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At least 739 records · Page 41Linked to original sources
PubMed1995

Characterization of hprt splicing mutations induced by the ultimate carcinogenic metabolite of benzo[a]pyrene in Chinese hamster V-79 cells.

The molecular basis for putative aberrant splicing of hypoxanthine (guanine) phosphoribosyltransferase (hprt) pre-mRNA in Chinese hamster V-79 cells was determined for 75 independent (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene [(+)-BPDE]-induced and 6 spontaneous 8-azaguanine-resistant mutant clones that had exon deletions in their hprt cDNA. Genomic DNA fragments corresponding to the missing exons and their flanking intron regions were amplified by PCR and sequenced. The results indicated that each of these mutants generated a normal-sized PCR product and resulted from aberrant splicing. For (+)-BPDE-induced aberrant splicing mutants, 81% (61 of 75 clones) had base substitution mutations, 5% (4 of 75 clones) had a single base deletion, and 13% (10 of 75 clones) lacked a detectable mutation in the skipped exon, its flanking intron sequences, or in the upstream donor site of the preceding intron. All mutations at a splice donor site resulted in skipping of the entire upstream neighboring exon, whereas alterations at a splice acceptor site caused skipping of the downstream neighboring exon or activation of a cryptic acceptor site in the downstream exon. Fifty-nine % of the splicing mutants had a mutation occurring at the splice site consensus sequence in the intron, and 28% of the splicing mutants had mutations within exon sequences. Among 21 aberrant splicing mutant clones with a mutation inside an exon sequence, seven were in exon 2, two were in exon 3, and twelve were in exon 4. Evidence is presented that a stemloop structure sequesters the splice donor site of exon 2 in pre-mRNA and plays a role in exon 2 skipping. Mutant clones with mutations stabilizing the proposed stemloop structure inhibited the use of the normal exon 2 splice site which resulted in exon 2 skipping in the hprt mRNA. These mutant clones expressed a mixed population of mRNAs, and both normal-sized and truncated mRNA were formed. Similar to our earlier finding that treatment of V-79 cells with (+)-BPDE resulted in a dose-dependent mutation profile within the coding region of the hprt gene, we also observed the presence of dose-dependence in the profile of (+)-BPDE-induced base substitutions in aberrant splicing mutants. As the dose of (+)-BPDE was decreased, the proportion of base substitution mutations at AT base pairs that affected RNA splicing was increased.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1997

Inhibition of mammalian spliceosome assembly and pre-mRNA splicing by peptide inhibitors of protein kinases.

Four peptides are shown to block mammalian spliceosome assembly and pre-mRNA splicing in vitro. Previously, these peptides have been shown to inhibit Ca2+-dependent calmodulin kinase II (CaMK II) via distinct mechanisms. One is a competitive inhibitor of the kinase, two interfere with autophosphorylation events, and one competes for binding to calmodulin, a CaMK II-activating protein. However, because EGTA does not inhibit splicing, the involvement of CaMK II itself in splicing is unlikely; rather, a protein similar to CaMK II may be involved in spliceosome assembly and splicing. Two of the inhibitory peptides, the calmodulin binding domain (CBD) and glycogen synthase (GS) fragment, block assembly of spliceosomal complex C. These peptides inhibited splicing if they were added to reactions any time within the first 10 min of splicing assays. No inhibition of spliceosome assembly or splicing occurred in the presence of randomized versions of the CBD or GS peptide. Additionally, the GS peptide inhibited splicing when added to assays at later time points, despite the fact that spliceosomal complex C had formed. Cumulatively, these analyses suggest that the peptides inhibit at least two distinct events in the spliceosomal cycle. The first event occurs early during in vitro splicing. For this event, prolonged incubations of splicing reactions do not result in a recovery of splicing activity. The second event occurs later and represents a slowing of an essential step, because splicing activity can be recovered in prolonged incubations. Peptides known to inhibit protein kinase A and protein kinase C had no effect on pre-mRNA splicing, underscoring the specificity of the observed inhibitory effects.

Amino Acid Sequence↗
PubMed1984

Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro.

