RNA secondary structure in mutually exclusive splicing

Abstract

Mutually exclusive splicing is a regulated means to generate protein diversity, but the underlying mechanisms are poorly understood. Here comparative genome analysis revealed the built-in intronic elements for controlling mutually exclusive splicing of the 14-3-3ξ pre-mRNA. These elements are clade specific but are evolutionarily conserved at the secondary structure level. Combined evidence revealed the triple functions of these inter-intronic RNA pairings in synergistically ensuring the selection of only one of multiple exons, through activation of the proximal variable exon outside the loop by the approximation of cis elements, and simultaneous repression of the exon within the loop, in combination with the physical competition of RNA pairing. Additionally, under this model, we also deciphered a similar structural code in exon clusters 4 and 9 of Dscam (38,016 isoforms) and Mhc (480 isoforms). Our findings suggest a broadly applicable mechanism to ensure mutually exclusive splicing.

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

“…23 Recent experiments have provided direct evidence for the formation and splicing-directed function of long-range secondary structures. 24 Taken together, these findings suggest that RNA secondary structure plays wider roles in gene regulation than was previously thought. This article reviews some new insights into the roles of RNA secondary structure in alternative splicing and the underlying mechanisms.…”

“…Recent experiments involving disruptive and compensatory mutations have provided direct evidence for the formation and splicing-directed function of long-range secondary structures. 24 Taken together, these studies imply the common occurrence of long-range RNA-RNA interactions in pre-mRNAs.…”

“…Recently, disruptive and compensatory mutagenesis revealed physical competition between secondary structures, which direct and regulate mutually exclusive splicing. 24 In this competitive pairing model, the strength of intronic RNA pairing was positively correlated with the frequency with which a given variant was selected, while the selection of exon 5b increased with decreasing distance between the two paired sites. 24 Competition between RNA secondary structures not only leads to mutual exclusivity, but also regulates the selection of alternative exons through pairing competitiveness.…”

“…45 A similar structural code has also been deciphered in exon clusters 4 and 9 of Dscam, 14-3-3ξ and Mhc. 24 A series of constructs were used to demonstrate that such inter-intronic RNA pairing may assist in bringing the elements closer together, suggesting that the RNA looping-facilitated interaction of upstream and downstream control sequences is critical for this activation. 24 RNA-RNA interactions may function not only to bring distant splice sites into closer proximity, but also upstream and downstream activator elements.…”

“…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. Thus, structure-modifying and compensatory structure-restoring mutations must typically be studied in vivo to verify the influence of the proposed structures on splicing.…”