Biomedical subjects

Julian P Venables

Publications and source records attributed to Julian P Venables.

10 recordsLinked to original sources

PubMed2007

Alternative RNA splicing complexes containing the scaffold attachment factor SAFB2.

The scaffold attachment factor SAFB1 and its recently discovered homologue SAFB2 might provide an important link between pre-mRNA splicing, intracellular signalling and transcription. Using novel mono-specific antisera, we found endogenous SAFB2 protein has a different spatial distribution from SAFB1 within the nucleus where it is found in much larger nuclear complexes (up to 670 kDa in size), and a distinct pattern of expression in adult human testis. By contrast, SAFB1 protein predominantly exists either as smaller complexes or as a monomeric protein. Our results suggest stable core complexes containing components comprised of SAFB1, SAFB2 and the RNA binding proteins Sam68 and hnRNPG exist in parallel with free SAFB1 protein. We found that SAFB2 protein, like SAFB1, acts as a negative regulator of a tra2beta variable exon. Despite showing an involvement in splicing, we detected no stable interaction between SAFB proteins and SR or SR-related splicing regulators, although these were also found in stable higher molecular mass complexes. Each of the detected alternative splicing regulator complexes exists independently of intact nucleic acids, suggesting they might be pre-assembled and recruited to nascent transcripts as modules to facilitate alternative splicing, and/or they represent nuclear storage compartments from which active proteins are recruited.

Adaptor Proteins, Signal Transducing↗

PubMed2006

EASI--enrichment of alternatively spliced isoforms.

Alternative splicing produces more than one protein from the majority of genes and the rarer forms can have dominant functions. Instability of alternative transcripts can also hinder the study of regulation of gene expression by alternative splicing. To investigate the true extent of alternative splicing we have developed a simple method of enriching alternatively spliced isoforms (EASI) from PCRs using beads charged with Thermus aquaticus single-stranded DNA-binding protein (T.Aq ssb). This directly purifies the single-stranded regions of heteroduplexes between alternative splices formed in the PCR, enabling direct sequencing of all the rare alternative splice forms of any gene. As a proof of principle the alternative transcripts of three tumour suppressor genes, TP53, MLH1 and MSH2, were isolated from testis cDNA. These contain missing exons, cryptic splice sites or include completely novel exons. EASI beads are stable for months in the fridge and can be easily combined with standard protocols to speed the cloning of novel transcripts.

Alternative Splicing↗

PubMed2006

Unbalanced alternative splicing and its significance in cancer.

Alternative pre-mRNA splicing leads to distinct products of gene expression in development and disease. Antagonistic splice variants of genes involved in differentiation, apoptosis, invasion and metastasis often exist in a delicate equilibrium that is found to be perturbed in tumours. In several recent examples, splice variants that are overexpressed in cancer are expressed as hyper-oncogenic proteins, which often correlate with poor prognosis, thus suggesting improved diagnosis and follow up treatment. Global gene expression technologies are just beginning to decipher the interplay between alternatively spliced isoforms and protein-splicing factors that will lead to identification of the mutations in these trans-acting factors responsible for pathogenic alternative splicing in cancer.

Alternative Splicing↗

PubMed2006

Atypical haemolytic uraemic syndrome associated with a hybrid complement gene.

BACKGROUND: Sequence analysis of the regulators of complement activation (RCA) cluster of genes at chromosome position 1q32 shows evidence of several large genomic duplications. These duplications have resulted in a high degree of sequence identity between the gene for factor H (CFH) and the genes for the five factor H-related proteins (CFHL1-5; aliases CFHR1-5). CFH mutations have been described in association with atypical haemolytic uraemic syndrome (aHUS). The majority of the mutations are missense changes that cluster in the C-terminal region and impair the ability of factor H to regulate surface-bound C3b. Some have arisen as a result of gene conversion between CFH and CFHL1. In this study we tested the hypothesis that nonallelic homologous recombination between low-copy repeats in the RCA cluster could result in the formation of a hybrid CFH/CFHL1 gene that predisposes to the development of aHUS. METHODS AND FINDINGS: In a family with many cases of aHUS that segregate with the RCA cluster we used cDNA analysis, gene sequencing, and Southern blotting to show that affected individuals carry a heterozygous CFH/CFHL1 hybrid gene in which exons 1-21 are derived from CFH and exons 22/23 from CFHL1. This hybrid encodes a protein product identical to a functionally significant CFH mutant (c.3572C>T, S1191L and c.3590T>C, V1197A) that has been previously described in association with aHUS. CONCLUSIONS: CFH mutation screening is recommended in all aHUS patients prior to renal transplantation because of the high risk of disease recurrence post-transplant in those known to have a CFH mutation. Because of our finding it will be necessary to implement additional screening strategies that will detect a hybrid CFH/CFHL1 gene.

