Competition between a noncoding exon and introns: Gomafu contains tandem UACUAAC repeats and associates with splicing factor-1.
Bottom Line: Unexpectedly, we found that all Gomafu RNA examined shared a distinctive feature: tandem repeats of UACUAAC, a sequence that has been identified as a conserved intron branch point in the yeast Saccharomyces cerevisiae.The tandem UACUAAC Gomafu RNA repeats bind to the SF1 splicing factor with a higher affinity than the divergent branch point sequence in mammals, which affects the kinetics of the splicing reaction in vitro.We propose that the Gomafu RNA regulates splicing efficiency by changing the local concentration of splicing factors within the nucleus.
Affiliation: Nakagawa Initiative Research Unit, RIKEN Advanced Science Institute, Hirosawa, Wako, Saitama, Japan.Show MeSH
Mentions: Considering the interaction between the Gomafu RNA and SF1, we hypothesized that the Gomafu RNA might regulate splicing by competing locally with the branch point sequences of pre-mRNAs for the splicing factor SF1. To confirm this hypothesis, we examined the effect of UACUAAC tandem repeats in the Gomafu RNA on the splicing reaction and spliceosome formation in vitro. We first used a model pre-mRNA substrate derived from mouse IgM (Watakabe et al. 1993), which possesses a predictably weak branch point with a degenerate sequence (Guth & Valcarcel 2000). As expected, the addition of the Gomafu repeat oligonucleotides when compared with the control oligonucleotides markedly delayed production of the spliced product (Fig. 4A). We then used another pre-mRNA substrate with strong intron consensus sequences derived from adenovirus (Zapp & Berget 1989). In this case, both the control and Gomafu repeat oligonucleotides inhibited the splicing reaction to some extent; however, no differences were found between the two conditions (Fig. 4B). These results were consistent with previous reports showing that BBP and SF1 are not essential for the splicing reaction itself but are required for optimal removal of introns with sub-optimal consensus sequences (Guth & Valcarcel 2000; Rutz & Seraphin 2000; Tanackovic & Kramer 2005). We then examined whether the Gomafu repeat oligonucleotides inhibit IgM pre-mRNA splicing in a dose-dependent manner. In this experiment, the splicing reaction was performed in an increasing amount of oligonucleotides for a fixed time (60 min). As expected, control oligonucleotides did not inhibit pre-mRNA splicing within a range of 1.25–10 pmol/reaction. On the other hand, inhibitory effect was recognizable with Gomafu repeat oligonucleotitdes as little as 2.5 pmol/reaction, which became clearer with an increased amount of the oligonucleotides (Fig. 4C). We also examined the effect of the Gomafu repeat oligonucleotides on the formation of a spliceosome complex using native gels. Although we did not observe obvious delay in the formation of H/E or A complex, the formation of B complex was markedly delayed in the presence of the Gomafu repeat oligonucleotides (Fig. 4D). Finally, we tested whether these inhibitory effects could be neutralized by an excess amount of SF1 using nuclear extracts prepared from HEK293T cells overexpressing SF1 (Fig. 4E, F). The overexpression resulted in approximately 4 times more SF1 compared with the control cells (Fig. 4E). As expected, the inhibitory effect of the Gomafu repeat oligonucleotides was rescued when using nuclear extracts prepared from SF1-overexpressing cells (Fig. 4F). Taken together, these results suggested that Gomafu RNAs potentially affect kinetics of splicing reaction by competing with endogenous introns for the branch point binding protein SF1.
Affiliation: Nakagawa Initiative Research Unit, RIKEN Advanced Science Institute, Hirosawa, Wako, Saitama, Japan.