modulates selection of a suboptimal 3' splice site at nucleotide 3225. The present study demonstrates that this pyrimidine-rich ESS inhibits utilization of upstream 3' splice sites by blocking early steps in spliceosome assembly. Analysis of the proteins that bind to the ESS showed that the U-rich 5' region binds U2AF65 and polypyrimidine tract binding protein, the C-rich central part binds 35- and 54-55-kDa serine/arginine-rich (SR) proteins, and the AG-rich 3' end binds alternative splicing factor/splicing factor 2. Mutational and functional studies indicated that the most critical region of the ESS maps to the central C-rich core (GGCUCCCCC). This core sequence, along with additional nonspecific downstream nucleotides, is sufficient for partial suppression of spliceosome assembly and splicing of BPV-1 pre-mRNAs. The inhibition of splicing by the ESS can be partially relieved by excess purified HeLa SR proteins, suggesting that the ESS suppresses pre-mRNA splicing by interfering with normal bridging and recruitment activities of SR proteins.

Alternative Splicing↗
PubMed1994

Disruption of pre-mRNA splicing in vivo results in reorganization of splicing factors.

We have examined the functional significance of the organization of pre-mRNA splicing factors in a speckled distribution in the mammalian cell nucleus. Upon microinjection into living cells of oligonucleotides or antibodies that inhibit pre-mRNA splicing in vitro, we observed major changes in the organization of splicing factors in vivo. Interchromatin granule clusters became uniform in shape, decreased in number, and increased in both size and content of splicing factors, as measured by immunofluorescence. These changes were transient and the organization of splicing factors returned to their normal distribution by 24 h following microinjection. Microinjection of these oligonucleotides or antibodies also resulted in a reduction of transcription in vivo, but the oligonucleotides did not inhibit transcription in vitro. Control oligonucleotides did not disrupt splicing or transcription in vivo. We propose that the reorganization of splicing factors we observed is the result of the inhibition of splicing in vivo.

Autoradiography↗
PubMed1999

The splicing factor-associated protein, p32, regulates RNA splicing by inhibiting ASF/SF2 RNA binding and phosphorylation.

The cellular protein p32 was isolated originally as a protein tightly associated with the essential splicing factor ASF/SF2 during its purification from HeLa cells. ASF/SF2 is a member of the SR family of splicing factors, which stimulate constitutive splicing and regulate alternative RNA splicing in a positive or negative fashion, depending on where on the pre-mRNA they bind. Here we present evidence that p32 interacts with ASF/SF2 and SRp30c, another member of the SR protein family. We further show that p32 inhibits ASF/SF2 function as both a splicing enhancer and splicing repressor protein by preventing stable ASF/SF2 interaction with RNA, but p32 does not block SRp30c function. ASF/SF2 is highly phosphorylated in vivo, a modification required for stable RNA binding and protein-protein interaction during spliceosome formation, and this phosphorylation, either through HeLa nuclear extracts or through specific SR protein kinases, is inhibited by p32. Our results suggest that p32 functions as an ASF/SF2 inhibitory factor, regulating ASF/SF2 RNA binding and phosphorylation. These findings place p32 into a new group of proteins that control RNA splicing by sequestering an essential RNA splicing factor into an inhibitory complex.

Carrier Proteins↗
PubMed2000

Interaction of the yeast DExH-box RNA helicase prp22p with the 3' splice site during the second step of nuclear pre-mRNA splicing.

Using site-specific incorporation of the photo-chemical cross-linking reagent 4-thiouridine, we demonstrate the previously unknown association of two proteins with yeast 3' splice sites. One of these is an unidentified approximately 122 kDa protein that cross-links to 3' splice sites during formation of the pre--spliceosome. The other factor is the DExH-box RNA helicase, Prp22p. With substrates functional in the second step of splicing, only very weak cross-linking of Prp22p to intron sequences at the 3' splice site is observed. In contrast, substrates blocked at the second step exhibit strong cross-linking of Prp22 to intron sequences at the 3' splice site, but not to adjacent exon sequences. In vitro reconstitution experiments also show that the association of Prp22p with intron sequences at the 3' splice site is dependent on Prp16p and does not persist when release of mature mRNA from the spliceosome is blocked. Taken together, these results suggest that the 3' splice site of yeast introns is contacted much earlier than previously envisioned by a protein of approximately 120 kDa, and that a transient association of Prp22p with the 3' splice site occurs between the first and second catalytic steps.

Actins↗
PubMed1996

Factors involved in the activation of pre-mRNA splicing from downstream splicing enhancers.

