Phosphorylation-dependent control of the pre-mRNA splicing machinery.
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Alternative Splicing↗
Biomedical subjects
J Soret
Publications and source records attributed to J Soret.
Specific inhibition of serine- and arginine-rich splicing factors phosphorylation, spliceosome assembly, and splicing by the antitumor drug NB-506.
Specific phosphorylation of serine- and arginine-rich pre-mRNA splicing factors (SR proteins) is one of the key determinants regulating splicing events. Several kinases involved in SR protein phosphorylation have been identified and characterized, among which human DNA topoisomerase I is known to have DNA-relaxing activity. In this study, we have investigated the mechanism of splicing inhibition by a glycosylated indolocarbazole derivative (NB-506), a potent inhibitor of both kinase and relaxing activities of topoisomerase I. NB-506 completely inhibits the capacity of topoisomerase I to phosphorylate, in vitro, the human splicing factor 2/alternative splicing factor (SF2/ASF). This inhibition is specific, because NB-506 does not demonstrate activity against other kinases known to phosphorylate SF2/ASF such as SR protein kinase 1 and cdc2 kinase. Importantly, HeLa nuclear extracts competent in splicing but not splicing-deficient cytoplasmic S100 extracts treated with the drug fail to phosphorylate SF2/ASF and to support splicing of pre-mRNA substrates containing SF2/ASF-target sequences. Native gel analysis of splicing complexes revealed that the drug affects the formation of the spliceosome, a dynamic ribonucleoprotein structure where splicing takes place. In the presence of the drug, neither pre-spliceosome nor spliceosome is formed, demonstrating that splicing inhibition occurs at early steps of spliceosome assembly. Splicing inhibition can be relieved by adding phosphorylated SF2/ASF, showing that extracts treated with NB-506 lack a phosphorylating activity required for splicing. Moreover, NB-506 has a cytotoxic effect on murine P388 leukemia cells but not on P388CPT5 camptothecin-resistant cells that carry two point mutations in conserved regions of topoisomerase I gene (Gly361Val and Asp709Tyr). After drug treatment, P388 cells accumulated hypophosphorylated forms of SR proteins and polyadenylated RNA in the nucleus. In contrast, neither SR protein phosphorylation nor polyadenylated mRNA distribution was affected in P388 CPT5-treated cells. Consistently, NB506 treatment altered the mRNA levels and/or splicing pattern of several tested genes (Bcl-X, CD 44, SC35, and Sty) in P388 cells but not in P388 CPT5 cells. The study shows for the first time that indolocarbazole drugs targeting topoisomerase I can affect gene expression by modulating pre-mRNA splicing through inhibition of SR proteins phosphorylation.
Animals↗
SC35 autoregulates its expression by promoting splicing events that destabilize its mRNAs.
SC35 belongs to the family of SR proteins that regulate alternative splicing in a concentration-dependent manner in vitro and in vivo. We previously reported that SC35 is expressed through alternatively spliced mRNAs with differing 3' untranslated sequences and stabilities. Here, we show that overexpression of SC35 in HeLa cells results in a significant decrease of endogenous SC35 mRNA levels along with changes in the relative abundance of SC35 alternatively spliced mRNAs. Remarkably, SC35 leads to both an exon inclusion and an intron excision in the 3' untranslated region of its mRNAs. In vitro splicing experiments performed with recombinant SR proteins demonstrate that SC35, but not ASF/SF2 or 9G8, specifically activates these alternative splicing events. Interestingly, the resulting mRNA is very unstable and we present evidence that mRNA surveillance is likely to be involved in this instability. SC35 therefore constitutes the first example of a splicing factor that controls its own expression through activation of splicing events leading to expression of unstable mRNA.
Alternative Splicing↗
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↗
Values for the free to total prostate-specific antigen ratio as a function of age: necessity of reference range validation.
OBJECTIVE: To measure the levels of free prostate-specific antigen (PSA), total PSA, and free to total PSA ratio in a population of men with no known prostate pathology aged from 20 to 70 years. PATIENTS AND METHODS: Serum total PSA and free PSA values were determined in 1,502 patients due for a systematic health examination. The digital rectal examination was only proposed for those over 50 years of age. The assays were determined on the AsXYM apparatus, from Abbott laboratories, by MEIA technology with monoclonal antibodies. RESULTS: 1,274 men were available for study. The mean age was 43.6 +/- 11 years (range 20-69 years). The total PSA level was stable up to 40 years. Beyond that, it increased with age. There was a linear regression between the age and the logarithm of the total PSA rate (r = 0.26, p < 0.0001) from 40 to 70 years. The upper limit of the normal value (95th percentile) increased from 1.07 for the 20- to 30-year age range to 2.82 for the 60- to 70-year range. The free PSA level was stable up to 50 years of age. It then significantly increased. The upper limit of the normal value was measured as 0.42 in the range of 20-30 years and as 0.53 in the range of 60-70 years with an annual average increase rate of roughly 0.5%. Overall there was a linear regression between age and the free PSA rate (r = 0.12, p < 0.0001). The upper limit of the free to total PSA ratio, measured as being 0.68 in the range of 20-29 years, dropped towards 60-69 years with an upper limit of the normal of 0.48. The average annual reduction rate was around 0.70%. There was a linear regression between the age and the free to total PSA ratio (r = 0.17, p < 0.0001). CONCLUSION: These total PSA levels are lower than the ones measured in other studies with other assay methods. These variations stress the importance of validating reference values of total PSA and free PSA as a function of the assay method and the population to which they are applied before using them as an aid in the diagnosis of prostate cancer.