Human beta-globin mRNA precursors (pre-mRNAs) synthesized in vitro from a bacteriophage SP6 promoter/beta-globin gene fusion are accurately and efficiently spliced when added to a HeLa cell nuclear extract. Under optimal conditions, the first intervening sequence (IVS 1) is removed by splicing in up to 90% of the input pre-mRNA. Splicing requires ATP and in its absence the pre-mRNA is neither spliced nor cleaved at splice junctions. Splicing does not require that the pre-mRNA contain a correct 5' or 3' end, a 3' poly A tail, or a 5'-terminal cap structure. However, capping of the pre-mRNA significantly affects the specificity of in vitro processing. In the absence of a cap approximately 30%-40% of the pre-mRNA is accurately spliced, and a number of aberrantly cleaved RNAs are also detected. In contrast, capped pre-mRNAs are spliced more efficiently and produce fewer aberrant RNA species. The specificity of splice-site selection in vitro was tested by analyzing pre-mRNAs that contain beta-thalassemia splicing mutations in IVS 1. Remarkably, these mutations cause the same abnormal splicing events in vitro and in vivo. The ability to synthesize mutant pre-mRNAs and study their splicing in a faithful in vitro system provides a powerful approach to determine the mechanisms of RNA splice-site selection.

Adenosine Triphosphate↗
PubMed2002

Early results of a prospective randomized trial of spliced vein versus polytetrafluoroethylene graft with a distal vein cuff for limb-threatening ischemia.

OBJECTIVE: Single-piece vein remains the conduit of choice in patients who need bypass grafting for limb salvage. When this option is not available, two of the remaining options are prosthetic bypass graft or several segments of vein spliced together. In this study, we compare spliced vein bypass grafting versus polytetrafluoroethylene grafting with a distal vein cuff in patients with limb-threatening ischemia. METHODS: Between 1996 and 2000, 39 bypass grafting procedures in 36 patients were performed for limb-threatening ischemia. These procedures were prospectively randomized to either spliced vein bypass grafting (spliced group, 19 bypass grafts) or polytetrafluoroethylene grafting with a distal vein cuff (cuff group, 20 bypass grafts). All the patients in the cuff group underwent anticoagulation therapy with warfarin sodium after surgery. The inclusion criteria included: no single-piece vein option for bypass grafting, adequate vein for splice, no composite sequential option, and limb-threatening ischemia. The demographics were similar between the two groups. RESULTS: The primary patency rate at 2 years was 44% and 49% for the spliced and cuff groups, respectively. In the spliced group, seven of 19 bypass grafts underwent revision in the follow-up period, and two of 20 cuffed bypass grafts were successfully revised. The secondary patency rate was 87% and 59% (P <.05), with limb salvage rates of 94% and 85% for spliced and cuff groups, respectively. Four patients in the spliced vein group needed reoperation for wound complications related to vein harvest. One polytetrafluoroethylene graft needed removal for infection. Two early mortalities occurred in the spliced group, one from myocardial infarction and one from stroke. The overall survival rate at 2 years between the two groups was 67% and 100% for the spliced and cuff groups, respectively (P <.05). CONCLUSION: Although this is a preliminary report, it appears that both spliced vein bypass grafting and polytetrafluoroethylene bypass grafting with a distal vein cuff produce acceptable limb salvage rates. The secondary patency rate for spliced vein is better, but these bypass grafts more often need revision or reoperation for wound complications.

Aged↗
PubMed2004

Splicing of a cap-proximal human Papillomavirus 16 E6E7 intron promotes E7 expression, but can be restrained by distance of the intron from its RNA 5' cap.

