The splicing factor PRP2, a putative RNA helicase, interacts directly with pre-mRNA.
The RNA helicase-like splicing factor PRP2 interacts only transiently with spliceosomes. To facilitate analysis of interactions of PRP2 with spliceosomal components, PRP2 protein was stalled in splicing complexes by two different methods. A dominant negative mutant form of PRP2 protein, which associates stably with spliceosomes, was found to interact directly with pre-mRNAs, as demonstrated by UV-crosslinking experiments. The use of various mutant and truncated pre-mRNAs revealed that this interaction requires a spliceable pre-mRNA and an assembled spliceosome; a 3' splice site is not required. To extend these observations to the wild-type PRP2 protein, spliceosomes were depleted of ATP; PRP2 protein interacts with pre-mRNA in these spliceosomes in an ATP-independent fashion. Comparison of RNA binding by PRP2 protein in the presence of ATP or gamma S-ATP showed that ATP hydrolysis rather than mere ATP binding is required to release PRP2 protein from pre-mRNA. As PRP2 is an RNA-stimulated ATPase, these experiments strongly suggest that the pre-mRNA is the native co-factor stimulating ATP hydrolysis by PRP2 protein in spliceosomes. Since PRP2 is a putative RNA helicase, we propose that the pre-mRNA is the target of RNA displacement activity of PRP2 protein, promoting the first step of splicing.
Teigelkamp
S
S
Institute of Cell and Molecular Biology, University of Edinburgh, UK.
McGarvey
M
M
Plumpton
M
M
Beggs
J D
JD
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D053487:DEAD-box RNA Helicases; D005656:Fungal Proteins / Q000378:metabolism* / Q000528:radiation effects; D006868:Hydrolysis; D008969:Molecular Sequence Data; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D012331:RNA, Fungal / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017381:Spliceosomes / Q000378:metabolism; D014466:Ultraviolet Rays
D005656:Fungal Proteins; D012322:RNA Precursors; D012331:RNA, Fungal; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D012316:RNA Nucleotidyltransferases; C066490:PRP2 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases