Distinct activities of the DExD/H-box splicing factor hUAP56 facilitate stepwise assembly of the spliceosome.
The essential splicing factor human UAP56 (hUAP56) is a DExD/H-box protein known to promote prespliceosome assembly. Here, using a series of hUAP56 mutants that are defective for ATP-binding, ATP hydrolysis, or dsRNA unwindase/helicase activity, we assess the relative contributions of these biochemical functions to pre-mRNA splicing. We show that prespliceosome assembly requires hUAP56's ATP-binding and ATPase activities, which, unexpectedly, are required for hUAP56 to interact with U2AF(65) and be recruited into splicing complexes. Surprisingly, we find that hUAP56 is also required for mature spliceosome assembly, which requires, in addition to the ATP-binding and ATPase activities, hUAP56's dsRNA unwindase/helicase activity. We demonstrate that hUAP56 directly contacts U4 and U6 snRNAs and can promote unwinding of the U4/U6 duplex, and that both these activities are dependent on U2AF(65). Our results indicate that hUAP56 first interacts with U2AF(65) in an ATP-dependent manner, and subsequently with U4/U6 snRNAs to facilitate stepwise assembly of the spliceosome.
Shen
Haihong
H
Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea. haihongshen@gist.ac.kr
Zheng
Xuexiu
X
Shen
Jingping
J
Zhang
Lingdi
L
Zhao
Rui
R
Green
Michael R
MR
D000255:Adenosine Triphosphate / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D012322:RNA Precursors / Q000235:genetics; D012326:RNA Splicing / Q000502:physiology*; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D017381:Spliceosomes / Q000502:physiology*; D000072668:Splicing Factor U2AF
D009687:Nuclear Proteins; D012322:RNA Precursors; D012330:RNA, Double-Stranded; D012342:RNA, Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human; C405486:U6 small nuclear RNA; D000255:Adenosine Triphosphate; C487886:DDX39B protein, human; D053487:DEAD-box RNA Helicases