Adult↗
Characterization of SRp46, a novel human SR splicing factor encoded by a PR264/SC35 retropseudogene.
The highly conserved SR family contains a growing number of phosphoproteins acting as both essential and alternative splicing factors. In this study, we have cloned human genomic and cDNA sequences encoding a novel SR protein designated SRp46. Nucleotide sequence analyses have revealed that the SRp46 gene corresponds to an expressed PR264/SC35 retropseudogene. As a result of mutations and amplifications, the SRp46 protein significantly differs from the PR264/SC35 factor, mainly at the level of its RS domain. Northern and Western blot analyses have established that SRp46 sequences are expressed at different levels in several human cell lines and normal tissues, as well as in simian cells. In contrast, sequences homologous to SRp46 are not present in mice. In vitro splicing studies indicate that the human SRp46 recombinant protein functions as an essential splicing factor in complementing a HeLa cell S100 extract deficient in SR proteins. In addition, complementation analyses performed with beta-globin or adenovirus E1A transcripts and different splicing-deficient extracts have revealed that SRp46 does not display the same activity as PR264/SC35. These results demonstrate, for the first time, that an SR splicing factor, which represents a novel member of the SR family, is encoded by a functional retropseudogene.
3T3 Cells↗
Characterization of multiple alternative RNAs resulting from antisense transcription of the PR264/SC35 splicing factor gene.
The PR264/SC35 splicing factor belongs to the family of SR proteins which function as essential and alternative splicing factors. Here, we report that the human PR264/SC35 locus is bidirectionally transcribed. Double in situ hybridization experiments have allowed simultaneous detection of sense and antisense RNA in human CCRF-CEM cells, suggesting that expression of the corresponding genes is not mutually exclusive. We have characterized three main classes of ET RNAs encoded by the opposite strand of the PR264/SC35 gene and containing PR264/SC35-overlapping sequences, PR264/SC35-non overlapping sequences or a combination of both. We show that their expression results from the use of alternative promoters, exons and polyadenylation signals. PR264/SC35-non overlapping ET mRNA species potentially encode two protein isoforms (449 and 397 amino acids) and are expressed from the PR264/SC35 promoting region. Northern blots and RNase protection analyses indicate that ET polyadenylated RNAs are differentially expressed in several human cell lines. Similar studies performed in the mouse have revealed that the bidirectional transcription of the PR264/SC35 locus is a conserved mechanism and that the open reading frame identified in a subset of human ET mRNAs is highly conserved (93% homology). Northern blot analyses performed with several murine tissues confirmed the differential expression of the ET gene and revealed that it is predominantly expressed in the testis.
Alternative Splicing↗
The PR264/c-myb connection: expression of a splicing factor modulated by a nuclear protooncogene.
We have previously reported that expression of the c-myb gene in normal avian thymic cells proceeds through the intermolecular recombination of ET (thymus-specific) and c-myb coding sequences, thereby generating a novel type of c-myb product. Antisense transcripts expressed from the ET locus encode the extremely well-conserved splicing factor PR264/SC35. We now show that the human PR264 promoter sequences contain several myb-recognition elements that efficiently interact in vitro with the c-myb DNA-binding domain. Moreover, expression from the PR264 promoter is transactivated, both in vitro and in cultured cells, by different c-myb products. Thus, the PR264 gene is most likely a physiological target for the c-myb family of transcription factors.
Animals↗
A potential splicing factor is encoded by the opposite strand of the trans-spliced c-myb exon.
We previously established that the expression of a thymic c-myb mRNA species requires the intermolecular recombination of coding sequences expressed from transcriptional units localized on different chromosomes, in both chicken and human. We now report that a putative splicing factor (PR264), extremely well conserved in chicken and human, is encoded by the opposite strand of the c-myb trans-spliced exon. The PR264 polypeptide, which contains a typical ribonucleoprotein 80 and an arginine/serine-rich domain, is highly homologous to the Drosophila splicing regulators tra, tra-2, and su(wa) and to the human alternative splicing factor ASF/SF2. Furthermore, we show that PR264-specific mRNAs are expressed in normal hematopoietic cells of chicken and human origin and that the relative proportion of the PR264 transcripts is developmentally regulated in chicken.
Amino Acid Sequence↗
Transforming potential of truncated v-myb and stimulation of replication by gag-myb fusion products.
