RNA structure replaces the need for U2AF2 in splicing

Abstract

RNA secondary structure plays an integral role in catalytic, ribosomal, small nuclear, micro, and transfer RNAs. Discovering a prevalent role for secondary structure in pre-mRNAs has proven more elusive. By utilizing a variety of computational and biochemical approaches, we present evidence for a class of nuclear introns that relies upon secondary structure for correct splicing. These introns are defined by simple repeat expansions of complementary AC and GT dimers that co-occur at opposite boundaries of an intron to form a bridging structure that enforces correct splice site pairing. Remarkably, this class of introns does not require U2AF2, a core component of the spliceosome, for its processing. Phylogenetic analysis suggests that this mechanism was present in the ancestral vertebrate lineage prior to the divergence of tetrapods from teleosts. While largely lost from land dwelling vertebrates, this class of introns is found in 10% of all zebrafish genes.[Supplemental material is available for this article.]RNA splicing is a process that removes an internal segment of RNA (i.e., the intron) and rejoins together the two flanking segments (exons). Distinct but evolutionarily related versions of this processing reaction are found in prokaryotes and eukaryotes in a variety of different contexts. In eukaryotes, the splicing of nuclear introns is catalyzed by a large riboprotein complex called the spliceosome (Matlin and Moore 2007). RNA encoded by genes in organelles and some bacterial genomes contain self-splicing group I and II introns which catalyze their own removal (Cech et al. 1981). A basic problem for all introns is the correct identification and pairing of the splice sites. In group I and II introns, this pairing function is performed by RNA secondary structure alone, whereas in spliceosomal introns, small nuclear ribonucleoproteins (snRNPs) recognize and pair together the correct 5 ′ splice site (5 ′ ss) and branchpoint site (BP). However, there are some examples where the pairing of sites is assisted by intramolecular secondary structure in the intron (Goguel and Rosbash 1993;Libri et al. 1995;Charpentier and Rosbash 1996;Howe and Ares 1997;Spingola et al. 1999). In addition, there are some fascinating examples of how secondary structures can regulate mutually exclusive alternative splicing (Warf and Berglund 2007;McManus and Graveley 2011): Several regions of the Dscam1 pre-mRNA undergo extensive alternative splicing. In one of these regions, an upstream "selector" sequence near exon 5 can select from an array of 48 complementary downstream "docking" sequences. Each "docking" sequence can potentially base-pair with the "selector" sequence, thereby bringing an alternate version of exon 6 to splice to exon 5 ( Secondary structure in RNA can be identified experimentally or computationally. There are currently around a thousand publicly available structures-53% determined by X-ray crystallography and 47% bysolution NMR (Bernstein et al. 1977). Therehavebeen a great many advances in computational approaches t...

RNA structure in splicing: An evolutionary perspective

“…54 In addition, in mouse, long-range RNA-RNA interactions that position Rbfox binding sites close to its regulated exon facilitate the exon inclusion. 55 These reports all together indicate that secondary structure plays a critical role throughout evolution in regulating the accessibility and the functional outcome of primary sequence elements for premRNA splicing.…”

The effects of structure on pre-mRNA processing and stability

“…Using pairwise comparisons of genomic sequences near the intron-exon junctions, lineage-specific k-mers around the splice site were discovered [12]. This unique distribution of k-mers suggests that these discovered sequence motifs regulate splicing in a lineage-specific manner.…”

“…(AC)m-(GT)n introns were shuffled and refolded, while preserving dinucleotide composition. The shuffled introns are less structured than their native sequence, demonstrating that their stable predicted structures are not due purely to dinucleotide composition[12]. To determine whether dinucleotide repeats can drive predicted secondary structure, an in silico analysis was performed using all possible dinucleotide repeats.…”

“…(AC)m-(GT)n introns were also found in lamprey, indicating it was an ancient splicing mechanism evolved before the divergence of Tetrapod and Teleost. The repeats were lost in mammals and other higher vertebrates; instead, exonic splicing enhancer-like sequences were found enriched in the human introns homologous to the (AC)m-(GT)n introns of zebrafish, suggesting a compensatory mechanism evolved to replace the dinucleotide repeats [12]. …”

“…Complimentary k-mers of length 12 can give maximal or near maximal bridging across an intron. Increases in k-mer length, did not result in appreciable increases in pairing (Figure 2) [12]. …”