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At least 37 records · Page 2Linked to original sources
PubMed2003

SC35 and heterogeneous nuclear ribonucleoprotein A/B proteins bind to a juxtaposed exonic splicing enhancer/exonic splicing silencer element to regulate HIV-1 tat exon 2 splicing.

Splicing of the human immunodeficiency virus, type 1, primary transcript is highly regulated. Maintaining the proper equilibrium among spliced, unspliced, and partially spliced isoforms is essential for the replication of the virus. Here we characterize a complex cis-acting element located in tat exon 2 that is required for the splicing regulation of the upstream intron. An exonic splicing enhancer (ESE) and an exonic splicing silencer (ESS) are both located within the regulatory element. Heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins bind the ESS to repress splicing, whereas the SR protein SC35 binds the ESE to activate it. We show that the SC35 and the hnRNP A1 binding sites overlap within the juxtaposed ESE/ESS. We propose that hnRNP A1 binding to the ESS inhibits splicing of the upstream intron by directly masking the SC35 binding site.

Base Sequence↗
PubMed1993

General splicing factor SF2/ASF promotes alternative splicing by binding to an exonic splicing enhancer.

The general splicing factor SF2/ASF binds in a sequence-specific manner to a purine-rich exonic splicing enhancer (ESE) in the last exon of bovine growth hormone (bGH) pre-mRNA. More importantly, SF2/ASF stimulates in vitro splicing of bGH intron D through specific interaction with the ESE sequences. However, another general splicing factor, SC35, does not bind the ESE sequences and has no effect on bGH intron D splicing. Thus, one possible function of SF2/ASF in alternative and, perhaps, constitutive pre-mRNA splicing is to recognize ESE sequences. The stimulation of bGH intron D splicing by SF2/ASF is counteracted by the addition of hnRNP A1. The relative levels of SF2/ASF and hnRNP A1 influence the efficiency of bGH intron D splicing in vitro and may be the underlying mechanism of this alternative pre-mRNA processing event in vivo.

Alternative Splicing↗
PubMed2005

Molecular recognition in a trans excision-splicing ribozyme: non-Watson-Crick base pairs at the 5' splice site and omegaG at the 3' splice site can play a role in determining the binding register of reaction substrates.

Trans excision-splicing (TES) ribozymes, derived from a Pneumocystis carinii group I intron, can catalyze the excision of targeted sequences from within RNAs. In this report, the sequence requirements of the splice sites are analyzed. These conserved sequences include a u-G wobble pair at the 5' splice site and a guanosine in the omega position at the 3' splice site (in the substrate). We report that 7 out of 16 base pair combinations at the 5' splice site produce appreciable TES product. This promiscuity is in contrast to results reported for analogous self-splicing reactions using a Tetrahymena ribozyme. At long reaction times TES products dissociate and rebind free ribozyme, at which point product degradation occurs via the 5' cleavage reaction. Unexpectedly, only in cases where Watson-Crick base pairs form at the 5'splice site do we see degradation of TES products at cryptic sites, suggesting that non-Watson-Crick base pairs at the 5' splice site are acting in concert with other factors to precisely determine the binding register of TES reaction substrates within the ribozyme. Moreover, cryptic site degradation does not occur with the corresponding reaction substrates, which additionally contain omegaG, suggesting that omegaG can play a similar role. We report that omegaG cannot be replaced by any other base, so TES substrates require a guanosine as the last (or only) base to be excised. Additionally, we demonstrate that P9.0 and P10 are expendable for TES reactions, suggesting that omegaG is sufficient as a 3' molecular recognition element.

Base Pairing↗
PubMed1991

Intramolecular base pairing between the nematode spliced leader and its 5' splice site is not essential for trans-splicing in vitro.

The spliced leader RNAs of both trypanosomes and nematodes can form similar secondary structures where the trans-splice donor site is involved in intramolecular base pairing with the spliced leader sequence. It has been proposed that this base pairing could serve to activate autonomously the SL RNA splice donor site. Here, we have examined exon requirements for trans-splicing in a nematode cell free system. Complete disruption of secondary structure interactions at and around the trans-splice donor site did not affect the ability of the SL RNA to function in trans-splicing. In addition, the highly conserved 22 nt sequence could be productively replaced by artificial exons ranging in size from 2 to 246 nucleotides. These results reinforce the view that the 'intron' portion of the SL RNA functions as an independent Sm snRNP whose role is to deliver exon sequences to the trans-spliceosome.

