title: A conserved intronic U1 snRNP-binding sequence promotes trans-splicing in Drosophila.
abstract: Unlike typical cis-splicing, trans-splicing joins exons from two separate transcripts to produce chimeric mRNA and has been detected in most eukaryotes. Trans-splicing in trypanosomes and nematodes has been characterized as a spliced leader RNA-facilitated reaction; in contrast, its mechanism in higher eukaryotes remains unclear. Here we investigate mod(mdg4), a classic trans-spliced gene in Drosophila, and report that two critical RNA sequences in the middle of the last 5' intron, TSA and TSB, promote trans-splicing of mod(mdg4). In TSA, a 13-nucleotide (nt) core motif is conserved across Drosophila species and is essential and sufficient for trans-splicing, which binds U1 small nuclear RNP (snRNP) through strong base-pairing with U1 snRNA. In TSB, a conserved secondary structure acts as an enhancer. Deletions of TSA and TSB using the CRISPR/Cas9 system result in developmental defects in flies. Although it is not clear how the 5' intron finds the 3' introns, compensatory changes in U1 snRNA rescue trans-splicing of TSA mutants, demonstrating that U1 recruitment is critical to promote trans-splicing in vivo. Furthermore, TSA core-like motifs are found in many other trans-spliced Drosophila genes, including lola. These findings represent a novel mechanism of trans-splicing, in which RNA motifs in the 5' intron are sufficient to bring separate transcripts into close proximity to promote trans-splicing.
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China;
authors.forename: Jun-Li
authors.initials: JL
authors.lastname: Gao
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China;
authors.forename: Yu-Jie
authors.initials: YJ
authors.lastname: Fan
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China;
authors.forename: Xiu-Ye
authors.initials: XY
authors.lastname: Wang
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China;
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China;
authors.forename: Jia
authors.initials: J
authors.lastname: Pu
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China;
authors.forename: Liang
authors.initials: L
authors.lastname: Li
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China;
authors.forename: Wei
authors.initials: W
authors.lastname: Shao
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China;
authors.forename: Shuai
authors.initials: S
authors.lastname: Zhan
authors.affiliation: Poochon Scientific, Frederick, Maryland 21704, USA.
authors.forename: Jianjiang
authors.initials: J
authors.lastname: Hao
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; yzxu@sibs.ac.cn.
authors.forename: Yong-Zhen
authors.initials: YZ
authors.lastname: Xu
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D017124:Conserved Sequence / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D004330:Drosophila / Q000235:genetics*; D029721:Drosophila Proteins / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D007438:Introns / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics*; D012342:RNA, Small Nuclear / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics; D020040:Trans-Splicing / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; C405483:U1 small nuclear RNA; C117962:mod protein, Drosophila
keywords: Drosophila; RNA motif; U1 snRNP; mod(mdg4); pseudo-5′ splice site; trans-splicing
references.citation: Nature. 2001 Feb 22;409(6823):1000
references.pmid: 11234000
references.citation: Dev Cell. 2014 Mar 31;28(6):685-96
references.pmid: 24631403
references.citation: Mol Cell Biol. 2002 Aug;22(15):5337-46
references.pmid: 12101229
references.citation: Proc Natl Acad Sci U S A. 2002 Sep 17;99(19):12203-7
references.pmid: 12215496
references.citation: Genetica. 2003 Mar;117(2-3):165-77
references.pmid: 12723696
references.citation: Nucleic Acids Res. 2003 Jul 1;31(13):3406-15
references.pmid: 12824337
references.citation: Genes Dev. 2003 Oct 15;17(20):2496-501
references.pmid: 14522953
references.citation: Gene. 2004 Apr 28;331:165-76
references.pmid: 15094203
references.citation: Cell. 1985 Aug;42(1):157-64
references.pmid: 3848347