The excision of introns with weak polypyrimidine tracts at their 3' splice sites can be enhanced by sequence elements in the downstream exon or by a downstream 5' splice site. The enhancers inside the exon do not conform to a strict consensus, but they are generally rich in purines. Here, we show that members of the family of SR proteins recognize these elements. Not only does SF2/ASF activate many different polypurine enhancers, but also at least one other SR protein, most likely SC35, is active as well. The degree of splicing activation varies with the polypurine enhancers and the SR proteins. Further, we show that the similar activation by downstream 5' splice sites requires U1 snRNP, which is not the case with purine-rich enhancers. These results are consistent with a model showing that U1 snRNP binds to the 5' splice site and SR proteins to exonic sequences upstream of the 5' splice site. Both interact with U2AF at the 3' splice site. This represents a molecular explanation for the exon recognition which is important for splice site selection in mammals.

Alpharetrovirus↗
PubMed1992

U1 snRNP targets an essential splicing factor, U2AF65, to the 3' splice site by a network of interactions spanning the exon.

A description of cellular factors that govern alternative splicing of pre-mRNA is largely incomplete. In the case of the rat preprotachykinin gene, splicing of the alternative exon E4 occurs by a poorly understood mechanism in which exon selection is under the positive control of U1 snRNP. Because the binding of U1 snRNP to the 5' splice site of E4 is coincident with the selection of the 3' splice site of E4, this mechanism would appear to involve interactions that bridge across the exon. In this work, a UV cross-linking strategy was used to identify possible RNA-protein interactions involved in the proposed exon-bridging model. Of particular interest is a prominent 61-kD protein, p61, that binds to the 3' splice site of E4 in a manner that is clearly facilitated by a downstream 5' splice site and U1 snRNP particles. The identity of p61 is the essential splicing factor U2AF65, on the basis of copurification and selective binding to polypyrimidine tracts. These results indicate a model in which exon selection is positively regulated by the communication of U1 snRNP and U2AF65. That is, a natural deficiency in binding U2AF65 to the 3' splice site that leads to exon skipping might be overcome by a mechanism in which U1 snRNP facilitates the binding of U2AF65 through a network of template-directed and exon-bridging interactions.

Base Sequence↗
PubMed1997

Pyrimidine tracts between the 5' splice site and branch point facilitate splicing and recognition of a small Drosophila intron.

The minimum size for splicing of a vertebrate intron is approximately 70 nucleotides. In Drosophila melanogaster, more than half of the introns are significantly below this minimum yet function well. Such short introns often lack the pyrimidine tract located between the branch point and 3' splice site common to metazoan introns. To investigate if small introns contain special sequences that facilitate their recognition, the sequences and factors required for the splicing of a 59-nucleotide intron from the D. melanogaster mle gene have been examined. This intron contains only a minimal region of interrupted pyrimidines downstream of the branch point. Instead, two longer, uninterrupted C-rich tracts are located between the 5' splice site and branch point. Both of these sequences are required for maximal in vivo and in vitro splicing. The upstream sequences are also required for maximal binding of factors to the 5' splice site, cross-linking of U2AF to precursor RNA, and assembly of the active spliceosome, suggesting that sequences upstream of the branch point influence events at both ends of the small mle intron. Thus, a very short intron lacking a classical pyrimidine tract between the branch point and 3' splice site requires accessory pyrimidine sequences in the short region between the 5' splice site and branch point.

Animals↗
PubMed2001

Distinctive features of Drosophila alternative splicing factor RS domain: implication for specific phosphorylation, shuttling, and splicing activation.

The human splicing factor 2, also called human alternative splicing factor (hASF), is the prototype of the highly conserved SR protein family involved in constitutive and regulated splicing of metazoan mRNA precursors. Here we report that the Drosophila homologue of hASF (dASF) lacks eight repeating arginine-serine dipeptides at its carboxyl-terminal region (RS domain), previously shown to be important for both localization and splicing activity of hASF. While this difference has no effect on dASF localization, it impedes its capacity to shuttle between the nucleus and cytoplasm and abolishes its phosphorylation by SR protein kinase 1 (SRPK1). dASF also has an altered splicing activity. While being competent for the regulation of 5' alternative splice site choice and activation of specific splicing enhancers, dASF fails to complement S100-cytoplasmic splicing-deficient extracts. Moreover, targeted overexpression of dASF in transgenic flies leads to higher deleterious developmental defects than hASF overexpression, supporting the notion that the distinctive structural features at the RS domain between the two proteins are likely to be functionally relevant in vivo.

Alternative Splicing↗
PubMed2005

Identification of splicing silencers and enhancers in sense Alus: a role for pseudoacceptors in splice site repression.

Auxiliary splicing signals in introns play an important role in splice site selection, but these elements are poorly understood. We show that a subset of serine/arginine (SR)-rich proteins activate a cryptic 3' splice site in a sense Alu repeat located in intron 4 of the human LST1 gene. Utilization of this cryptic splice site is controlled by juxtaposed Alu-derived splicing silencers and enhancers between closely linked short tandem repeats TNFd and TNFe. Systematic mutagenesis of these elements showed that AG dinucleotides that were not preceded by purine residues were critical for repressing exon inclusion of a chimeric splicing reporter. Since the splice acceptor-like sequences are present in excess in exonic splicing silencers, these signals may contribute to inhibition of a large number of pseudosites in primate genomes.