Human papillomavirus 16 (HPV16) E6E7 pre-mRNA is bicistronic and has an intron in the E6 coding region with one 5' splice site and two alternative 3' splice sites, which produce E6(*)I and E6(*)II, respectively. If this intron remains unspliced, the resulting E6E7 mRNA expresses oncogenic E6. We found for the first time that the E6E7 pre-mRNA was efficiently spliced in vitro only when capped and that cellular cap-binding factors were involved in the splicing. The cap-dependent splicing of the E6E7 pre-mRNA was extremely efficient in cervical cancer-derived cells, producing mostly E6(*)I, but inefficient in cells transfected with a common retrovirus expression vector, pLXSN16E6E7, due to the large size of this vector's exon 1. Further studies showed that efficient splicing of the E6E7 pre-mRNA depends on the distance of the cap-proximal intron from the RNA 5' cap, with an optimal distance of less than 307nt in order to facilitate better association of U1 small nuclear RNA with the intron 5' splice site. The same was true for splicing of human beta-globin RNA. Splicing of the E6E7 RNA provided more E7 RNA templates and promoted E7 translation, whereas a lack of RNA splicing produced a low level of E7 translation. Together, our data indicate that the distance between the RNA 5' cap and cap-proximal intron is rate limiting for RNA splicing. HPV16 E6E7 pre-mRNA takes advantage of its small cap-proximal exon to confer efficient splicing for better E7 expression.

3' Flanking Region↗
PubMed2005

Structural genomics analysis of alternative splicing and application to isoform structure modeling.

Alternative splicing is a sophisticated nuclear process that regulates gene expression. It represents an important mechanism for enhancing the functional diversity of proteins. Our current knowledge of alternatively spliced variants is derived mainly from mRNA transcripts, and very little is known about their protein tertiary structures. We carried out a large-scale analysis of known alternatively spliced variants at both protein sequence and structure levels and have shown that threading is, in general, a viable approach for modeling structures of alternatively spliced variants. An examination of alternative splicing at the protein sequence level revealed that the size of splicing events follows the power law distribution and the majority of splicing isoforms harbor only one or two alternations. We examined alternative splicing in the context of protein 3D structures and found that the boundaries of alternative splicing events generally happen in coil regions of secondary structures and exposed residues and the majority of the sequences involved in splicing are located on the surface of proteins. In light of these findings, we then proceeded to demonstrate that threading represents a useful tool for structure prediction of alternative splicing isoforms and addressed the fold stability issue of threading-based structure prediction by molecular dynamics simulation. Our analysis and the insights gained have helped to establish a viable method for structure prediction of alternatively spliced isoforms at the genome scale.

Alternative Splicing↗
PubMed2002

tau Exon 10 expression involves a bipartite intron 10 regulatory sequence and weak 5' and 3' splice sites.

tau mutations that deregulate alternative exon 10 (E10) splicing cause frontotemporal dementia with parkinsonism chromosome 17-type by several mechanisms. Previously we showed that E10 splicing involved exon splicing enhancer sequences at the 5' and 3' ends of E10, an exon splicing silencer, a weak 5' splice site, and an intron splicing silencer (ISS) within intron 10 (I10). Here, we identify additional regulatory sequences in I10 using both non-neuronal and neuronal cells. The ISS sequence extends from I10 nucleotides 11-18, which is sufficient to inhibit use of a weakened 5' splice site of a heterologous exon. Furthermore, ISS function is location-independent but requires proximity to a weak 5' splice site. Thus, the ISS functions as a linear sequence. A new cis-acting element, the intron splicing modulator (ISM), was identified immediately downstream of the ISS at I10 positions 19-26. The ISM and ISS form a bipartite regulatory element, within which the ISM functions when the ISS is present, mitigating E10 repression by the ISS. Additionally, the 3' splice site of E10 is weak and requires exon splicing enhancer elements for efficient E10 inclusion. Thus far, tau FTDP-17 splicing mutations affect six predicted cis-regulatory sequences.

Alternative Splicing↗
PubMed2002

CDK11 complexes promote pre-mRNA splicing.