Animals↗
PubMed2000

An intronic splicing enhancer binds U1 snRNPs to enhance splicing and select 5' splice sites.

Intronic G triplets are frequently located adjacent to 5' splice sites in vertebrate pre-mRNAs and have been correlated with splicing efficiency and specificity via a mechanism that activates upstream 5' splice sites in exons containing duplicated sites (26). Using an intron dependent upon G triplets for maximal activity and 5' splice site specificity, we determined that these elements bind U1 snRNPs via base pairing with U1 RNA. This interaction is novel in that it uses nucleotides 8 to 10 of U1 RNA and is independent of nucleotides 1 to 7. In vivo functionality of base pairing was documented by restoring activity and specificity to mutated G triplets through compensating U1 RNA mutations. We suggest that the G-rich region near vertebrate 5' splice sites promotes accurate splice site recognition by recruiting the U1 snRNP.

Base Pairing↗
PubMed1990

Splicing factor SF4 is dispensable for the assembly of a functional splicing complex and participates in the subsequent steps of the splicing reaction.

The splicing of nuclear messenger RNA precursors (pre-mRNA) can be reconstituted in vitro with factors partially purified from HeLa cell nuclear extracts. Splicing complexes are assembled in the presence of the small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U4, U5 and U6 and the protein factors SF1, SF2, SF3 and U2AF. However, the complexes thus formed are inactive, i.e. they only contain unprocessed pre-mRNA. The intermediates and products of the splicing reaction are generated after addition of SF4. This splicing factor is a heat-labile protein which requires sulfhydryl groups for its activity. SF4 appears to participate, directly or indirectly, in the conversion of a functional but inactive splicing complex to the active spliceosome.

Cell Nucleus↗
PubMed2003

Binding of a candidate splice regulator to a calcitonin-specific splice enhancer regulates calcitonin/CGRP pre-mRNA splicing.

The calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA is alternatively processed in a tissue-specific manner leading to the production of calcitonin mRNA in thyroid C cells and CGRP mRNA in neurons. A candidate calcitonin/CGRP splice regulator (CSR) isolated from rat brain was shown to inhibit calcitonin-specific splicing in vitro. CSR specifically binds to two regions in the calcitonin-specific exon 4 RNA previously demonstrated to function as a bipartate exonic splice enhancer (ESE). The two regions, A and B element, are necessary for inclusion of exon 4 into calcitonin mRNA. A novel RNA footprinting method based on the UV cross-linking assay was used to define the site of interaction between CSR and B element RNA. Base changes at the CSR binding site prevented CSR binding to B element RNA and CSR was unable to inhibit in vitro splicing of pre-mRNAs containing the mutated CSR binding site. When expressed in cells that normally produce predominantly CGRP mRNA, a calcitonin/CGRP gene containing the mutated CSR binding site expressed predominantly calcitonin mRNA. These observations demonstrate that CSR binding to the calcitonin-specific ESE regulates calcitonin/CGRP pre-mRNA splicing.

Animals↗
PubMedDate not supplied

Cleavage of 5' splice site and lariat formation are independent of 3' splice site in yeast mRNA splicing.

Analysis of messenger RNA splicing in yeast and in metazoa has led to the identification of an RNA molecule in a lariat conformation. This structure has been found as an mRNA splicing intermediate in vitro and identical molecules have been identified in vivo. Lariat formation involves cleavage of the precursor at the 5' splice site (5' SS) and the formation of a 2'-5' phosphodiester bond between the guanosine residue at the 5' end of the intron and an adenosine within the intron. The yeast branchpoint is located within the absolutely conserved TACTAAC box (that is, the last A of the TACTAAC box is the site of formation of the 2'-5' phosphodiester bond with the 5' end of the intron)3,4. Moreover, efficient 5' SS cleavage and lariat formation require proper sequences at the 5' splice junction and within the TACTAAC box. Here we demonstrate that 5' SS cleavage and lariat formation take place in vitro in the absence of the 3' SS and much of the 3' junction. These results are discussed in light of possible differences between yeast and metazoan mRNA splicing.

