Biomedical subjects

Paul Ajuh

Publications and source records attributed to Paul Ajuh.

5 recordsLinked to original sources

PubMed2005

SNEV is an evolutionarily conserved splicing factor whose oligomerization is necessary for spliceosome assembly.

We have isolated the human protein SNEV as downregulated in replicatively senescent cells. Sequence homology to the yeast splicing factor Prp19 suggested that SNEV might be the orthologue of Prp19 and therefore might also be involved in pre-mRNA splicing. We have used various approaches including gene complementation studies in yeast using a temperature sensitive mutant with a pleiotropic phenotype and SNEV immunodepletion from human HeLa nuclear extracts to determine its function. A human-yeast chimera was indeed capable of restoring the wild-type phenotype of the yeast mutant strain. In addition, immunodepletion of SNEV from human nuclear extracts resulted in a decrease of in vitro pre-mRNA splicing efficiency. Furthermore, as part of our analysis of protein-protein interactions within the CDC5L complex, we found that SNEV interacts with itself. The self-interaction domain was mapped to amino acids 56-74 in the protein's sequence and synthetic peptides derived from this region inhibit in vitro splicing by surprisingly interfering with spliceosome formation and stability. These results indicate that SNEV is the human orthologue of yeast PRP19, functions in splicing and that homo-oligomerization of SNEV in HeLa nuclear extract is essential for spliceosome assembly and that it might also be important for spliceosome stability.

Amino Acid Sequence↗

PubMed2005

Interaction of U-box E3 ligase SNEV with PSMB4, the beta7 subunit of the 20 S proteasome.

Recognition of specific substrates for degradation by the ubiquitin-proteasome pathway is ensured by a cascade of ubiquitin transferases E1, E2 and E3. The mechanism by which the target proteins are transported to the proteasome is not clear, but two yeast E3s and one mammalian E3 ligase seem to be involved in the delivery of targets to the proteasome, by escorting them and by binding to the 19 S regulatory particle of the proteasome. In the present study, we show that SNEV (senescence evasion factor), a protein with in vitro E3 ligase activity, which is also involved in DNA repair and splicing, associates with the proteasome by directly binding to the beta7 subunit of the 20 S proteasome. Upon inhibition of proteasome activity, SNEV does not accumulate within the cells although its co-localization with the proteasome increases significantly. Since immunofluorescence microscopy also shows increased co-localization of SNEV with ubiquitin after proteasome inhibition, without SNEV being ubiquitinated by itself, we suggest that SNEV shows E3 ligase activity not only in vitro but also in vivo and escorts its substrate to the proteasome. Since the yeast homologue of SNEV, Prp19, also interacts with the yeast beta7 subunit of the proteasome, this mechanism seems to be conserved during evolution. Therefore these results support the hypothesis that E3 ligases might generally be involved in substrate transport to the proteasome. Additionally, our results provide the first evidence for a physical link between components of the ubiquitin-proteasome system and the spliceosome.

Amino Acid Sequence↗

PubMed2003

Identification of peptide inhibitors of pre-mRNA splicing derived from the essential interaction domains of CDC5L and PLRG1.

CDC5L and PLRG1 are both spliceosomal proteins that are highly conserved across species. They have both been shown to be part of sub- spliceosomal protein complexes that are essential for pre-mRNA splicing in yeast and humans. CDC5L and PLRG1 interact directly in vitro. This interaction is mediated by WD40 regions in PLRG1 and the C-terminal domain of CDC5L. In order to determine whether this interaction is important for the splicing mechanism, we have designed peptides corresponding to highly conserved sequences in the interaction domains of both proteins. These peptides were used in in vitro splicing experiments as competitors to the cognate sequences in the endogenous proteins. Certain peptides derived from the binding domains of both proteins were found to inhibit in vitro splicing. This splicing inhibition could be prevented by preincubating the peptides with the corresponding partner protein that had been expressed in Escherichia coli. The results from this study indicate that the interaction between CDC5L and PLRG1 is essential for pre-mRNA splicing and further demonstrate that small peptides can be used as effective splicing inhibitors.

Amino Acid Sequence↗

PubMed2003

hLodestar/HuF2 interacts with CDC5L and is involved in pre-mRNA splicing.

hLodestar/HuF2 belongs to the SNF2 family of proteins. This family of proteins has been shown to play a critical role in altering protein-DNA interactions in a variety of cellular contexts. We have identified an unexpected interaction between hLodestar/HuF2 and CDC5L in both the yeast two-hybrid system and HeLa nuclear extract. CDC5L is a well-characterized pre-mRNA splicing factor in yeast and humans. Our findings demonstrate that hLodestar/HuF2 associates with human splicing complexes. We also found that a truncated hLodestar/HuF2 polypeptide that overlaps with the CDC5L-binding region can inhibit pre-mRNA splicing by disrupting spliceosome assembly. These findings indicate that hLodestar/HuF2 may have a role in pre-mRNA splicing. These data are consistent with a close co-ordination of the transcription and splicing pathways in eukaryotes. Although many members of the DExH/D helicase superfamily have been linked to pre-mRNA splicing, this is the first SNF2 family member to be implicated in this pathway.

Adenosine Triphosphatases↗

PubMed2002

A novel function for human factor C1 (HCF-1), a host protein required for herpes simplex virus infection, in pre-mRNA splicing.

Human factor C1 (HCF-1) is needed for the expression of herpes simplex virus 1 (HSV-1) immediate-early genes in infected mammalian cells. Here, we provide evidence that HCF-1 is required for spliceosome assembly and splicing in mammalian nuclear extracts. HCF-1 interacts with complexes containing splicing snRNPs in uninfected mammalian cells and is a stable component of the spliceosome complex. We show that a missense mutation in HCF-1 in the BHK21 hamster cell line tsBN67, at the non-permissive temperature, inhibits the protein's interaction with U1 and U5 splicing snRNPs, causes inefficient spliceosome assembly and inhibits splicing. Transient expression of wild-type HCF-1 in tsBN67 cells restores splicing at the non-permissive temperature. The inhibition of splicing in tsBN67 cells correlates with the temperature-sensitive cell cycle arrest phenotype, suggesting that HCF-1-dependent splicing events may be required for cell cycle progression.

Cell Nucleus↗