The DEAH box ATPases Prp16 and Prp43 cooperate to proofread 5' splice site cleavage during pre-mRNA splicing.
To investigate the mechanisms underlying accurate pre-mRNA splicing, we developed an in vitro assay sensitive to proofreading of 5' splice site cleavage. We inactivated spliceosomes by disrupting a metal-ligand interaction at the catalytic center and discovered that, when the DEAH box ATPase Prp16 was disabled, these spliceosomes catalyzed 5' splice site cleavage but at a reduced rate. Although Prp16 does not promote splicing of a genuine substrate until after 5' splice site cleavage, we found that Prp16 can associate with spliceosomes before 5' splice site cleavage, consistent with a role for Prp16 in proofreading 5' splice site cleavage. We established that Prp16-mediated rejection is reversible, necessitating a downstream discard pathway that we found requires the DEAH box ATPase Prp43, a spliceosome disassembly factor. These data indicate that spliceosomes distinguish slow substrates and that the mechanisms for establishing the fidelity of 5' splice site cleavage and exon ligation share a common ATP-dependent framework.
Koodathingal
Prakash
P
Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637, USA.
Novak
Thaddeus
T
Piccirilli
Joseph A
JA
Staley
Jonathan P
JP
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000235:genetics / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism*; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D022821:RNA Splice Sites / Q000502:physiology*; D012326:RNA Splicing / Q000502:physiology*; D000072260:RNA Splicing Factors; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017381:Spliceosomes / Q000235:genetics / Q000378:metabolism
D012322:RNA Precursors; D022821:RNA Splice Sites; D000072260:RNA Splicing Factors; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C075669:PRP16 protein, S cerevisiae; C109034:PRP43 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases