Stoichiometries of U2AF35, U2AF65 and U2 snRNP reveal new early spliceosome assembly pathways.
The selection of 3΄ splice sites (3΄ss) is an essential early step in mammalian RNA splicing reactions, but the processes involved are unknown. We have used single molecule methods to test whether the major components implicated in selection, the proteins U2AF35 and U2AF65 and the U2 snRNP, are able to recognize alternative candidate sites or are restricted to one pre-specified site. In the presence of adenosine triphosphate (ATP), all three components bind in a 1:1 stoichiometry with a 3΄ss. Pre-mRNA molecules with two alternative 3΄ss can be bound concurrently by two molecules of U2AF or two U2 snRNPs, so none of the components are restricted. However, concurrent occupancy inhibits splicing. Stoichiometric binding requires conditions consistent with coalescence of the 5΄ and 3΄ sites in a complex (I, initial), but if this cannot form the components show unrestricted and stochastic association. In the absence of ATP, when complex E forms, U2 snRNP association is unrestricted. However, if protein dephosphorylation is prevented, an I-like complex forms with stoichiometric association of U2 snRNPs and the U2 snRNA is base-paired to the pre-mRNA. Complex I differs from complex A in that the formation of complex A is associated with the loss of U2AF65 and 35.
University of Leicester, Leicester Institute for Structural and Chemical Biology and Department of Molecular and Cell Biology, Leicester LE1 9HN, UK.
Li
L
Chen
University of Leicester, Leicester Institute for Structural and Chemical Biology and Department of Molecular and Cell Biology, Leicester LE1 9HN, UK.
Robert
R
Weinmeister
University of Southampton, Faculty of Medicine, Southampton SO16 6YD, UK.
Jana
J
Kralovicova
University of Leicester, Leicester Institute for Structural and Chemical Biology and Department of Molecular and Cell Biology, Leicester LE1 9HN, UK.
Lucy P
LP
Eperon
University of Southampton, Faculty of Medicine, Southampton SO16 6YD, UK.
Igor
I
Vorechovsky
University of Leicester, Leicester Institute for Structural and Chemical Biology and Department of Chemistry, Leicester LE1 7RH, UK.
Andrew J
AJ
Hudson
University of Leicester, Leicester Institute for Structural and Chemical Biology and Department of Molecular and Cell Biology, Leicester LE1 9HN, UK.
Ian C
IC
Eperon
D000255:Adenosine Triphosphate / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D007438:Introns; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D011485:Protein Binding; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D022821:RNA Splice Sites; D012326:RNA Splicing; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism; D017381:Spliceosomes / Q000378:metabolism*; D000072668:Splicing Factor U2AF / Q000378:metabolism*; D055540:Survival of Motor Neuron 2 Protein / Q000378:metabolism; D014335:Tropomyosin / Q000378:metabolism
D046912:Multiprotein Complexes; D012322:RNA Precursors; D022821:RNA Splice Sites; D017412:Ribonucleoprotein, U1 Small Nuclear; D000072668:Splicing Factor U2AF; D055540:Survival of Motor Neuron 2 Protein; C490775:TPM1 protein, human; D014335:Tropomyosin; D000255:Adenosine Triphosphate; D010744:Phosphoric Monoester Hydrolases
Mol Cell Biol. 1989 May;9(5):2007-17
2546057
Cell. 2015 Oct 22;163(3):712-23
26496610
J Cell Biol. 2008 Jun 16;181(6):921-34
18559666
Proc Natl Acad Sci U S A. 1989 Dec;86(23):9243-7
2531895
Cell. 1997 May 2;89(3):403-12
9150140
Mol Cell Biol. 1998 Dec;18(12):7510-20
9819436
Cold Spring Harb Perspect Biol. 2011 Jul 01;3(7):null
21441581
Cell. 1990 Oct 19;63(2):293-302
2170025
Mol Cell Biol. 1998 Aug;18(8):4752-60
9671485
Mol Cell Biol. 1987 Jan;7(1):281-93
2951586
RNA. 2007 Aug;13(8):1214-23
17592047
Genes Dev. 1998 May 15;12 (10 ):1409-14
9585501
Nucleic Acids Res. 1992 Oct 25;20(20):5263-9
1331983
Nat Struct Biol. 2002 Aug;9(8):576-81
12091875
Nature. 2013 Jul 18;499(7458):360-3
23792564
RNA. 2001 Jun;7(6):806-18
11421359
Genes Dev. 1993 Jun;7(6):1008-20
8504926
Nature. 1993 Mar 11;362(6416):171-5
7680770
Biochem Soc Trans. 2012 Aug;40(4):815-20
22817740
Genes Dev. 1989 Oct;3(10):1562-71
2612905
Mol Cell Biol. 1994 May;14(5):2994-3005
8164655
Annu Rev Biochem. 1985;54:1151-93
2411210
Proc Natl Acad Sci U S A. 2003 Apr 1;100(7):4114-9
12642665
Mol Cell. 2004 Aug 13;15(3):477-83
15304226
Nat Struct Mol Biol. 2014 Nov;21(11):997-1005
25326705
Genes Dev. 2014 Nov 15;28(22):2518-31
25403181
Structure. 2013 Feb 5;21(2):197-208
23273425
Cell. 2012 Jul 6;150(1):53-64
22770214
Mol Cell Biol. 2007 Feb;27(3):830-41
17101796
Nucleic Acids Res. 2013 May;41(9):4765-82
23511971
Nucleic Acids Res. 2015 Dec 2;43(21):10456-73
26420826
Mol Biol Cell. 2005 Mar;16(3):1366-77
15647371
Proc Natl Acad Sci U S A. 2014 Dec 9;111(49):17420-5
25422459
J Biol Chem. 2003 Dec 12;278(50):50572-7
14506271
Proc Natl Acad Sci U S A. 2013 Apr 23;110(17):6783-8
23569281
Nucleic Acids Res. 2013 Jan;41(2):1343-54
23175611
Nucleic Acids Symp Ser. 1995;(33):218-9
8643375
Genes Dev. 1999 Feb 15;13(4):462-71
10049361
Genome Biol. 2015 Jun 06;16:119
26047612
Mol Cell Biol. 2000 Apr;20(7):2317-25
10713155
Genes Dev. 2000 Jan 1;14(1):97-107
10640279
Genes Dev. 1991 Apr;5(4):642-55
2010089
J Biol Chem. 1995 Feb 24;270(8):4031-6
7876151
Nature. 2011 Jul 13;475(7356):408-11
21753750
RNA. 2010 Dec;16(12 ):2384-403
20980672
RNA. 1995 Mar;1(1):21-35
7489484
Cell. 2013 Jan 31;152(3):453-66
23374342
Hum Genet. 2010 Oct;128(4):383-400
20628762
Genes Dev. 1998 Mar 15;12 (6):858-67
9512519
Cell. 1985 Oct;42(3):737-50
2996775
Nature. 1999 Dec 16;402(6763):832-5
10617206
Cell. 2015 Jun 4;161(6):1474-e1
26046445
EMBO J. 1993 Sep;12(9):3607-17
8253084
Genes Dev. 2000 Dec 15;14 (24):3166-78
11124808
Nucleic Acids Res. 2012 Aug;40(14):6850-62
22505580
Nucleic Acids Res. 2013 Apr 1;41(6):3859-73
23376934
Mol Cell Biol. 1987 Aug;7(8):2877-83
2959853
J Biol Chem. 1999 Dec 3;274(49):35074-9
10574987
Mol Cell. 2007 Feb 9;25(3):399-411
17289587
Cell Rep. 2013 Oct 17;5(1):151-65
24075986
Nature. 1999 Dec 16;402(6763):838-41
10617208
Mol Cell Biol. 2009 Feb;29(4):1072-82
19064642
Mol Cell. 2010 May 14;38(3):416-27
20471947
Mol Cell. 2004 Nov 5;16(3):363-73
15525510
Genes Dev. 2015 Aug 1;29(15):1649-60
26215567
Science. 1982 Jul 30;217(4558):456-8
6178162
RNA. 1999 Nov;5(11):1504-8
10580479
RNA. 2009 Apr;15(4):515-23
19244360
Science. 1995 May 26;268(5214):1173-6
7761834
Mol Cell. 2005 Aug 19;19(4):485-96
16109373
RNA. 1998 Jan;4(1):85-100
9436911
Mol Cell Biol. 1992 Oct;12(10):4279-87
1383687
Genome Biol. 2006;7(1):R1
16507133
Genes Dev. 1991 Dec;5(12B):2534-46
1836445
RNA. 2005 Aug;11(8):1201-14
16043505
J Cell Biol. 1997 Jun 2;137(5):975-87
9166400
Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8769-73
1388271
Mol Cell Biol. 2001 Nov;21(22):7673-81
11604503
FEBS Lett. 2013 Mar 18;587(6):555-61
23395799
Cell Rep. 2014 Oct 9;9(1):193-205
25263560
Cell. 1993 Dec 17;75(6):1061-70
8261509
Nucleic Acids Res. 2012 Mar;40(6):2639-52
22110043
RNA. 2012 Jun;18(6):1244-56
22535589
Nucleic Acids Res. 2015 Apr 20;43(7):3747-63
25779042
EMBO J. 2010 Jul 7;29(13):2161-72
20502437
Cell. 1988 Jan 29;52(2):207-19
2963698
Genes Dev. 1997 Jul 15;11(14 ):1864-72
9242493
Genes Dev. 1996 Jun 1;10 (11):1356-68
8647433
Nature. 1999 Dec 16;402(6763):835-8
10617207
Mol Cell. 2005 Aug 19;19(4):475-84
16109372
Science. 1996 Sep 20;273(5282):1706-9
8781232
Mol Cell Biol. 1995 Oct;15(10 ):5750-6
7565727
Mol Cell. 2000 May;5(5):779-87
10882114
Genes Dev. 1992 Dec;6(12B):2554-68
1285125
Science. 1992 Sep 25;257(5078):1918-25
1411506
Nucleic Acids Res. 2011 Mar;39(5):1868-79
21062807
Cell. 1986 Aug 29;46(5):681-90
2427200
Nature. 2010 Dec 2;468(7324):664-8
20881964
Mol Cell Biol. 2006 Dec;26(23 ):8755-69
16982681