references.citation: Cell. 1985 Aug;42(1):165-71
references.pmid: 3848348
references.citation: Cell. 1986 Nov 21;47(4):517-25
references.pmid: 3779835
references.citation: Cell. 1986 Nov 21;47(4):527-35
references.pmid: 3022935
references.citation: Cell. 1987 Jan 16;48(1):111-9
references.pmid: 3791410
references.citation: Cell. 1987 Jul 17;50(2):147-8
references.pmid: 3594568
references.citation: Nature. 1988 Oct 6;335(6190):559-62
references.pmid: 2971142
references.citation: Cell. 1990 May 4;61(3):459-66
references.pmid: 1692263
references.citation: Cell. 1990 Jun 29;61(7):1247-55
references.pmid: 2163760
references.citation: Cell. 1990 Sep 7;62(5):889-99
references.pmid: 2168293
references.citation: J Biol Chem. 1991 Dec 5;266(34):22792-5
references.pmid: 1835972
references.citation: Plant Cell. 1991 Dec;3(12):1363-78
references.pmid: 1726722
references.citation: Science. 1992 Dec 11;258(5089):1775-80
references.pmid: 1465612
references.citation: Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11376-80
references.pmid: 8248257
references.citation: Cell. 1993 Dec 17;75(6):1049-59
references.pmid: 8261508
references.citation: Nature. 1994 Nov 17;372(6503):270-2
references.pmid: 7969472
references.citation: Cell. 1995 Aug 25;82(4):587-97
references.pmid: 7664338
references.citation: RNA. 1995 Oct;1(8):815-27
references.pmid: 7493327
references.citation: RNA. 1996 Aug;2(8):735-45
references.pmid: 8752084
references.citation: RNA. 1996 Aug;2(8):746-55
references.pmid: 8752085
references.citation: Genetics. 2000 May;155(1):141-57
references.pmid: 10790390
references.citation: J Immunol. 1996 Oct 15;157(8):3450-9
references.pmid: 8871644
references.citation: Microb Pathog. 1996 Sep;21(3):149-55
references.pmid: 8878012
references.citation: Nat Biotechnol. 1999 Mar;17(3):246-52
references.pmid: 10096291
references.citation: Genetics. 2005 Feb;169(2):723-36
references.pmid: 15520256
references.citation: Genetics. 2005 Jun;170(2):661-74
references.pmid: 15802507
references.citation: Methods. 2005 Dec;37(4):292-305
references.pmid: 16314258
references.citation: RNA. 2005 Dec;11(12):1869-83
references.pmid: 16314458
references.citation: RNA. 2007 Apr;13(4):511-20
references.pmid: 17283210
references.citation: RNA Biol. 2008 Jan-Mar;5(1):17-9
references.pmid: 18388487
references.citation: Nature. 2008 Sep 25;455(7212):491-6
references.pmid: 18784652
references.citation: Nucleic Acids Res. 2009 May;37(9):e67
references.pmid: 19339519
references.citation: EMBO J. 2010 Feb 17;29(4):749-60
references.pmid: 20094034
references.citation: Genome Res. 2010 May;20(5):646-54
references.pmid: 20305017
references.citation: Proc Natl Acad Sci U S A. 2010 Jul 20;107(29):12975-9
references.pmid: 20615941
references.citation: J Biol Chem. 2010 Aug 27;285(35):27457-67
references.pmid: 20566628
references.citation: Nature. 2010 Dec 2;468(7324):664-8
references.pmid: 20881964
references.citation: Curr Biol. 2011 Feb 22;21(4):311-5
references.pmid: 21315596
references.citation: Cell Stem Cell. 2011 May 6;8(5):580-93
references.pmid: 21549331
references.citation: Cold Spring Harb Perspect Biol. 2011 Jul;3(7). pii: a003707. doi: 10.1101/cshperspect.a003707
references.pmid: 21441581
references.citation: Wiley Interdiscip Rev RNA. 2011 May-Jun;2(3):417-34
references.pmid: 21957027
references.citation: Nucleic Acids Res. 2012 Jan;40(1):1-10
references.pmid: 21908400
references.citation: Trends Biochem Sci. 2012 May;37(5):179-88
references.pmid: 22480731
references.citation: RNA. 2012 Jul;18(7):1395-407
references.pmid: 22627775
references.citation: Apoptosis. 2012 Dec;17(12):1327-39
references.pmid: 23011181
references.citation: Nucleic Acids Res. 2013 Apr;41(8):4660-70
references.pmid: 23462954
references.citation: Cell Res. 2013 Aug;23(8):1007-24
references.pmid: 23835473
references.citation: Proc Natl Acad Sci U S A. 2013 Nov 19;110(47):19012-7
references.pmid: 24191015
references.citation: Mol Oncol. 2013 Dec;7(6):1056-68
references.pmid: 23998959
references.citation: RNA Biol. 2014;11(2):134-45
references.pmid: 24526065
references.citation: Nature. 2002 Jun 6;417(6889):667-70
references.pmid: 12050671