The PITSLRE protein kinases, hereafter referred to as CDK11 because of their association with the cyclin L regulatory partner, belong to large molecular weight protein complexes that contain RNA polymerase II. These CDK11(p110) complexes have been reported to influence transcription as well as interact with the general pre-mRNA-splicing factor RNPS1. Some of these complexes may also play a role in pre-mRNA splicing. Using a two-hybrid interactive screen, the splicing protein 9G8 was identified as an in vivo partner for CDK11(p110). The identification of several splicing-related factors as CDK11(p110) interactors along with the close relationship between transcription and splicing indicated that CDK11(p110) might influence splicing activity directly. Immunodepletion of CDK11(p110) from splicing extracts greatly reduced the appearance of spliced products using an in vitro assay system. Moreover, the re-addition of these CDK11(p110) immune complexes to the CDK11(p110)-immunodepleted splicing reactions completely restored splicing activity. Similarly, the addition of purified CDK11(p110) amino-terminal domain protein was sufficient to inhibit the splicing reaction. Finally, 9G8 is a phosphoprotein in vivo and is a substrate for CDK11(p110) phosphorylation in vitro. These data are among the first demonstrations showing that a CDK activity is functionally coupled to the regulation of pre-mRNA-splicing events and further support the hypothesis that CDK11(p110) is in a signaling pathway that may help to coordinate transcription and RNA-processing events.

Base Sequence↗
PubMed2004

Genome-wide analysis of pre-mRNA splicing: intron features govern the requirement for the second-step factor, Prp17 in Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Removal of pre-mRNA introns is an essential step in eukaryotic genome interpretation. The spliceosome, a ribonucleoprotein performs this critical function; however, precise roles for many of its proteins remain unknown. Genome-wide consequences triggered by the loss of a specific factor can elucidate its function in splicing and its impact on other cellular processes. We have employed splicing-sensitive DNA microarrays, with yeast open reading frames and intron sequences, to detect changes in splicing efficiency and global expression. Comparison of expression profiles, for intron-containing transcripts, among mutants of two second-step factors, Prp17 and Prp22, reveals their unique and shared effects on global splicing. This analysis enabled the identification of substrates dependent on Prp17. We find a significant Prp17 role in splicing of introns which are longer than 200nts and note its dispensability when introns have a < or =13-nucleotide spacing between their branch point nucleotide and 3 ' splice site. In vitro splicing of substrates with varying branch nucleotide to 3 ' splice site distances supports the differential Prp17 dependencies inferred from the in vivo analysis. Furthermore, we tested the predicted dispensability of Prp17 for splicing short introns in the evolutionarily distant yeast, Schizosaccharomyces pombe, where the genome contains predominantly short introns. SpPrp17 was non-essential at all growth temperatures implying that functional evolution of splicing factors is integrated with genome evolution. Together our studies point to a role for budding yeast Prp17 in splicing of subsets of introns and have predictive value for deciphering the functions of splicing factors in gene expression and regulation in other eukaryotes.

Cell Cycle Proteins↗
PubMed2006

In silico and in vivo splicing analysis of MLH1 and MSH2 missense mutations shows exon- and tissue-specific effects.

BACKGROUND: Abnormalities of pre-mRNA splicing are increasingly recognized as an important mechanism through which gene mutations cause disease. However, apart from the mutations in the donor and acceptor sites, the effects on splicing of other sequence variations are difficult to predict. Loosely defined exonic and intronic sequences have been shown to affect splicing efficiency by means of silencing and enhancement mechanisms. Thus, nucleotide substitutions in these sequences can induce aberrant splicing. Web-based resources have recently been developed to facilitate the identification of nucleotide changes that could alter splicing. However, computer predictions do not always correlate with in vivo splicing defects. The issue of unclassified variants in cancer predisposing genes is very important both for the correct ascertainment of cancer risk and for the understanding of the basic mechanisms of cancer gene function and regulation. Therefore we aimed to verify how predictions that can be drawn from in silico analysis correlate with results obtained in an in vivo splicing assay. RESULTS: We analysed 99 hMLH1 and hMSH2 missense mutations with six different algorithms. Transfection of three different cell lines with 20 missense mutations, showed that a minority of them lead to defective splicing. Moreover, we observed that some exons and some mutations show cell-specific differences in the frequency of exon inclusion. CONCLUSION: Our results suggest that the available algorithms, while potentially helpful in identifying splicing modulators especially when they are located in weakly defined exons, do not always correspond to an obvious modification of the splicing pattern. Thus caution must be used in assessing the pathogenicity of a missense or silent mutation with prediction programs. The variations observed in the splicing proficiency in three different cell lines suggest that nucleotide changes may dictate alternative splice site selection in a tissue-specific manner contributing to the widely observed phenotypic variability in inherited cancers.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Alternative splicing of the human estrogen receptor alpha primary transcript: mechanisms of exon skipping.

