Biomedical subjects

Gil Ast

Publications and source records attributed to Gil Ast.

22 records · Page 2Linked to original sources

PubMed2003

The U1 snRNP base pairs with the 5' splice site within a penta-snRNP complex.

Recognition of the 5' splice site is an important step in mRNA splicing. To examine whether U1 approaches the 5' splice site as a solitary snRNP or as part of a multi-snRNP complex, we used a simplified in vitro system in which a short RNA containing the 5' splice site sequence served as a substrate in a binding reaction. This system allowed us to study the interactions of the snRNPs with the 5' splice site without the effect of other cis-regulatory elements of precursor mRNA. We found that in HeLa cell nuclear extracts, five spliceosomal snRNPs form a complex that specifically binds the 5' splice site through base pairing with the 5' end of U1. This system can accommodate RNA-RNA rearrangements in which U5 replaces U1 binding to the 5' splice site, a process that occurs naturally during the splicing reaction. The complex in which U1 and the 5' splice site are base paired sediments in the 200S fraction of a glycerol gradient together with all five spliceosomal snRNPs. This fraction is functional in mRNA spliceosome assembly when supplemented with soluble nuclear proteins. The results argue that U1 can bind the 5' splice site in a mammalian preassembled penta-snRNP complex.

Alternative Splicing↗

PubMed2003

Drug-targeting strategies for prostate cancer.

Prostate cancer is the most frequently diagnosed cancer in North American men and accounts for 10% of cancer-related deaths in men. Despite advances in early detection and aggressive treatment of early disease, the overall mortality rate has not appear to have fallen, indicating that the current therapies are not beneficial for life expectancy and new strategies are required. Prostate cancer is a dynamic evolving process that develops in distinct steps, with each step liable to additional genetic hits that change the cancer cell phenotype and alter the patterns of gene expression. The molecular events in prostate cancer are beginning to be understood, including altered expression of tumor suppressor genes, pro- and anti-apoptotic genes, and oncogenes associated with the progression of the disease; and specific genes that are expressed predominantly or exclusively in prostate cells, prostate cancer cells, and prostate metastasis cells. These latter genes on the level of DNA, RNA and protein products are the targets of several new approaches to prostate cancer therapy and are the focus of this review.

Antisense Elements (Genetics)↗

PubMed2002

Reversible inhibition of the second step of splicing suggests a possible role of zinc in the second step of splicing.

A multicomponent complex of proteins and RNA is assembled on the newly synthesized pre-mRNA to form the spliceosome. This complex catalyzes a two-step transesterification reaction required to remove the introns and ligate the exons. To date, only six proteins have been found necessary for the second step of splicing in yeast, and their human homologs have been identified. We demonstrate that the addition of the selective chelator of zinc, 1,10-phenanthroline, to an in vitro mRNA splicing reaction causes a dose-dependent inhibition of the second step of splicing. This inhibition is accompanied by the accumulation of spliceosomes paused before completion of step two of the splicing reaction. The inhibition effect on the second step is due neither to snRNA degradation nor to direct binding to the mRNA, and is reversible by dialysis or add-back of zinc, but not of other divalent metals, at the beginning of the reaction. These findings suggest that the activity of a putative zinc-dependent metalloprotein(s) involved in the second step of splicing is affected. This study outlines a new method for specific reversible inhibition of the second step of splicing using external reagents, and suggests a possible role of divalent cations in the second step of mRNA splicing, most likely zinc.

Chelating Agents↗

PubMed2002

Alu-containing exons are alternatively spliced.

Alu repetitive elements are found in approximately 1.4 million copies in the human genome, comprising more than one-tenth of it. Numerous studies describe exonizations of Alu elements, that is, splicing-mediated insertions of parts of Alu sequences into mature mRNAs. To study the connection between the exonization of Alu elements and alternative splicing, we used a database of ESTs and cDNAs aligned to the human genome. We compiled two exon sets, one of 1176 alternatively spliced internal exons, and another of 4151 constitutively spliced internal exons. Sixty one alternatively spliced internal exons (5.2%) had a significant BLAST hit to an Alu sequence, but none of the constitutively spliced internal exons had such a hit. The vast majority (84%) of the Alu-containing exons that appeared within the coding region of mRNAs caused a frame-shift or a premature termination codon. Alu-containing exons were included in transcripts at lower frequencies than alternatively spliced exons that do not contain an Alu sequence. These results indicate that internal exons that contain an Alu sequence are predominantly, if not exclusively, alternatively spliced. Presumably, evolutionary events that cause a constitutive insertion of an Alu sequence into an mRNA are deleterious and selected against.

Alternative Splicing↗