Novel splice variant of mouse insulin2 mRNA: Implications for insulin expression

Insulin is a secreted peptide that controls glucose homeostasis in mammals, and insulin biosynthesis is regulated by glucose at many levels. Rodent insulin is encoded by two non-allelic genes. We have identified a novel splice variant of the insulin2 gene in mice that constitutes about 75% of total insulin2 mRNA. The alternate splicing does not alter the ORF but reduces the 5′UTR by 12 bases. A reporter gene containing the novel short 5′UTR, is more efficiently expressed in cells, suggesting that alternative splicing of insulin mRNA in mice could result in an additional level of regulation in insulin biosynthesis.     

Interaction with Polyglutamine Aggregates Reveals a Q/N-rich Domain in TDP-43

The identification of pathologic TDP-43 aggregates in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration, followed by the discovery of dominantly inherited point mutations in TDP-43 in familial ALS, have been critical insights into the mechanism of these untreatable neurodegenerative diseases. However, the biochemical basis of TDP-43 aggregation and the mechanism of how mutations in TDP-43 lead to disease remain enigmatic. In efforts to understand how TDP-43 alters its cellular localization in response to proteotoxic stress, we found that TDP-43 is sequestered into polyglutamine aggregates. Furthermore, we found that binding to polyglutamine aggregates requires a previously uncharacterized glutamine/asparagine (Q/N)-rich region in the C-terminal domain of TDP-43. Sequestration into polyglutamine aggregates causes TDP-43 to be cleared from the nucleus and become detergent-insoluble. Finally, we observed that sequestration into polyglutamine aggregates led to loss of TDP-43-mediated splicing in the nucleus and that polyglutamine toxicity could be partially rescued by increasing expression of TDP-43. These data indicate pathologic sequestration into polyglutamine aggregates, and loss of nuclear TDP-43 function may play an unexpected role in polyglutamine disease pathogenesis. Furthermore, as Q/N domains have a strong tendency to self-aggregate and in some cases can function as prions, the identification of a Q/N domain in TDP-43 has important implications for the mechanism of pathologic aggregation of TDP-43 in ALS and other neurodegenerative diseases.     

Silent mutations in the gene encoding the p53 protein are preferentially located in conserved amino acid positions and splicing enhancers

The last release of p53 somatic mutation database contains more than 20,000 of mutation among which 951 are silent (synonymous). This striking amount of silent mutations is much more than what would be expected if synonymous mutations were effectively neutral. The prevalent explanation to reconcile this vast amount of silent mutations with the neutral expectation is that they are just the subproduct of the hypermutability process that affect cancer cells. Some evidences have been presented in this direction, and the explanation has been taken as granted. Assuming that silent mutations are effectively neutral has major implication in the investigation of mutational processes that affect the gene encoding the p53 protein, since on the basis of this assumption they are considered the Null hypothesis, for instance for measuring and comparing among tissues the endogenous mutability. From this it follows that determining whether silent mutations in the p53 gene, and in all disease genes in general, are or not basically mutational noise, is of paramount importance.

In this paper we readdress this topic by testing whether there is a relationship between the spatial distribution of silent mutations inside the p53 gene and functional significant features of the gene. For this purpose we divided the population of silent mutations in three groups: those that are found accompanied by other mutations (doublets and multiplest), those that were isolated as singlets, but the same mutation was also isolated as being part of a doublet (or multiplet) in another individual. And the last group is composed by those that were always found as singlets and never as being part of a doublet or a multiplet. This last group was expected to be enriched in functionally significant silent mutations. We found that all silent mutations, but particularly those of the last group, are preferentially located in conserved amino acid positions (i.e. functionally important amino acids) and also tend to be located inside suspected splicing enhancers. Noteworthy, this association remains even after eliminating the possible contribution of mutation hotspots. Besides, we present additional evidence in the direction that these putative splicing enhancers are real functional enhancers.     

Archaeal pre-mRNA splicing: a connection to hetero-oligomeric splicing endonuclease

Eukaryotic Cbf5 is a protein subunit of the small nucleolar RNA-protein complex. Previously, we identified, in archaeal homologs of cbf5 of the crenarchaea, Aeropyrum pernix, Sulfolobus solfataricus, and Sulfolobus tokodaii, the first examples of introns of archaeal protein-coding genes. Here, we report the immunological detection of Cbf5 protein of S. tokodaii, the product of the spliced cbf5 mRNA. The hetero-oligomeric splicing endonuclease activity from recombinant S. tokodaii subunits cleaved at the exon-intron boundaries of cbf5 pre-mRNA fragments,suggesting that synthesis of full-length Cbf5 protein requires this activity. Database searches and PCR screens identified additional cbf5 introns in some, but not all sequenced crenarchaeal genomes. The predicted secondary structures of exon-intron boundaries of many of the newly identified intron-containing cbf5 pre-mRNAs contained relaxed forms of the bulge-helix-bulge motif similar to that of S. tokodaii. These observations are consistent with previous reports indicating that subunit composition of the splicing endonuclease contributes to substrate specificity.     

Regulation of Vascular Endothelial Growth Factor (VEGF) Splicing from Pro-angiogenic to Anti-angiogenic Isoforms: A NOVEL THERAPEUTIC STRATEGY FOR ANGIOGENESIS*

Vascular endothelial growth factor (VEGF) is produced either as a pro-angiogenic or anti-angiogenic protein depending upon splice site choice in the terminal, eighth exon. Proximal splice site selection (PSS) in exon 8 generates pro-angiogenic isoforms such as VEGF₁₆₅, and distal splice site selection (DSS) results in anti-angiogenic isoforms such as VEGF₁₆₅b. Cellular decisions on splice site selection depend upon the activity of RNA-binding splice factors, such as ASF/SF2, which have previously been shown to regulate VEGF splice site choice. To determine the mechanism by which the pro-angiogenic splice site choice is mediated, we investigated the effect of inhibition of ASF/SF2 phosphorylation by SR protein kinases (SRPK1/2) on splice site choice in epithelial cells and in in vivo angiogenesis models. Epithelial cells treated with insulin-like growth factor-1 (IGF-1) increased PSS and produced more VEGF₁₆₅ and less VEGF₁₆₅b. This down-regulation of DSS and increased PSS was blocked by protein kinase C inhibition and SRPK1/2 inhibition. IGF-1 treatment resulted in nuclear localization of ASF/SF2, which was blocked by SPRK1/2 inhibition. Pull-down assay and RNA immunoprecipitation using VEGF mRNA sequences identified an 11-nucleotide sequence required for ASF/SF2 binding. Injection of an SRPK1/2 inhibitor reduced angiogenesis in a mouse model of retinal neovascularization, suggesting that regulation of alternative splicing could be a potential therapeutic strategy in angiogenic pathologies.