The 1785 nucleotides of the coding region of the estrogen receptor alpha (ER-alpha) are dispersed over a region of more than 300,000 nucleotides in the primary transcript. Splicing of this precursor RNA frequently leads to variants lacking one or more exons that have been associated to breast cancer progression. The most frequent splice variant lacks exon 4 and is expressed in the human mammary carcinoma cell line MCF-7 at a level similar to that of the full-length messenger. The in silico analysis of ER-alpha splice sites by Hamming clustering, a self learning method trained on more than 28,000 experimentally proved splice sites, reveals high relevance for the 5' and 3' splice sites of exon 4. The splicing analysis of transfected mini-gene constructs containing drastically shortened introns excludes that weak splice sites, intron or exon lengths or splice enhancers are responsible for exon skipping. Exon 6 is never skipped in MCF-7 cells but is spliced out from mini-gene derived primary transcripts if inserted between exons 3 and 5 instead of exon 4. As a consequence, it appears that a particular splice site affinity of exon 3 donor (5' splice site) and exon 5 acceptor sites (3' splice site) is responsible for skipping of the exon in between.

Alternative Splicing↗
PubMed1998

Regulated tissue-specific expression of antagonistic pre-mRNA splicing factors.

The SR proteins are essential metazoan pre-mRNA splicing factors that can also influence the selection of alternative 5' splice sites in a concentration-dependent manner. Their activity in alternative splicing in vitro is antagonized by members of the hnRNP A/B family of proteins. The opposite effects of members of these two families of antagonistic splicing factors in vitro and upon overexpression in vivo suggest that changes in their relative levels may be a natural mechanism for the regulation of alternative splicing in vivo. One prediction of this model is that the ratios of these antagonists should vary in different cell types and in other situations in which cellular or viral transcripts are differentially spliced. We raised monoclonal antibodies specific for SF2/ASF and used them to measure the abundance of SF2/ASF protein and its isoforms, its phosphorylation state in vivo and during splicing in vitro, and its association with the spliceosome. SF2/ASF exists predominantly or exclusively in a highly phosphorylated state in vivo in all cell types examined, and unphosphorylated protein was not detectable. Unphosphorylated recombinant SF2/ASF becomes rapidly phosphorylated under splicing conditions in HeLa cell extracts and associates stably with one or more exons of beta-globin pre-mRNA. This interaction appears to persist through the splicing reaction and SF2/ASF remains bound to spliced mRNA. We compared the distribution of SF2/ASF to that of its antagonist, hnRNP A1, in different rat tissues and in immortal and transformed cell lines. We found that the protein levels of these antagonistic splicing factors vary naturally over a very wide range, supporting the notion that changes in the ratio of these proteins can affect alternative splicing of a variety of pre-mRNAs in vivo.

Adenoviridae↗
PubMed2000

Pre-mRNA splicing in the absence of an SR protein RS domain.

SR proteins are essential pre-mRNA splicing factors that act at the earliest stages of splice-site recognition and spliceosome assembly, as well as later in the splicing pathway. SR proteins consist of one or two RNA-recognition motifs and a characteristic arginine/serine-rich C-terminal RS domain. The RS domain, which is extensively phosphorylated, mediates the subcellular localization of individual SR proteins and also functions as a splicing activation module, apparently by engaging in protein-protein interactions. The RS domain of SF2/ASF is dispensable for the concentration-dependent effects of this SR protein on alternative splice-site selection. However, this RS domain is highly conserved phylogenetically, and was shown to be required for constitutive splicing in vitro and for cell viability. Here, we demonstrate that the RS domain of SF2/ASF is, in fact, dispensable for splicing of several substrates, including constitutive and enhancer-dependent pre-mRNAs. The requirement for this RS domain is substrate specific, and correlates with the strength of the splicing signals. When the 3' splice site is weak, both the SF2/ASF RS domain and U2AF(35) are required for splicing. These results show the existence of an RS domain-independent function of SR proteins in constitutive and enhancer-dependent splicing, and suggest mechanisms for their role in enhancer function besides U2AF recruitment.

