A T-TO-G-MUTATION IN THE POLYPYRIMIDINE TRACT OF THE 2ND INTRON OF THE HUMAN BETA-GLOBIN GENE REDUCES IN-VITRO SPLICING EFFICIENCY - EVIDENCE FOR AN INCREASED HNRNP-C INTERACTION

In a patient with a beta-thalassemia intermedia, a mutation was identi fied in the second intron of the human beta-globin gene, The U-->G mut ation is located within the polypyrimidine tract at position -8 upstre am of the 3' splice site, In vivo, this mutation leads to decreased le vels of the hemoglobin protein, Because of the location of the mutatio n and the role of the polypyrimidine tract in the splicing process, we performed in vitro splicing assays on the pre-messenger RNA (pre-RNA) , We found that the splicing efficiency of the mutant pre-mRNA is redu ced compared to the wild type and that no cryptic splice sites are act ivated, Analysis of splicing complex formation shows that the U-->G mu tation affects predominantly the progression of the H complex towards the pre-spliceosome complex, By cross-linking and immunoprecipitation assays, we show that the hnRNP C protein interacts more efficiently wi th the mutant precursor than with the wild-type, This stronger interac tion could play a role, directly or indirectly, in the decreased splic ing efficiency.