We have previously reported that truncated forms of the v-myb oncogene of avian myeloblastosis virus (AMV) are expressed in transformed chicken embryo fibroblasts (CEF). In this paper, we show that deletion mutants encoding v-myb products altered in either the DNA-binding or the negative regulatory domains are able to induce CEF transformation. In addition, we report that recombinant plasmids expressing gag-myb fusion proteins are maintained as extrachromosomal forms in transfected cells. This observation provides an important clue for a possible role of myb in the DNA replication processes.
Animals↗
A novel type of RNA-binding protein is potentially encoded by the opposite strand of the trans-spliced c-myb coding exon.
Recently, we reported evidence suggesting that expression of c-myb thymic mRNA species involves the intermolecular recombination of coding sequences (ET and c-myb) localized on two different chromosomes, both in chicken and human. Our present studies demonstrate that the ET locus encodes, in the antisense orientation, a novel member of the RNA binding protein family in these two species.
Amino Acid Sequence↗
C-myb proto-oncogene: evidence for intermolecular recombination of coding sequences.
We have characterized a novel chicken c-myb exon whose sequences are specifically expressed in thymic cells. In situ hybridization experiments indicate that this thymus-specific coding exon is localized on a small chromosome, distinct from the large acrocentric chromosome 3 on which we recently mapped the bulk of 15 exons, common to the c-myb mRNA species expressed in hematopoietic cells of both B and T lineages. These observations indicate that intermolecular recombination is required for the tissue-specific expression of the c-myb proto-oncogene. We also show that these thymus-specific sequences are conserved in human DNA and lie on chromosome 17q25, whereas the human c-myb locus is localized on chromosome 6q22-23. Sequencing data obtained from genomic DNA and PCR analyses performed with c-myb mRNA species expressed in chicken thymic cells strongly suggest that a repeated decameric sequence plays a key role in the recombination process.
Animals↗
Chromosomal reallocation of the chicken c-myb locus and organization of 3'-proximal coding exons.
In the course of our studies concerning the tissue-specific expression of the c-myb proto-oncogene, we have established the nucleotide sequence of the chicken c-myb 3'-proximal coding exons. In situ hybridization performed with different genomic DNA probes corresponding to nearly all the c-myb gene allowed us to localize the corresponding locus on the large acrocentric chromosome 3 in chicken. Our sequencing data also indicate that the 3'-proximal noncoding sequences represented in c-myb mRNA species are derived from non-contiguous exons.
Animals↗
Induction of nephroblastoma by myeloblastosis-associated virus type 1: state of proviral DNAs in tumor cells.
Myeloblastosis-associated virus type 1 (MAV1) derived from a molecular clone of infectious proviral DNA (B. Perbal, J. S. Lipsick, J. Svoboda, R. F. Silva, and M. A. Baluda, J. Virol. 56:240-244, 1985) was shown to specifically induce nephroblastoma in chickens and therefore belongs to the MAV-N class. We show that nephroblastomas are polyclonal tumors containing rearranged proviral genomes. Rearrangements occur preferentially in the gag-pol region of the MAV1 proviral genome, and similar rearrangements can be detected in well-developed independent tumors.
Animals↗
Organization of 5'-proximal c-myb exons in chicken DNA. Implications for c-myb tissue-specific transcription.
The organization of 5'-proximal c-myb exons in chicken DNA has been established by restriction enzyme mapping and nucleotide sequencing. Hybridization studies performed with cDNA probes revealed that yolk sac and thymic c-myb RNAs differ in their 5'-termini. A comparison of the genomic c-myb sequence with that of cDNAs isolated from normal thymic and lymphoma avian cells suggests that different promoter regions are used to initiate c-myb transcription in hematopoietic cells of different origins.
Amino Acid Sequence↗
Expression of a truncated v-myb product in transformed chicken embryo fibroblasts.
Transformed cells have been isolated after transfection of chicken embryo fibroblasts (CEF) with the DNA of a recombinant clone (KXA 3457) in which the v-myb sequences are flanked by the two AMV-LTRs. Abnormal myb-specific RNA species and myb-related polypeptides were found to be expressed in these cells, suggesting that transformation of CEF by v-myb might require alterations of the oncogene product.
Animals↗
myb-specific probes.
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Avian Myeloblastosis Virus↗
A cryptic transcription promoter in the myb oncogene of avian myeloblastosis virus.
The potential regulatory signals contained in the v-myb oncogene of avian myeloblastosis virus have been inserted upstream to the herpes simplex type 1 thymidine kinase gene in order to test their promoter activity. The isolation of TK+ transformants after transfection of clone 1D(TK-) mouse cells with the resulting recombinant DNAs indicated that the expression of the TK gene was made possible by the myb-derived sequences. Analysis of the TK specific RNA expressed in different TK+ transformants revealed that the regulatory signals contained in v-myb correspond to a weak functional promoter.
Avian Leukosis Virus↗