Higher order RNA structures can mask splicing signals, loop out exons, or constitute riboswitches all of which contributes to the complexity of splicing regulation. We identified a G to A substitution between branch point (BP) and 3' splice site (3'ss) of Saccharomyces cerevisiae COF1 intron, which dramatically impaired its splicing. RNA structure prediction and in-line probing showed that this mutation disrupted a stem in the BP-3'ss region. Analyses of various COF1 intron modifications revealed that the secondary structure brought about the reduction of BP to 3'ss distance and masked potential 3'ss. We demonstrated the same structural requisite for the splicing of UBC13 intron. Moreover, RNAfold predicted stable structures for almost all distant BP introns in S. cerevisiae and for selected examples in several other Saccharomycotina species. The employment of intramolecular structure to localize 3'ss for the second splicing step suggests the existence of pre-mRNA structure-based mechanism of 3'ss recognition.

Department of Cell Biology Faculty of Science Charles University Prague Prague Czech Republic

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Chen M, Manley JL. Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches. Nat. Rev. Mol. Cell Biol. 2009;10:741–754. PubMed PMC

Munroe SH. Secondary structure of splice sites in adenovirus mRNA precursors. Nucleic Acids Res. 1984;12:8437–8456. PubMed PMC

Solnick D. Alternative splicing caused by RNA secondary structure. Cell. 1985;43:667–676. PubMed

Buratti E, Baralle FE. Influence of RNA secondary structure on the pre-mRNA splicing process. Mol.Cell. Biol. 2004;24:10505–10514. PubMed PMC

Warf MB, Berglund JA. Role of RNA structure in regulating pre-mRNA splicing. Trends Biochem. Sci. 2010;35:169–178. PubMed PMC

Singh NN, Singh RN, Androphy EJ. Modulating role of RNA structure in alternative splicing of a critical exon in the spinal muscular atrophy genes. Nucleic Acids Res. 2007;35:371–389. PubMed PMC

Howe KJ, Ares M., Jr Intron self-complementarity enforces exon inclusion in a yeast pre-mRNA. Proc. Natl Acad. Sci. USA. 1997;94:12467–12472. PubMed PMC

Raker VA, Mironov AA, Gelfand MS, Pervouchine DD. Modulation of alternative splicing by long-range RNA structures in Drosophila. Nucleic Acids Res. 2009;37:4533–4544. PubMed PMC

Blouin S, Mulhbacher J, Penedo JC, Lafontaine DA. Riboswitches: ancient and promising genetic regulators. Chembiochem. 2009;10:400–416. PubMed

Macias S, Bragulat M, Tardiff DF, Vilardell J. L30 binds the nascent RPL30 transcript to repress U2 snRNP recruitment. Mol. Cell. 2008;30:732–742. PubMed

Scannell DR, Butler G, Wolfe KH. Yeast genome evolution—the origin of the species. Yeast. 2007;24:929–942. PubMed

Irimia M, Roy SW. Evolutionary convergence on highly-conserved 3′ intron structures in intron-poor eukaryotes and insights into the ancestral eukaryotic genome. PLoS Genet. 2008;4:e1000148. PubMed PMC

Libri D, Stutz F, McCarthy T, Rosbash M. RNA structural patterns and splicing: molecular basis for an RNA-based enhancer. RNA. 1995;1:425–436. PubMed PMC

Charpentier B, Rosbash M. Intramolecular structure in yeast introns aids the early steps of in vitro spliceosome assembly. RNA. 1996;2:509–522. PubMed PMC

Rogic S, Montpetit B, Hoos HH, Mackworth AK, Ouellette BF, Hieter P. Correlation between the secondary structure of pre-mRNA introns and the efficiency of splicing in Saccharomyces cerevisiae. BMC Genomics. 2008;9:355. PubMed PMC

Cellini A, Felder E, Rossi JJ. Yeast pre-messenger RNA splicing efficiency depends on critical spacing requirements between the branch point and 3' splice site. EMBO J. 1986;5:1023–1030. PubMed PMC

Golemis EA, Gyuris J, Brent R. Interaction trap/two-hybrid system to identify interacting proteins. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JD, Smith JA, Struhl K, editors. Current Protocols in Molecular Biology. 1996. Wiley, New York, Unit 20.1.

