Spliceosome Activation by PRP2 ATPase prior to the First Transesterification Reaction of Pre-mRNA Splicing

Abstract

In addition to small nuclear RNAs and spliceosomal proteins, ATP hydrolysis is needed for nuclear pre-mRNA splicing. A number of RNA-dependent ATPases which are involved in several distinct ATPdependent steps in splicing have been identified in Saccharomyces cerevisiae and mammals. These so-called DEAD/H ATPases contain conserved RNA helicase motifs, although RNA unwinding activity has not been demonstrated in purified proteins. Here we report the role of one such DEAH protein, PRP2 of S. cerevisiae, in spliceosome activation. PRP2 bound to a precatalytic spliceosome prior to the first step of splicing. By blocking the activity of a novel splicing factor(s), HP, which was involved in a post-PRP2 step, we found that PRP2 hydrolyzed ATP to cause a change in the spliceosome without the occurrence of splicing. The change was quite dramatic and could account for the previously reported differences between the precatalytic, pre-mRNAcontaining spliceosome and the "active," intermediate-containing spliceosome. The post-PRP2-ATP spliceosome was further isolated and could carry out the subsequent reaction apparently in the absence of PRP2 and ATP. We hypothesize that PRP2 functions as a molecular motor, similar to some DExH ATPases in transcription, in the activation of the precatalytic spliceosome for the transesterification reaction.Several RNA-dependent ATPases which are essential for pre-mRNA splicing have been identified in Saccharomyces cerevisiae (reviewed in references 3, 17, 40, and 41). These proteins contain the conserved RNA helicase motifs, including the signature DEAD or DEAH sequence (reviewed in references 13, 15, 43, and 52). For example, PRP5 and PRP28 are involved in spliceosome assembly, PRP2 and PRP16 are required for the catalytic steps, and PRP22 is required for the release of mRNA from the spliceosome. Recently, a human PRP22 homolog was isolated and characterized (33,34), and a mammalian RNA-dependent ATPase activity was found to associate with the U5 small nuclear ribonucleoprotein particle (26). This work was an attempt to address the role of one such ATPase, PRP2, in pre-mRNA splicing.A considerable amount of work has been done on the role of PRP16 in splicing (reviewed in reference 50). PRP16 is required for an ATP-dependent reaction during the second step of splicing (44). ATP hydrolysis by PRP16 apparently can be uncoupled from the second transesterification reaction (reaction 2). This was shown by depleting splicing factors, such as PRP18 (20) or SLU7 (2, 21), which are needed after the PRP16 step from extracts. The 3Ј splice site becomes resistant to RNase H after ATP hydrolysis by PRP16, suggesting that some changes to the spliceosome occur (45). Interestingly, PRP16 also plays a proofreading role in splicing (7).Previous results from our own and other laboratories indicate that PRP2 also interacts with the spliceosome transiently (22, 24, 37). The protein binds to the pre-mRNA-containing spliceosome in the absence of ATP in vitro and is released from the spliceosome upon ATP hydrolysis a...

The DEAD Box Protein eIF4A. 2. A Cycle of Nucleotide and RNA-Dependent Conformational Changes

“…Other DEAD box proteins could perform functions distinct from RNA unwinding. These possible functions include (1) mediating larger scale RNA structural rearrangements (Figure 10b) (38)(39)(40); (2) altering protein-RNA or protein-protein interactions, analogous to the disruption of protein-DNA contacts in the nucleosome by the Swi/Snf complex ( Figure 10c; the ATPase of the Swi/Snf complex, Swi2/Snf2, is related to the DEAD box family (41,(42)(43)(44)(45)); and (3) functioning, as the G-protein EF-Tu does, as a guarantor of fidelity in RNA-RNA interactions and rearrangements (46). There is no reason to assume a priori that all members of a sequence family of proteins perform the same function.…”

Prp43 Is an Essential RNA-dependent ATPase Required for Release of Lariat-Intron from the Spliceosome

“…Prp2, Prp16, and Prp22 associate with the spliceosome at distinct stages and dissociate after they hydrolyze ATP (15,16,32). Their functional specificity is determined at least in part by unique interactions with the spliceosome.…”

“…The samples were incubated on ice for 10 min, and the charcoal was recovered by centrifugation. 32 P radioactivity in the supernatant was quantified by liquid scintillation counting. All the values are averages from at least two reaction mixtures, with a variation of less than 10% between experiments.…”

The 100-kDa U5 snRNP protein (hPrp28p) contacts the 5′ splice site through its ATPase site

“…Splicing of pre-mRNA involves two successive transesterification reactions carried out by a large ribonucleoprotein (RNP) spliceosome complex+ This complex is composed of five snRNAs (U1, U2, U4, U5, and U6) and nearly 100 proteins (Moore et al+, 1993)+ Spliceosome formation involves a complex network of specific and conserved interactions among snRNAs, as well as between snRNAs and pre-mRNA, that lead to formation of the catalytic center+ During this process, an initial interaction of U1 snRNP with the 59 splice site (59SS) is followed by recognition of the branch site by U2 snRNP, yielding splicing complex A+ At the stage of dissociation of the 59SS:U1 snRNA duplex and release of U1 snRNP, U4/U5/U6 tri-snRNP joins complex A to form splicing complex B, which subsequently rearranges into the catalytically active complex C+ Several DExD/ H-box proteins (ATPases/RNA helicases, or unwindases) identified both in yeast and mammalian systems are thought to mediate multiple RNA conformational changes occurring throughout the splicing process (reviewed in Staley & Guthrie, 1998;Murray & Jarrell, 1999)+ One of these factors, yPrp28p, has been implicated in the switch between the initial 59SS:U1 snRNA duplex and the subsequent 59SS:U6 snRNA pairing (Staley & Guthrie, 1998, 1999)+ However, a number of questions concerning the mechanism of action, recognition of RNA substrates, and interactions of these putative RNA helicases with other spliceosomal proteins remain to be answered+ Gorbalenya and Koonin (1993) classified helicases into three major superfamilies based on characteristic motifs shared by these proteins+ Crystal structures of two DNA helicases; PcrA and Rep (Subramanya et al+, 1996;Korolev et al+, 1997;Velankar et al+, 1999), and two RNA helicases; HCV NS3 and eIF4A (Yao et al+, 1997;Kim et al+, 1998), demonstrate that the conserved motifs required for ATP binding and hydrolysis are located at similar positions along the cleft separating two structurally similar protein domains+ Helicases are known to be involved in several biological processes, including replication, translation, transcription, and pre-mRNA splicing (Schmid & Linder, 1992;Lüking et al+, 1998)+ Among the DExD/H-box proteins involved in splicing, several exhibit single-stranded RNAdependent ATPase activity in vitro (Schwer & Guthrie, 1991;Kim & Lin, 1996;O'Day et al+, 1996;Raghunathan & Guthrie, 1998;Schwer & Gross, 1998;Wagner et al+, 1998), but only in a few cases has the ATPdependent RNA unwinding been demonstrated in vitro …”