Adult↗

PubMed2005

Up-regulation of the ubiquitous alternative splicing factor Tra2beta causes inclusion of a germ cell-specific exon.

We have discovered a new exon of the homeodomain-interacting kinase HipK3 that incorporates a premature stop codon and is included only in the human testis. To investigate this, we tested the effects of transfecting cells with green fluorescent protein fusions of RNA-binding proteins implicated in spermatogenesis using a novel assay based on multi-fraction fluorescence-activated cell sorting (MF-FACS). This allows the effect of a controlled titration of any splicing factor on the splicing of endogenous genes to be studied in vivo. We found that Tra2beta recapitulates testis-specific splicing of endogenous HipK3 in a concentration-dependent manner and binds specifically to a long purine-rich sequence in the novel exon. This sequence was also specifically bound by hnRNP A1, hnRNP H, ASF/SF2 and SRp40, but not by 9G8. Consistent with these observations, in vitro studies showed that this sequence shifts splicing to a downstream 5' splice site within a heterologous pre-mRNA substrate in the presence of Tra2beta, ASF/SF2 and SRp40, whereas hnRNP A1 specifically inhibits this choice. By mutating the purine-rich sequence in the context of the HipK3 gene, we also show that it is the major determinant of Tra2beta- and hnRNP A1-mediated regulation. Tra2 is essential for sex determination and spermatogenesis in flies, and Tra2beta protein was most highly expressed in testis out of six mouse tissues, whereas hnRNP A1 is down-regulated during germ cell development. Therefore, our data imply an evolutionarily conserved role for Tra2 proteins in spermatogenesis and suggest that an elevated concentration of Tra2beta may convert it into a tissue-specific splicing factor.

Alternative Splicing↗

PubMed2004

Aberrant and alternative splicing in cancer.

Pre-mRNA splicing is a sophisticated and ubiquitous nuclear process, which is a natural source of cancer-causing errors in gene expression. Intronic splice site mutations of tumor suppressor genes often cause exon-skipping events that truncate proteins just like classical nonsense mutations. Also, many studies over the last 20 years have reported cancer-specific alternative splicing in the absence of genomic mutations. Affected proteins include transcription factors, cell signal transducers, and components of the extracellular matrix. Antibodies against alternatively spliced products on cancer cells are currently in clinical trials, and competitive reverse transcription-PCR across regions of alternative splicing is being used as a simple diagnostic test. As well as being associated with cancer, the nature of the alternative gene products is usually consistent with an active role in cancer; therefore, the alternative splicing process itself is a potential target for gene therapy.

Alternative Splicing↗

PubMed2004

SIAH1 targets the alternative splicing factor T-STAR for degradation by the proteasome.

T-STAR is one of three members of the SAM68 family of RNA-binding proteins that have been shown to be involved in various gene expression pathways including the control of pre-mRNA splicing. We employed a two-hybrid screen to identify proteins that interact with human T-STAR. The predominant interacting proteins were the E3 ubiquitin ligases SIAH1 and SIAH2. We found that SIAH1 bound to an octapeptide sequence in T-STAR targeting it for proteasome-dependent degradation. Rodent T-STAR orthologues (also known as etoile or SLM2) were not targeted for degradation by SIAH1. However a double amino acid substitution of mouse T-STAR that mimics the human SIAH1-binding site brought mouse T-STAR under in vivo control of SIAH1. Using a minigene transfection assay for alternative splicing activity we showed that human T-STAR, like its rodent orthologues can influence splice site choice and that human, but not mouse, T-STAR-dependent alternative splicing is modulated by SIAH1. Western blots of protein from purified germ cells indicated that SIAH1 protein expression peaks in meiosis. In mouse, T-STAR is co-expressed with SIAH1 during meiosis but, in humans, T-STAR is only strongly expressed after meiosis. Comparative sequence analysis showed SIAH-mediated proteasomal degradation of T-STAR has evolved in the primate lineage. Collectively these data suggest that SIAH-mediated down regulation of alternative splicing may be an important developmental difference between otherwise highly conserved T-STAR proteins.

Alternative Splicing↗

PubMed2004

Ca(2+)-promoted cyclin B1 degradation in mouse oocytes requires the establishment of a metaphase arrest.

CDK1-cyclin B1 is a universal cell cycle kinase required for mitotic/meiotic cell cycle entry and its activity needs to decline for mitotic/meiotic exit. During their maturation, mouse oocytes proceed through meiosis I and arrest at second meiotic metaphase with high CDK1-cyclin B1 activity. Meiotic arrest is achieved by the action of a cytostatic factor (CSF), which reduces cyclin B1 degradation. Meiotic arrest is broken by a Ca2+ signal from the sperm that accelerates it. Here we visualised degradation of cyclin B1::GFP in oocytes and found that its degradation rate was the same for both meiotic divisions. Ca2+ was the necessary and sufficient trigger for cyclin B1 destruction during meiosis II; but it played no role during meiosis I and furthermore could not accelerate cyclin B1 destruction during this time. The ability of Ca2+ to trigger cyclin B1 destruction developed in oocytes following a restabilisation of cyclin B1 levels at about 12 h of culture. This was independent of actual first polar body extrusion. Thus, in metaphase I arrested oocytes, Ca2+ would induce cyclin B1 destruction and the first polar body would be extruded. In contrast to some reports in lower species, we found no evidence that oocyte activation was associated with an increase in 26S proteasome activity. We therefore conclude that Ca2+ mediates cyclin B1 degradation by increasing the activity of an E3 ubiquitin ligase. However, this stimulation occurs only in the presence of the ubiquitin ligase inhibitor CSF. We propose a model in which Ca2+ directly stimulates destruction of CSF during mammalian fertilisation.

Animals↗

PubMed2003

Tr-kit promotes the formation of a multimolecular complex composed by Fyn, PLCgamma1 and Sam68.

Tr-kit is a truncated form of the tyrosine kinase receptor c-kit expressed in the haploid phase of spermatogenesis. Upon microinjection, tr-kit triggers metaphase-to-anaphase transition in mouse eggs by the sequential activation of Fyn and PLCgamma1. Here, we show that tr-kit promotes the interaction of several tyrosine-phosphorylated proteins with the SH3 domain of PLCgamma1. Western blot analysis indicates that one of these proteins is Sam68, an RNA-binding protein that is known to interact with and be phosphorylated by Src-like kinases in mitosis. tr-kit promotes the association of Sam68 with PLCgamma1 and Fyn in a multimolecular complex, as demonstrated by co-immunoprecipitation of the phosphorylated forms of these proteins using antibodies directed to anyone of the partners of the complex. Expression of tr-kit potentiates the interaction of endogenous Sam68 also with the SH3 domain of Fyn. Furthermore, the subcellular localization of Sam68 is affected by tr-kit through activation of Fyn in live cells. Lastly, we show that interaction with the SH3 domain of Fyn triggers the release of Sam68 from bound RNA. Thus, our data suggest that tr-kit promotes the formation of a multimolecular complex composed of Fyn, PLCgamma1 and Sam68, which allows phosphorylation of PLCgamma1 by Fyn, and may modulate RNA metabolism.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Alternative splicing in the testes.

Germ-cell differentiation is an ideal process for studying the effects of alternative splicing and there are examples of alternative splicing of genes involved in gene regulation and signal transduction at every stage of the spermatogenic pathway. A network of testes-specific splicing factor interactions has been uncovered and combining our knowledge of these RNAs and proteins should lead to an understanding of the regulation of alternative splicing and male fertility.

Alternative Splicing↗