Lesser CF, Guthrie C. Mutational analysis of pre-mRNA splicing in Saccharomyces cerevisiae using a sensitive new reporter gene, CUP1. Genetics. 1993;133:851–863. PubMed PMC

Regulski EE, Breaker RR. In-line probing analysis of riboswitches. Methods Mol. Biol. 2008;419:53–67. PubMed

Gruber AR, Lorenz R, Bernhart SH, Neubock R, Hofacker IL. The Vienna RNA websuite. Nucleic Acids Res. 2008;36:W70–W74. PubMed PMC

Steffen P, Voss B, Rehmsmeier M, Reeder J, Giegerich R. RNAshapes: an integrated RNA analysis package based on abstract shapes. Bioinformatics. 2006;22:500–503. PubMed

Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003;31:3406–3415. PubMed PMC

Parisien M, Major F. The MC-Fold and MC-Sym pipeline infers RNA structure from sequence data. Nature. 2008;452:51–55. PubMed

Deshler JO, Rossi JJ. Unexpected point mutations activate cryptic 3' splice sites by perturbing a natural secondary structure within a yeast intron. Genes Dev. 1991;5:1252–1263. PubMed

Wahl MC, Will CL, Luhrmann R. The spliceosome: design principles of a dynamic RNP machine. Cell. 2009;136:701–718. PubMed

Rymond BC, Torrey DD, Rosbash M. A novel role for the 3' region of introns in pre-mRNA splicing of Saccharomyces cerevisiae. Genes Dev. 1987;1:238–246. PubMed

Cheng SC. Formation of the yeast splicing complex A1 and association of the splicing factor PRP19 with the pre-mRNA are independent of the 3' region of the intron. Nucleic Acids Res. 1994;22:1548–1554. PubMed PMC

Wu S, Romfo CM, Nilsen TW, Green MR. Functional recognition of the 3' splice site AG by the splicing factor U2AF35. Nature. 1999;402:832–835. PubMed

Anderson K, Moore MJ. Bimolecular exon ligation by the human spliceosome bypasses early 3' splice site AG recognition and requires NTP hydrolysis. RNA. 2000;6:16–25. PubMed PMC

Robberson BL, Cote GJ, Berget SM. Exon definition may facilitate splice site selection in RNAs with multiple exons. Mol. Cell Biol. 1990;10:84–94. PubMed PMC

Berget SM. Exon recognition in vertebrate splicing. J. Biol. Chem. 1995;270:2411–2414. PubMed

Gooding C, Clark F, Wollerton MC, Grellscheid SN, Groom H, Smith CW. A class of human exons with predicted distant branch points revealed by analysis of AG dinucleotide exclusion zones. Genome Biol. 2006;7:R1. PubMed PMC

Smith CW, Chu TT, Nadal-Ginard B. Scanning and competition between AGs are involved in 3' splice site selection in mammalian introns. Mol. Cell Biol. 1993;13:4939–4952. PubMed PMC

Hallegger M, Sobala A, Smith CW. Four exons of the serotonin receptor 4 gene are associated with multiple distant branch points. RNA. 2010;16:839–851. PubMed PMC

Patterson B, Guthrie C. A U-rich tract enhances usage of an alternative 3' splice site in yeast. Cell. 1991;64:181–187. PubMed

Luukkonen BG, Seraphin B. The role of branchpoint-3' splice site spacing and interaction between intron terminal nucleotides in 3' splice site selection in Saccharomyces cerevisiae. EMBO J. 1997;16:779–792. PubMed PMC

Liu ZR, Laggerbauer B, Luhrmann R, Smith CW. Crosslinking of the U5 snRNP-specific 116-kDa protein to RNA hairpins that block step 2 of splicing. RNA. 1997;3:1207–1219. PubMed PMC

Collins L, Penny D. Complex spliceosomal organization ancestral to extant eukaryotes. Mol. Biol. Evol. 2005;22:1053–1066. PubMed

Introns provide a platform for intergenic regulatory feedback of RPL22 paralogs in yeast