BETA-TROPOMYOSIN PRE-MESSENGER-RNA FOLDING AROUND A MUSCLE-SPECIFIC EXON INTERFERES WITH SEVERAL STEPS OF SPLICEOSOME ASSEMBLY

The chicken beta-tropomyosin pre-mRNA is spliced in a tissue-specific manner. Internal exons 6B and 6A are specifically used in skeletal mus cle and non-skeletal muscle cells, respectively. Pre-mRNA secondary st ructure around exon 6B has been shown to be part of the mechanism tl;a t inhibits exon 6B to 7 splicing in HeLa nuclear extract. We analyse t he influence of pre-mRNA folding on the different steps of spliceosome assembly under different conditions. At 3 mM MgCl2, conditions that f avour RNA structure formation, the interactions of U1, U2, U4, U5 and U6 small nuclear ribonucleoprotein particles (snRNPs) with the pre-mRN A are all affected. The study of several mutants destabilising some pr oposed stem-loop structures shows that the in vitro splicing activatio n is correlated with an increased binding of snRNPs on pre-mRNA molecu les. At 1 mM MgCl2, conditions that allow a partial relaxation of the inhibitory structure, U1 snRNP binding on exon 6B 5' splice site occur s very efficiently Nonetheless, if this first step of spliceosome asse mbly is derepressed, U2, U4, U5 and U6 snRNP interaction processes rem ain inhibited. Altogether, these results suggest that the choice betwe en exon 6A and 6B donor sites is a complex process not simply directed by a difference in the efficiency of interaction between U1 snRNP and alternative 5' splice sites. (C) 1995 Academic Press Limited