Base Sequence↗
PubMed1999

Redefinition of exon 7 in the COL1A1 gene of type I collagen by an intron 8 splice-donor-site mutation in a form of osteogenesis imperfecta: influence of intron splice order on outcome of splice-site mutation.

Most splice-site mutations lead to a limited array of products, including exon skipping, use of cryptic splice-acceptor or -donor sites, and intron inclusion. At the intron 8 splice-donor site of the COL1A1 gene, we identified a G+1-->A transition that resulted in the production of several splice products from the mutant allele. These included one in which the upstream exon 7 was extended by 96 nt, others in which either intron 8 or introns 7 and 8 were retained, one in which exon 8 was skipped, and one that used a cryptic donor site in exon 8. To determine the mechanism by which exon-7 redefinition might occur, we examined the order of intron removal in the region of the mutation by using intron/exon primer pairs to amplify regions of the precursor nuclear mRNA between exon 5 and exon 10. Removal of introns 5, 6, and 9 was rapid. Removal of intron 8 usually preceded removal of intron 7 in the normal gene, although, in a small proportion of copies, the order was reversed. The proportion of abnormal products suggested that exon 7 redefinition, intron 7 plus intron 8 inclusion, and exon 8 skipping all represented products of the impaired rapid pathway, whereas the intron-8 inclusion product resulted from use of the slow intron 7-first pathway. The very low-abundance cryptic exon 8 donor site product could have arisen from either pathway. These results suggest that there is commitment of the pre-mRNA to the two pathways, independent of the presence of the mutation, and that the order and rate of intron removal are important determinants of the outcome of splice-site mutations and may explain some unusual alterations.

Amino Acid Sequence↗
PubMed2002

The human XPC DNA repair gene: arrangement, splice site information content and influence of a single nucleotide polymorphism in a splice acceptor site on alternative splicing and function.

XPC DNA repair gene mutations result in the cancer-prone disorder xeroderma pigmentosum. The XPC gene spans 33 kb and has 16 exons (82-882 bp) and 15 introns (0.08-5.4 kb). A 1.6 kb intron was found within exon 5. Sensitive real- time quantitative reverse transcription-polymerase chain reaction methods were developed to measure full-length XPC mRNA (the predominant form) and isoforms that skipped exons 4, 7 or 12. Exon 7 was skipped in approximately 0.07% of XPC mRNAs, consistent with the high information content of the exon 7 splice acceptor and donor sites (12.3 and 10.4 bits). In contrast, exon 4 was skipped in approximately 0.7% of the XPC mRNAs, consistent with the low information content of the exon 4 splice acceptor (-0.1 bits). A new common C/A single nucleotide polymorphism in the XPC intron 11 splice acceptor site (58% C in 97 normals) decreased its information content from 7.5 to 5.1 bits. Fibroblasts homozygous for A/A had significantly higher levels (approximately 2.6-fold) of the XPC mRNA isoform that skipped exon 12 than those homozygous for C/C. This abnormally spliced XPC mRNA isoform has diminished DNA repair function and may contribute to cancer susceptibility.

Alternative Splicing↗
PubMed2006

A 6-bp deletion at the splice donor site of the first intron resulted in aberrant splicing using a cryptic splice site within exon 1 in a patient with succinyl-CoA: 3-Ketoacid CoA transferase (SCOT) deficiency.

Succinyl-CoA: 3-ketoacid-CoA transferase (SCOT; locus symbol OXCT, EC 2.8.3.5) deficiency is a rare genetic disorder affecting ketone body utilization in extra-hepatic tissues. A 6-bp deletion at the splice donor site of intron 1 resulted in the absence of a full-length mature SCOT mRNA with faint amounts of aberrantly spliced transcripts using a cryptic splice donor site within exon 1, which was located just 7 bases upstream from the authentic site in a SCOT deficient patient.

Base Pairing↗
PubMed2000

Optimization of a weak 3' splice site counteracts the function of a bovine papillomavirus type 1 exonic splicing suppressor in vitro and in vivo.

