MECHANISM OF SPLICING REGULATION BY THE MEIOSIS ENHANCER FACTOR Mer1p IN YEAST Saccharomyces cerevisiae

by Armisen Garrido, Javier

In eukaryotes, genes are presented in a series of coding and non-coding DNA regions (exons/introns) that are transcribed into a premature RNA (pre-mRNA). Introns can be removed from the mature premRNA, before its translation into proteins, in a process called splicing. The splicing reaction occurs in two highly regulated transesterification reactions inside of the cell nucleus, and it is catalyzed by the Spliceosome, involving the binding and release of five small nuclear ribonucleoprotein particles (snRNPs). While some introns are constitutively spliced, others can be alternatively spliced, giving different exon combinations and therefore different proteins, increasing the protein diversity of the species. In humans, misregulation of alternative splicing can result in the production of aberrant proteins, some of which may produce cancer or other severe diseases. In yeast, alternative splicing is regulated by different splicing factors, such as Mer1p. Mer1p is expressed during meiosis in the yeast Saccharomyces cerevisiae and activates the splicing in at least three different genes (AMA1, MER2, and MER3), which contain a conserved intronic splicing enhancer sequence. Previous results have shown that Mer1p is able to interact with the pre-mRNA and with specific proteins of the U1 and U2 snRNPs. However, the specific molecular mechanisms by which Mer1p activates splicing remained unknown. The objective of this work is to determine how Mer1p regulates the splicing of its targets, and how different splicing factors modulate Mer1p activity. II Using biochemistry and genetics, the data presented in this work indicate that Mer1p recruits the snRNPs U1, U2 and U6, to pre-mRNA. This recruitment of the snRNPs is dependent of the U1 snRNP protein Nam8p and the U2 snRNP protein Snu17p, but independent on the branchpoint region or ATP. Furthermore, Mer1p accelerates and stabilizes the formation of the early complexes of the spliceosome. Finally, U1 and U2 are recruited to the pre-mRNA at the same time, emerging a new alternative hypothesis of splicing regulation that can be applied to other enhancer regulators and that differs from the classical model of stepwise assembly of the snRNP. III Dedicado con mucho cariño a mis padres Enrique y Cristina y a mis hermanos, Enrique, Carolina, Cristina, Guillermo, David, Juan Jose, Rocio y Virginia, por estar siempre conmigo y apoyarme en todo momento. También se lo dedico a Marie-Chistine, por su cariño y dedicación A ellos GRACIAS. IV