Modulation of alternative splicing by long-range RNA structures in Drosophila

Abstract

Accurate and efficient recognition of splice sites during pre-mRNA splicing is essential for proper transcriptome expression. Splice site usage can be modulated by secondary structures, but it is unclear if this type of modulation is commonly used or occurs to a significant degree with secondary structures forming over long distances. Using phlyogenetic comparisons of intronic sequences among 12 Drosophila genomes, we elucidated a group of 202 highly conserved pairs of sequences, each at least nine nucleotides long, capable of forming stable stem structures. This set was highly enriched in alternatively spliced introns and introns with weak acceptor sites and long introns, and most occurred over long distances (>150 nucleotides). Experimentally, we analyzed the splicing of several of these introns using mini-genes in Drosophila S2 cells. Wild-type splicing patterns were changed by mutations that opened the stem structure, and restored by compensatory mutations that re-established the base-pairing potential, demonstrating that these secondary structures were indeed implicated in the splice site choice. Mechanistically, the RNA structures masked splice sites, brought together distant splice sites and/or looped out introns. Thus, base-pairing interactions within introns, even those occurring over long distances, are more frequent modulators of alternative splicing than is currently assumed.

Conservation and regulation of alternative splicing by dynamic inter- and intra-intron base pairings in Lepidoptera 14-3-3z pre-mRNAs

“…34 Indeed, some examples have indicated that RNA secondary structures are involved in splicing regulation. 7,8,19,21,[35][36][37][38] In yeast, new discoveries using high-throughput sequencing have lent strong support to the general hypothesis that the information encoded in large mRNAs is regulated and organized by RNA structure. 39 Therefore, RNA secondary structure strongly contributes to alternative splicing regulation in higher organisms.…”

New insights into RNA secondary structure in the alternative splicing of pre-mRNAs

“…36 Several potential RNA secondary structures are phylogenetically conserved in the 12 Drosophila genomes spanning 40 million years, with most proposed stems having extensive loops stretching over 1,000 bases. 29 Nevertheless, more research is needed to verify these structures and to determine how ligands. 25 For both chemical and enzymatic probing, RNA accessibility is the major criterion for reactivity.…”

“…Computational structure prediction methods have been developed to facilitate fast, large-scale analyses of many RNA sequences. 28,29 Table 1 summarizes some programs for RNA secondary structure prediction. They mostly rely on thermodynamic parameters, based on experimentally derived sets of base pairs, to compute the minimum free energy of secondary structures.…”

“…30 The evolutionary conservation of structures and co-variation of base pairs (e.g., changing GC to CG) can be investigated across species to efficiently determine the importance of a given RNA structure. 29,31 However, if they are old, the signs of conserved elements might have been partially erased. 24 Although prediction methods are generally powerful and achieve accuracies of ~40-70%, 32,33 they are often limited by the complexity of important modeling parameters, such as longdistance interactions or pseudoknots.…”

IRBIS: a systematic search for conserved complementarity

“…Recent studies on RSSs in eukaryotic genes revealed widespread occurrence of long-range RRI with diverse functions such as riboswitches (Li and Breaker 2013) and mediators of exon skipping (Lovci et al 2013), mutually exclusive exon choice (Kreahling and Graveley 2005;Yang et al 2011), and other types of alternative splicing events (Raker et al 2009;Pervouchine et al 2012). Many of these structures are located in regions lacking reliable sequence alignments and contain long, ultraconserved stretches of complementary nucleotides, in which the interacting bases can be separated by distances as large as 10 kb.…”