Base Sequence↗
PubMed2024

Alternative splicing in ovarian cancer.

Ovarian cancer is the second leading cause of gynecologic cancer death worldwide, with only 20% of cases detected early due to its elusive nature, limiting successful treatment. Most deaths occur from the disease progressing to advanced stages. Despite advances in chemo- and immunotherapy, the 5-year survival remains below 50% due to high recurrence and chemoresistance. Therefore, leveraging new research perspectives to understand molecular signatures and identify novel therapeutic targets is crucial for improving the clinical outcomes of ovarian cancer. Alternative splicing, a fundamental mechanism of post-transcriptional gene regulation, significantly contributes to heightened genomic complexity and protein diversity. Increased awareness has emerged about the multifaceted roles of alternative splicing in ovarian cancer, including cell proliferation, metastasis, apoptosis, immune evasion, and chemoresistance. We begin with an overview of altered splicing machinery, highlighting increased expression of spliceosome components and associated splicing factors like BUD31, SF3B4, and CTNNBL1, and their relationships to ovarian cancer. Next, we summarize the impact of specific variants of CD44, ECM1, and KAI1 on tumorigenesis and drug resistance through diverse mechanisms. Recent genomic and bioinformatics advances have enhanced our understanding. By incorporating data from The Cancer Genome Atlas RNA-seq, along with clinical information, a series of prognostic models have been developed, which provided deeper insights into how the splicing influences prognosis, overall survival, the immune microenvironment, and drug sensitivity and resistance in ovarian cancer patients. Notably, novel splicing events, such as PIGV|1299|AP and FLT3LG|50,941|AP, have been identified in multiple prognostic models and are associated with poorer and improved prognosis, respectively. These novel splicing variants warrant further functional characterization to unlock the underlying molecular mechanisms. Additionally, experimental evidence has underscored the potential therapeutic utility of targeting alternative splicing events, exemplified by the observation that knockdown of splicing factor BUD31 or antisense oligonucleotide-induced BCL2L12 exon skipping promotes apoptosis of ovarian cancer cells. In clinical settings, bevacizumab, a humanized monoclonal antibody that specifically targets the VEGF-A isoform, has demonstrated beneficial effects in the treatment of patients with advanced epithelial ovarian cancer. In conclusion, this review constitutes the first comprehensive and detailed exposition of the intricate interplay between alternative splicing and ovarian cancer, underscoring the significance of alternative splicing events as pivotal determinants in cancer biology and as promising avenues for future diagnostic and therapeutic intervention.

Humans↗
PubMed1998

Information analysis of human splice site mutations.

Splice site nucleotide substitutions can be analyzed by comparing the individual information contents (Ri, bits) of the normal and variant splice junction sequences [Rogan and Schneider, 1995]. In the present study, we related splicing abnormalities to changes in Ri values of 111 previously reported splice site substitutions in 41 different genes. Mutant donor and acceptor sites have significantly less information than their normal counterparts. With one possible exception, primary mutant sites with <2.4 bits were not spliced. Sites with Ri values > or = 2.4 bits but less than the corresponding natural site usually decreased, but did not abolish splicing. Substitutions that produced small changes in Ri probably do not impair splicing and are often polymorphisms. The Ri values of activated cryptic sites were generally comparable to or greater than those of the corresponding natural splice sites. Information analysis revealed preexisting cryptic splice junctions that are used instead of the mutated natural site. Other cryptic sites were created or strengthened by sequence changes that simultaneously altered the natural site. Comparison between normal and mutant splice site Ri values distinguishes substitutions that impair splicing from those which do not, distinguishes null alleles from those that are partially functional, and detects activated cryptic splice sites.

Base Sequence↗
PubMed2005

Alternative splicing of MDM2 mRNA in lung carcinomas and lung cell lines.