Alternative splicing is a critical component of the early to late switch in papillomavirus gene expression. In bovine papillomavirus type 1 (BPV-1), a switch in 3' splice site utilization from an early 3' splice site at nucleotide (nt) 3225 to a late-specific 3' splice site at nt 3605 is essential for expression of the major capsid (L1) mRNA. Three viral splicing elements have recently been identified between the two alternative 3' splice sites and have been shown to play an important role in this regulation. A bipartite element lies approximately 30 nt downstream of the nt 3225 3' splice site and consists of an exonic splicing enhancer (ESE), SE1, followed immediately by a pyrimidine-rich exonic splicing suppressor (ESS). A second ESE (SE2) is located approximately 125 nt downstream of the ESS. We have previously demonstrated that the ESS inhibits use of the suboptimal nt 3225 3' splice site in vitro through binding of cellular splicing factors. However, these in vitro studies did not address the role of the ESS in the regulation of alternative splicing. In the present study, we have analyzed the role of the ESS in the alternative splicing of a BPV-1 late pre-mRNA in vivo. Mutation or deletion of just the ESS did not significantly change the normal splicing pattern where the nt 3225 3' splice site is already used predominantly. However, a pre-mRNA containing mutations in SE2 is spliced predominantly using the nt 3605 3' splice site. In this context, mutation of the ESS restored preferential use of the nt 3225 3' splice site, indicating that the ESS also functions as a splicing suppressor in vivo. Moreover, optimization of the suboptimal nt 3225 3' splice site counteracted the in vivo function of the ESS and led to preferential selection of the nt 3225 3' splice site even in pre-mRNAs with SE2 mutations. In vitro splicing assays also showed that the ESS is unable to suppress splicing of a pre-mRNA with an optimized nt 3225 3' splice site. These data confirm that the function of the ESS requires a suboptimal upstream 3' splice site. A surprising finding of our study is the observation that SE1 can stimulate both the first and the second steps of splicing.

Alternative Splicing↗
PubMed1988

Exploitation of a thermosensitive splicing event to study pre-mRNA splicing in vivo.

The spliced form of MuSVts110 viral RNA is approximately 20-fold more abundant at growth temperatures of 33 degrees C or lower than at 37 to 41 degrees C. This difference is due to changes in the efficiency of MuSVts110 RNA splicing rather than selective thermolability of the spliced species at 37 to 41 degrees C or general thermosensitivity of RNA splicing in MuSVts110-infected cells. Moreover, RNA transcribed from MuSVts110 DNA introduced into a variety of cell lines is spliced in a temperature-sensitive fashion, suggesting that the structure of the viral RNA controls the efficiency of the event. We exploited this novel splicing event to study the cleavage and ligation events during splicing in vivo. No spliced viral mRNA or splicing intermediates were observed in MuSVts110-infected cells (6m2 cells) at 39 degrees C. However, after a short (about 30-min) lag following a shift to 33 degrees C, viral pre-mRNA cleaved at the 5' splice site began to accumulate. Ligated exons were not detected until about 60 min following the initial detection of cleavage at the 5' splice site, suggesting that these two splicing reactions did not occur concurrently. Splicing of viral RNA in the MuSVts110 revertant 54-5A4, which lacks the sequence -AG/TGT- at the usual 3' splice site, was studied. Cleavage at the 5' splice site in the revertant viral RNA proceeded in a temperature-sensitive fashion. No novel cryptic 3' splice sites were activated; however, splicing at an alternate upstream 3' splice site used at low efficiency in normal MuSVts110 RNA was increased to a level close to that of 5'-splice-site cleavage in the revertant viral RNA. Increased splicing at this site in 54-5A4 viral RNA is probably driven by the unavailability of the usual 3' splice site for exon ligation. The thermosensitivity of this alternate splice event suggests that the sequences governing the thermodependence of MuSVts110 RNA splicing do not involve any particular 3' splice site or branch point sequence, but rather lie near the 5' end of the intron.

Animals↗
PubMed1994

Alternative splicing of beta-tropomyosin pre-mRNA: multiple cis-elements can contribute to the use of the 5'- and 3'-splice sites of the nonmuscle/smooth muscle exon 6.

