THE HUMAN U5-2OOKD DEXH-BOX PROTEIN UNWINDS U4 U6 RNA DUPLICES IN-VITRO/

Splicing of nuclear precursors of mRNA (pre-mRNA) involves dynamic int eractions between the RNA constituents of the spliceosome. The rearran gement of RNA-RNA interactions, such as the unwinding of the U4/U6 dup lex, is believed to be driven by ATP-dependent RNA helicases, We recen tly have shown that spliceosomal U5 small nuclear ribonucleoproteins ( snRNPs) from HeLa cells contain two proteins, U5-200kD and U5-100kD, w hich share homology with the DEAD/DEXH-box families of RNA helicases, Here we demonstrate that purified U5 snRNPs exhibit ATP-dependent unwi nding of U4/U6 RNA duplices in vitro. To identify the protein responsi ble for this activity, U5 snRNPs were depleted of a subset of proteins under high salt concentrations and assayed for RNA unwinding, The act ivity was retained in U5 snRNPs that contain the U5-200kD protein but lack U5-100kD, suggesting that the U5-200kD protein could mediate U4/U 6 duplex unwinding. Finally, U5-200kD was purified to homogeneity by g lycerol gradient centrifugation of U5 snRNP proteins in the presence o f sodium thiocyanate, followed by ion exchange chromatography. The RNA unwinding activity was found to reside exclusively with the U5-200kD DEXH-box protein. Our data raise the interesting possibility that this RNA helicase catalyzes unwinding of the U4/U6 RNA duplex in the splic eosome.