The MDM2 gene is overexpressed in several human tumors and its product may be processed into various isoforms. Recently, alternative splicing forms of MDM2 mRNA have been detected in various types of tumors. In this study, lung tissue from human non small cell lung cancers was examined for MDM2 mRNA splicing variants by nested RT-PCR. Of the 117 lung cancer tissue samples analyzed, a total of 31 (26.5%) had splice variants for the MDM2 gene, while 59 (50.4%) had undetectable levels of MDM2 transcript. Further analysis indicated that the predominant variant for 26 of the 31 samples with alternative MDM2 splicing products was MDM2-657, a splice variant lacking exons 3-11. Significant associations were found between the frequency of alternative splicing and the gender and smoking habits of the patients. Approximately 36% of male patients had alternative splicing of MDM2 compared with only 9.5% of female patients (P = 0.008); 44.2% of the smoker patients had alternative MDM2 splice forms versus 16.2% of nonsmokers (P = 0.003). Furthermore, most normal lung cell lines examined possessed only full-length MDM2 mRNA, while among several lung cancer cell lines, only H1355 and CaLu-1 cells lacked alternatively spliced MDM2 transcripts. When H1355 cells were treated in vitro with the cigarette smoke carcinogen benzo[a]pyrene (B[a]P) or the B[a]P metabolite benzo[a]pyrene diolepoxide (BPDE), three MDM2 splicing products were detected by nested RT-PCR. Finally, with the use of several specific inhibitors, we found that BPDE-induced MDM2 mRNA alternative splicing in H1355 cells may occur through the PI3K or MAPK pathway. Overall, our results suggest that carcinogens present in cigarette smoke increase the risk of alternative MDM2 splicing, which is highly associated with lung cancer.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2006

Splicing modulation as a modifier of the CFTR function.

A significant fraction of CF-causing mutations affects pre-mRNA splicing. These mutations can generate both aberrant and correct transcripts, the level of which varies among different patients. An inverse correlation was found between this level and disease severity, suggesting a role for splicing regulation as a genetic modifier. Subsequent studies showed that overexpression of splicing factors modulated the level of correctly spliced RNA, transcribed from minigenes carrying CF-causing splicing mutations. Overexpression of splicing factors also modulated the level of normal CFTR transcripts, transcribed from the endogenous CFTR allele carrying splicing mutations, in CF-derived epithelial cells. Several of the factors promoted higher level of correct CFTR transcripts. The increased level of normal transcripts led to activation of the CFTR channel and restoration of its function. Restoration was also obtained by sodium butyrate, a histone deacetylase inhibitor, known to up-regulate the expression of splicing factors. These results highlight the role of the splicing machinery as a modifier of disease severity in patients carrying splicing mutations and shed a new light on the therapeutic potential of splicing modulation for genetic diseases caused by splicing mutations.

Alternative Splicing↗
PubMed1991

Splicing of the Petunia cytochrome oxidase subunit II intron.

A comparative analysis of the plant intron-containing mitochondrial cytochrome oxidase subunit II (coxII) genes provides an indication that four conserved sequence motifs, present in exon 1 (intron-binding sequences; IBS), and complementary motifs (exon-binding sequences; EBS), present in domain I of the group II intron, may be involved in splicing of the intron. Two of these potential IBS motifs (IBS1 and IBS2) have been previously discussed. Two further potential IBS motifs (IBSa and IBSb), which occur twice within exon 1, could be involved in specification of the 5' splice site and of a 5' cryptic splice site. Nuclease-protection experiments and DNA sequence analysis of a spliced coxII cDNA have confirmed the predicted positions of the petunia coxII 5' and 3' splice sites. Evidence for the occurrence of splicing in vivo at the putative 5' cryptic splice site in petunia is provided by the detection of a nuclease-protected fragment corresponding to the size which is predicted if splicing at the proposed cryptic splice site occurs. The existence and location of a cryptic splice site, upstream of the normal coxII 5' splice site, is consistent with the proposed derivation of the cytoplasmic male sterility (CMS)-associated pcf gene from an abnormally spliced coxII transcript (Pruitt and Hanson 1989).

Base Sequence↗