We previously found that the splicing of exon 5 to exon 6 in the rat beta-TM gene required that exon 6 first be joined to the downstream common exon 8 (Helfman et al., Genes and Dev. 2, 1627-1638, 1988). Pre-mRNAs containing exon 5, intron 5 and exon 6 are not normally spliced in vitro. We have carried out a mutational analysis to determine which sequences in the pre-mRNA contribute to the inability of this precursor to be spliced in vitro. We found that mutations in two regions of the pre-mRNA led to activation of the 3'-splice site of exon 6, without first joining exon 6 to exon 8. First, introduction of a nine nucleotide poly U tract upstream of the 3'-splice site of exon 6 results in the splicing of exon 5 to exon 6 with as little as 35 nucleotides of exon 6. Second, introduction of a consensus 5'-splice site in exon 6 led to splicing of exon 5 to exon 6. Thus, three distinct elements can act independently to activate the use of the 3'-splice site of exon 6: (1) the sequences contained within exon 8 when joined to exon 6, (2) a poly U tract in intron 5, and (3) a consensus 5'-splice site in exon 6. Using biochemical assays, we have determined that these sequence elements interact with distinct cellular factors for 3'-splice site utilization. Although HeLa cell nuclear extracts were able to splice all three types of pre-mRNAs mentioned above, a cytoplasmic S100 fraction supplemented with SR proteins was unable to efficiently splice exon 5 to exon 6 using precursors in which exon 6 was joined to exon 8. We also studied how these elements contribute to alternative splice site selection using precursors containing the mutually exclusive, alternatively spliced cassette comprised of exons 5 through 8. Introduction of the poly U tract upstream of exon 6, and changing the 5'-splice site of exon 6 to a consensus sequence, either alone or in combination, facilitated the use of exon 6 in vitro, such that exon 6 was spliced more efficiently to exon 8. These data show that intron sequences upstream of an exon can contribute to the use of the downstream 5'-splice, and that sequences surrounding exon 6 can contribute to tissue-specific alternative splice site selection.

Alternative Splicing↗
PubMed1991

Mutations in the regions of the Rous sarcoma virus 3' splice sites: implications for regulation of alternative splicing.

Retrovirus RNA is synthesized as a single primary transcript that is differentially processed by RNA splicing. Three species of viral RNA (spliced env, spliced src, and unspliced full-length RNA) are produced in chicken embryo fibroblasts infected with Rous sarcoma virus, an avian retrovirus. The env and src mRNAs are synthesized by the alternative use of two 3' splice sites. The mechanism by which balanced splicing at the two sites is maintained was investigated in this report. Mutants that increase or decrease splicing at one of the two 3' splice sites were analyzed for the effect on splicing at the other site. The two splice sites differed in their response. Mutations that caused a specific increase in the level of spliced env mRNA were associated with reciprocal changes in the levels of src mRNA but with no change in overall splicing. In contrast, mutants in which the src 3' splice site was inactivated demonstrated a decreased overall level of spliced RNA but had little or no effect on the level of spliced env mRNA. Mutations that caused specific increases in the level of spliced src mRNA had variable effects on env mRNA levels. Deletion of regions in gag, which was previously shown to contain a cis-acting negative regulator of splicing, resulted in a corresponding increase of both spliced viral mRNAs and a decrease in unspliced RNA, suggesting that this element suppressed both env and src splicing. Several models are discussed which are possible mechanisms for regulation of alternative splicing of Rous sarcoma virus RNA, but none of these models appear to be consistent with all of the data.

Animals↗
PubMed1986

In vitro splicing of influenza viral NS1 mRNA and NS1-beta-globin chimeras: possible mechanisms for the control of viral mRNA splicing.