Alternative Splicing↗
PubMed2004

Principles of 3' splice site selection and alternative splicing for an unusual group II intron from Bacillus anthracis.

We investigated the self-splicing properties of two introns from the bacterium Bacillus anthracis. One intron (B.a.I1) splices poorly in vitro despite having typical structural motifs, while the second (B.a.I2) splices well while having apparently degenerated features. The spliced exons of B.a.I2 were sequenced, and splicing was found to occur at a 3' site shifted one nucleotide from the expected position, thus restoring missing gamma-gamma' and IBS3-EBS3 pairings, but leaving the two conserved exonic ORFs out of frame. Because of the unexpected splice site, the principles for 3' intron definition were examined, which showed that the 3' splice site is flexible but contingent on gamma-gamma' and IBS3-EBS3 pairings, and can be as far away as four nucleotides from the wild-type site. Surprisingly, alternative splicing occurs at position +4 for wild-type B.a.I2 intron, both in vitro and in vivo, and the alternative event fuses the two conserved exon ORFs, presumably leading to translation of the downstream ORF. The finding suggests that the structural irregularities of B.a.I2 may be an adaptation to facilitate gene expression in vivo.

Alternative Splicing↗
PubMed2005

Two novel arginine/serine (SR) proteins in maize are differentially spliced and utilize non-canonical splice sites.

The serine-arginine (SR)-rich splicing proteins are highly conserved RNA binding nuclear phosphor-proteins that play important roles in both regular and alternative splicing. Here we describe two novel putative SR genes from maize, designated zmRSp31A and zmRSp31B. Both genes contain characteristic RNA binding motifs RNP-1 and RNP-2, a serine/arginine-rich (RS) domain and share significant sequence similarity to the Arabidopsis atRSp31 family of SR proteins. Both zmRSp31A and zmRSp31B produce multiple transcripts by alternative splicing, of which majority of the alternatively spliced transcripts utilize non-canonical splice sites. zmRSp31A and zmRSp31B produce at least six and four transcripts, respectively, of which only one corresponds to the wild type proteins for each gene. All the alternatively spliced transcripts of both the genes, with one exception, are predicted to encode small truncated proteins containing only the RNP-2 domain of their first RNA recognition motif and completely lack the carboxyl terminal RS domain. We provide evidence that some of the alternatively spliced transcripts of both genes are associated with polysomes and interact with the translational machinery.

Alternative Splicing↗
PubMed1999

Point mutations in the dystrophin gene: evidence for frequent use of cryptic splice sites as a result of splicing defects.

Ten different mutations have been identified in patients with Becker (n = 1) or Duchenne (n = 9) muscular dystrophy using reverse transcription of total RNA, polymerase chain reaction amplification of the whole coding region of the gene and protein truncation test (PTT) analysis. Seven mutations had not been reported previously, and these consist in three nonsense mutations (Q2522X, E2726X, R3381X), three frameshifting deletions (3686-3687delGT, 5126delA, 5759delC), and four splicing defects of which the effects on the muscle dystrophin mRNA transcripts have been analyzed. In one case, a 3' splice-site mutation (IVS74-2A-->G) resulted in a complex pattern of exon skipping involving exons of the C-terminal domain. In the three other cases, nucleotide substitutions in splice donor (IVS26+2T-->A, IVS65+1G-->A) or acceptor (IVS8-15A-->G) recognition sequences led to the use of cryptic splice sites, with consequent insertions of intronic sequences in the processed mRNA. Up to 34% (70/203) of the point mutations reported to date in the dystrophin database (http://www.dmd.nl) affect splice sites of the dystrophin gene. However, altered mRNA splicing has been confirmed experimentally in only 23% of cases (16/70). Combined with PTT, the transcript analysis protocol defined in this study permits direct determination of the impact of intronic variations on the structure of dystrophin mRNA and of the resulting consequences on the translational reading frame. We present evidence for a frequent use of cryptic splice sites as a result of splicing defects.

Base Sequence↗
PubMed1995

cis-splicing and polyadenylation of actin RNA can precede 5' trans-splicing in nematodes.

In nematodes many precursor RNAs undergo both cis-splicing (intron removal) and trans-splicing (spliced leader addition) via molecular mechanisms thought to be very similar. We examined the temporal order of trans- and cis-splicing by analyzing cDNAs of partially processed transcripts from an actin gene of the parasitic nematode, Onchocerca volvulus, which has five short introns. We found that in this model gene system trans-splicing can follow cis-splicing and polyadenylation, and we obtained no evidence for alternative pathways of splicing. This result is in contrast to the tubulin genes of African trypanosomes where trans-splicing precedes polyadenylation (1).

Actins↗