L30 Binds the Nascent RPL30 Transcript to Repress U2 snRNP Recruitment

Abstract

The mechanisms of pre-mRNA splicing regulation are poorly understood. Here we dissect how the Saccharomyces cerevisiae ribosomal L30 protein blocks splicing of its pre-mRNA upon binding a kink-turn structure including the 5' splice site. We show that L30 binds the nascent RPL30 transcript without preventing recognition of the 5' splice site by U1 snRNP but blocking U2 snRNP association with the branch site. Interaction of the factors BBP and Mud2 with the intron, relevant for U2 snRNP recruitment, is not affected by L30. Furthermore, the functions of neither the DEAD-box protein Sub2 in the incipient spliceosome nor the U2 snRNP factor Cus2 on branch site recognition are required for L30 inhibition. These findings contrast with the effects caused by binding a heterologous protein to the same region, completely blocking intron recognition. Collectively, our data suggest that L30 represses a spliceosomal rearrangement required for U2 snRNP association with the transcript.

RPL30 regulation of splicing reveals distinct roles for Cbp80 in U1 and U2 snRNP cotranscriptional recruitment

“…compatible (Macías et al 2008). Figure 1D shows similar levels of U1 coimmunoprecipitation in wt (lane 4) and 5A C9U transcripts (lane 5), consistent with the initial intron recognition by U1 snRNP being compatible with L30 binding.…”

“…There is lack of U2 coimmunoprecipitation in both transcripts, but not +12 (lane 6). This indicates repression of splicing in wt RNA (Macías et al 2008); however, since RPL30 5A C9U is spliced even in presence of L30; lack of U2 coimmunoprecipitation is consistent with L30 removal during U2 association with RPL30. Accordingly, we detect U1 cross-linking with the 5A C9U 59SS, as well as association of this intron with BBP, under repressive conditions (Supplemental Fig.…”

“…+12 combines 5A with moving the 59SS 12 nucleotides (nt) downstream. We have previously shown that in this transcript L30 binding and splicing are compatible, due to the added space between L30 and the intron (Macías et al 2008), and we use this RNA as a control for the experiments described below. As expected, C9U disrupts L30 binding in vitro (Fig.…”

“…L30 interacts with a kink-turn structure (Klein et al 2001) that mimics the L30 rRNA binding site (Vilardell et al 2000). This prevents association of U2 snRNP with the BS by a distinct mechanism likely to involve an interference with conformational changes that occur during spliceosome assembly (Macías et al 2008). Here we follow a genetic approach to address the molecular mechanisms involved in this regulation.…”

Promoter-driven splicing regulation in fission yeast

“…Macías et al (Macias et al, 2008). To study this possibility, we mutated the ATG start codon to a TAG codon disabling the possibility to translate Rem1 (ATGless mutant).…”