In influenza virus-infected cells, the splicing of the viral NS1 mRNA catalyzed by host nuclear enzymes is controlled so that the steady-state amount of the spliced NS2 mRNA is only 5-10% of that of the unspliced NS1 mRNA. Here we examine the splicing of NS1 mRNA in vitro, using nuclear extracts from HeLa cells. We show that in addition to its consensus 5' and 3' splice sites, NS1 mRNA has an intron branch-point adenosine residue that was functional in lariat formation. Nonetheless, this RNA was not detectably spliced in vitro under conditions in which a human beta-globin precursor was efficiently spliced. Using chimeric RNA precursors containing both NS1 and beta-globin sequences, we show that the NS1 5' splice site was effectively utilized by the beta-globin branch-point sequence and 3' splice site to form a spliced RNA, whereas the NS1 3' splice site did not function in detectable splicing in vitro, even in the presence of the beta-globin branch-point sequence or in the presence of both the branch-point sequence and 5' exon and splice site from beta-globin. With the chimeric precursors that were not detectably spliced, as with NS1 mRNA itself, a low level of a lariat structure containing only intron and not 3' exon sequences was formed. The inability of the consensus 3' splice site of NS1 mRNA to function effectively in in vitro splicing suggests that this site is structurally inaccessible to components of the splicing machinery. Based on these results, we propose two mechanisms whereby NS1 mRNA splicing in infected cells is controlled via the accessibility of its 3' splice site.

DNA, Recombinant↗
PubMed1990

A Sequential splicing mechanism promotes selection of an optimal exon by repositioning a downstream 5' splice site in preprotachykinin pre-mRNA.

To explore the structural basis of alternative splicing, we have analyzed the splicing of pre-mRNAs containing an optional exon, E4, from the preprotachykinin gene. This gene encodes substance P and related tachykinin peptides by alternative splicing of a common pre-mRNA. We have shown that alternative splicing of preprotachykinin pre-mRNA occurs by preferential skipping of optional E4. The competing mechanism that incorporates E4 into the final spliced RNA is constrained by an initial block to splicing of the immediate upstream intervening sequence (IVS), IVS3. This block is relieved by sequential splicing, in which the immediate downstream IVS4 is removed first. The structural change resulting from the first splicing event is directly responsible for activation of IVS3 splicing. This structural rearrangement replaces IVS4 sequences with E5 and its adjacent IVS5 sequences. To determine how this structural change promoted IVS3 splicing, we asked what structural change(s) would restore activity of IVS3 splicing-defective mutants. The most significant effect was observed by a 2-nucleotide substitution that converted the 5' splice site of E4 to an exact consensus match, GUAAGU. Exon 5 sequences alone were found not to promote splicing when present in one or multiple copies. However, when a 15-nucleotide segment of IVS5 containing GUAAGU was inserted into a splicing-defective mutant just downstream of the hybrid exon segment E4E5, splicing activity was recovered. Curiously, the 72-nucleotide L2 exon of adenovirus, without its associated 5' splice site, activates splicing when juxtaposed to E4. Models for the activation of splicing by an RNA structural change are discussed.

Base Sequence↗
PubMed1992

Intronic sequences and 3' splice sites control Rous sarcoma virus RNA splicing.

cis-acting sequences of Rous sarcoma virus (RSV) RNA involved in control of the incomplete splicing that is part of the retroviral life cycle have been studied. The 5' and two alternative 3' splice sites, as well as negative regulator of splicing element in the intron, have been introduced into chimeric constructs, and their responsive roles in splicing inhibition have been evaluated by transient transfection experiments. Although the RSV 5' splice site was used efficiently in these assays, substrates containing either the RSV env or the RSV src 3' splice site were not spliced completely, resulting in 40 to 50% unspliced RNA. Addition of the negative regulator of splicing element to substrates containing RSV 3' splice sites resulted in greater inhibition of splicing (70 to 80% unspliced RNA), suggesting that the two elements function independently and additively. Deletion of sequences more than 70 nucleotides upstream of the src 3' splice site resulted in efficient splicing at this site, suggesting that inefficient usage is not inherent in this splice site but is instead due to to sequences upstream of it. Insertion of these upstream sequences into the intron of a heterologous pre-mRNA resulted in partial inhibition of its splicing. In addition, secondary structure interactions were predicted to occur between the src 3' splice site and the inhibitory sequences upstream of it. Thus, RSV splicing control involves both intronic sequences and 3' splice sites, with different mechanisms involved in the underutilization of the env and src splice acceptor sites.

Avian Sarcoma Viruses↗