Identification of an RNA-dependent ATPase activity in mammalian U5 snRNPs

Nuclear pre-mRNA splicing requires ATP at several steps from spliceosome assembly to product release. Here, we demonstrate that an integral component of the 20S U5 snRNP is an RNA-dependent ATPase. The ATPase activity of 20S US and 25S [U4/U6,U5] snRNPs purified by glycerol gradient centrifugation is strongly stimulated by homopolymeric RNA but not ssDNA, Purified 12S U1 and U2 snRNPs do not exhibit ATPase activity. Moreover the US-associated NTPase specifically hydrolyzes ATP and dATP The additional purification of 20S U5 snRNPs by Mono Q chromatography does not affect the efficiency of ATP hydrolysis. Both US and tri-snRNPs bind ATP stoichiometrically in an RNA-independent manner. A candidate ATPase was identified by UV-irradiation of purified snRNPs with radiolabeled ATP. In the presence of homopolymeric RNA, the 200 kDa US-specific protein is the major crosslinked protein, even in Mono Q-purified U5 snRNPs. The correlation between RNA-dependent ATPase activity in the U5 snRNP and the RNA-dependent onset of this crosslink strongly suggests that the 200 kDa protein is an RNA-dependent ATPase. Furthermore, both the formation of the crosslink and ATPase activity appear with a similar substrate specificity for ATP.