Spliceosome discards intermediates via the DEAH box ATPase Prp43p.
To promote fidelity in nuclear pre-mRNA splicing, the spliceosome rejects and discards suboptimal substrates that have engaged the spliceosome. Whereas DExD/H box ATPases have been implicated in rejecting suboptimal substrates, the mechanism for discarding suboptimal substrates has remained obscure. Corroborating evidence that suboptimal, mutated lariat intermediates can be exported to the cytoplasm for turnover, we have found that the ribosome can translate mutated lariat intermediates. By glycerol gradient analysis, we have found that the spliceosome can dissociate mutated lariat intermediates in vivo in a manner that requires the DEAH box ATPase Prp43p. Through an in vitro assay, we demonstrate that Prp43p promotes the discard of suboptimal and optimal 5' exon and lariat intermediates indiscriminately. Finally, we demonstrate a requirement for Prp43p in repressing splicing at a cryptic splice site. We propose a model for the fidelity of exon ligation in which the DEAH box ATPase Prp22p slows the flow of suboptimal intermediates through exon ligation and Prp43p generally promotes discard of intermediates, thereby establishing a pathway for turnover of stalled intermediates. Because Prp43p also promotes spliceosome disassembly after exon ligation, this work establishes a parallel between the discard of suboptimal intermediates and the dissociation of a genuine excised intron product.
Mayas
Rabiah M
RM
Graduate Program in Biochemistry and Molecular Biophysics, University of Chicago, Chicago, IL 60637, USA.
Maita
Hiroshi
H
Semlow
Daniel R
DR
Staley
Jonathan P
JP
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003593:Cytoplasm / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D005091:Exons; D007438:Introns; D008954:Models, Biological; D009154:Mutation; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D022821:RNA Splice Sites; D012326:RNA Splicing; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism*
D012322:RNA Precursors; D022821:RNA Splice Sites; D012331:RNA, Fungal; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; C109034:PRP43 protein, S cerevisiae; D053487:DEAD-box RNA Helicases
Mol Cell. 2007 Oct 26;28(2):240-52
17964263
Mol Cell Biol. 2006 Aug;26(16):6016-23
16880513
Genes Dev. 2007 Sep 15;21(18):2312-25
17875666
Mol Cell. 2007 Dec 14;28(5):838-49
18082608
EMBO J. 2000 Apr 17;19(8):1873-86
10775271
Proc Natl Acad Sci U S A. 2001 Nov 6;98(23):12972-7
11687653
Nat Struct Mol Biol. 2006 Jun;13(6):482-90
16680161
Mol Cell. 2006 Aug 4;23(3):389-99
16885028
Nature. 2008 Dec 18;456(7224):910-4
19052544
Mol Cell. 2004 May 7;14(3):343-54
15125837
Cell. 1989 May 19;57(4):573-83
2655924
Nucleic Acids Res. 2000 Mar 15;28(6):1313-21
10684925
Proc Natl Acad Sci U S A. 1997 Oct 28;94(22):11798-802
9342317
Mol Cell Biol. 2006 Jan;26(2):513-22
16382143
Genetics. 1993 Apr;133(4):851-63
8462846
EMBO J. 1997 Apr 1;16(7):1759-71
9130720
Mol Cell Biol. 2006 Jan;26(2):523-34
16382144
J Biol Chem. 2002 May 17;277(20):17743-50
11886864
Cell. 2000 Sep 15;102(6):765-75
11030620
Science. 2005 Sep 2;309(5740):1584-7
16141078
Genes Dev. 2007 Apr 1;21(7):811-20
17403781
Nature. 2008 Jan 17;451(7176):359-62
18202663
EMBO J. 1992 Dec;11(13):5033-9
1464325
Mol Cell. 2009 May 15;34(3):333-43
19450531
Genes Dev. 2005 Dec 15;19(24):2991-3003
16357217
Mol Cell. 2003 Dec;12(6):1453-65
14690599
Genes Dev. 2007 Apr 1;21(7):821-34
17403782
EMBO J. 1998 May 15;17(10):2926-37
9582286
Nat Rev Mol Cell Biol. 2004 Mar;5(3):232-41
14991003
Cell. 2009 Feb 20;136(4):701-18
19239890
EMBO J. 1998 Apr 1;17(7):2086-94
9524130
RNA. 2002 Aug;8(8):1068-77
12212850
Mol Cell. 2009 Nov 25;36(4):593-608
19941820
J Biol Chem. 1994 Aug 12;269(32):20613-21
7519612
Cell. 1993 Jul 2;73(7):1377-91
8324826
Proc Natl Acad Sci U S A. 2006 Sep 12;103(37):13700-5
16945917
Genes Dev. 1993 Oct;7(10):1909-25
8405998
Mol Cell Biol. 2005 Nov;25(21):9269-82
16227579
RNA. 2009 Jul;15(7):1386-97
19458033
Cell. 2004 Jan 9;116(1):63-73
14718167
Nat Struct Mol Biol. 2007 Jun;14(6):519-26
17486100