La elongación por la RNA polimerasa II afecta las decisiones de splicing alternativo

DE LA MATA, MANUEL; ALONSO, CLAUDIO R.; KORNBLIHTT, ALBERTO R.

Carlos Paz, Córdoba, Argentina

Congreso; XXXVIII Reunión Anual de la SOCIEDAD ARGENTINA DE INVESTIGACION BIOQUIMICA Y BIOLOGIA MOLECULAR (SAIB); 2002

SOCIEDAD ARGENTINA DE INVESTIGACION BIOQUIMICA Y BIOLOGIA MOLECULAR (SAIB)

Alternative splicing is a highly regulated process which is dependent upon transcriptional regulation. In this study we demonstrate that splice site usage is modified by changes in RNAPII elongation capacity, and that this effect  is observed in genes subjected to different modes of alternative splicing. Our work is based on a strategy that allows us to express different mutants of RNAPII in cells whose endogenous RNAPII is inhibited by a-amanitin. The RNAPII  mutant assesed  in this study (C4 mutant) carries a point mutation in the RNAPII major subunit conferring a reduced transcriptional elongation capacity and a second point mutation conferring a-amanitin resistance. Hepatoma Hep3B cells were co-transfected with different minigenes and either the C4 mutant or a "wt" RNAPII carrying only the a-amanitin resistance point mutation. The C4 mutant was able to modify the exon skipping-exon inclusion pattern of a fibronectin minigene alternative exon as well as the adenovirus E1a isoform pattern by altering the 5´ splice site selection.  Additionaly, Drosophila mutants for RNAPII (C4 mutant) or TFIIH (haywire) loci that display the Ubx effect  show changes in the UBX  alternative splicing pattern.Taken together our results support a kinetic coupling model where transcript elongation determines the decisions between  multiple competing splicing sites.  Slow RNAPII elongation favors exon skipping  and distal 5´ splice site usage in genes subjected  to exon definition and alternative 5´ ss splicing respectively.