The C-terminal heptad repeat domain (CTD) of RNA polymerase II (pol II) is proposed to target pre-mRNA processing enzymes to nascent pol II transcripts, but this idea has not been directly tested in vivo. In vitro, the yeast mRNA capping enzymes Ceg1 and Abd1 bind specifically to the phosphorylated CTD. Here we show that yeast capping enzymes cross-link in vivo to the 5 ends of transcribed genes and that this localization requires the CTD. Both the extent of CTD phosphorylation at Ser 5 of the heptad repeat and the binding of capping enzymes decreased as polymerase moved from the 5 to the 3 ends of the ACT1, ENO2, TEF1, GAL1, and GAL10 genes. Ceg1 is released early in elongation, but Abd1 can travel with transcribing pol II as far as the 3 end of a gene. The CTD kinase, Kin28, is required for binding, and the CTD phosphatase, Fcp1, is required for dissociation of capping enzymes from the elongation complex. CTD phosphorylation and dephosphorylation therefore control the association of capping enzymes with pol II as it transcribes a gene. The conserved CTD of the pol II large subunit has dual functions in controlling transcriptional responses (Scafe et al. 1990;Gerber et al. 1995) and in coordinating premRNA synthesis with processing. It has been suggested that the CTD serves as a landing pad for processing factors and thereby targets them specifically to transcripts made by pol II and not by other RNA polymerases (Yuryev et

In order to assess the role of Prp22 in yeast pre-mRNA splicing, we have purified the 130 kDa Prp22 protein and developed an in vitro depletion/reconstitution assay. We show that Prp22 is required for the second step of actin pre-mRNA splicing. Prp22 can act on preassembled spliceosomes that are arrested after step 1 in an ATP-independent fashion. The requirement for Prp22 during step 2 depends on the distance between the branchpoint and the 3Ј splice site, suggesting a previously unrecognized role for Prp22 in splice site selection. We characterize the biochemical activities of Prp22, a member of the DExH-box family of proteins, and we show that purified recombinant Prp22 protein is an RNA-dependent ATPase and an ATP-dependent RNA helicase. Prp22 uses the energy of ATP hydrolysis to effect the release of mRNA from the spliceosome. Thus, Prp22 has two distinct functions in yeast premRNA splicing: an ATP-independent role during the second catalytic step and an ATP-requiring function in disassembly of the spliceosome.

The assembly of the spliceosome is an ATP-dependent process. The splicing factor PRP16 contains variations of several motifs that define the eIF-4A-like ATP-dependent RNA helicase family. The protein has now been purified and shown to exhibit RNA-dependent ATPase activity. PRP16 is required specifically for the second catalytic step of the splicing reaction in vitro. This function requires ATP binding and/or hydrolysis, which appears to be concomitant with release of the protein from the spliceosome. PRP16 may be the prototype for a set of splicing factors which use ATP to drive a cycle of conformational changes.

Members of the family of DEXH/D-box proteins are involved in all major RNA transactions, including transcription, translation, ribosome biogenesis, and pre-mRNA splicing (1, 2). DEXH/D-box proteins can hydrolyze NTP to NDP in a reaction that is stimulated by, or dependent on, a nucleic acid cofactor. Although several DEXH/D family members exhibit RNA helicase activity in vitro, the action of DEXH/D-box NTPases may not be limited to the unwinding of RNA duplexes. Recent studies suggest that they can act as "RNPases" to displace proteins from nucleic acids (3-6). DEXH-box proteins are defined by conserved motifs I (GXGKT), II (DEXH), III (S/TAT), and VI (QRXGRXGR), which are important for ATP hydrolysis and RNA unwinding (7,8).The DEXH/D-box ATPases Prp5, Brr2, Prp28, Sub2/UAP56, Prp2, Prp16, and Prp22 are involved in pre-mRNA splicing (9). Removal of introns from precursor RNAs is catalyzed by the spliceosome, which is formed by the assembly of U1, U2, and U4/U6/U5 snRNPs and non-snRNP 1 proteins onto the precursor RNA (10, 11). Splicing entails two successive transesterification reactions: in step 1, the 5Ј splice site is cleaved and the branched lariat-intermediate is formed; in step 2, the 3Ј splice site is cleaved and the exons are joined. Mature mRNA is then released, and the spliceosome components are presumed to recycle for the next round of splicing (10). Splice site recognition and positioning of the reactive nucleotides for catalysis requires dynamic remodeling of an intricate network of RNA-RNA and RNA-protein interactions (12, 13). In vitro studies have established that ATP is required for many steps in the splicing cycle and that DEXH/D-box proteins act at those ATPdependent steps (9, 10). For example: Prp28, Brr2, Prp5, and Sub2/UAP56 are important for spliceosome assembly; Prp2 promotes step 1 transesterification; Prp16 is required for the second transesterification step; and Prp22 triggers the release of mature mRNA from the spliceosome (9, 14 -16). Prp2, Prp16, and Prp22 mutants that are defective for ATP hydrolysis are also defective in executing their ATP-dependent functions in pre-mRNA splicing in vitro (16 -19). Such mutations are also invariably lethal in vivo (18,20,21). Moreover, overexpression of non-functional Prp2, Prp16, and Prp22 mutants impairs the growth of wild-type cells (18,20,21). The dominant-negative Prp16 and Prp22 phenotypes can be recapitulated in vitro with purified proteins; for example, inactive Prp16 proteins block step 2 transesterification chemistry and dominant-negative Prp22 proteins block release of mature mRNA from the spliceosome in trans (19). Thus, the steps arrested by the dominant-negative mutants illuminate the function of the wild-type proteins during pre-mRNA splicing. S. cerevisiae PRP43 and its mammalian homologue mDEAH9 were isolated in PCR-based screens for DEAH-box proteins (22,23). Yeast PRP43 is an essential gene that encodes a 767-amino acid polypeptide with a predicted molecular mass of 88 kDa. Arenas and Abelson (22) isolated a temperature-sensit...

DEAD/H-box NTPases remodel the spliceosome at multiple steps during the pre-mRNA splicing cycle. The RNA-dependent NTPase Prp43 catalyzes dissociation of excised lariat-intron from the spliceosome, but it is unclear how Prp43 couples the energy of ATP hydrolysis to intron release. Here, we report that activation of Prp43's inherently feeble helicase activity by the splicing factor Ntr1 is required for lariat-intron release. Lethal Prp43 mutants T384A and T384V, which are active for ATP hydrolysis and fail to dissociate lariat-intron from spliceosomes, are refractory to stimulation of RNA unwinding by Ntr1. An N-terminal 120-amino-acid segment of Ntr1 suffices for binding to Prp43 and for stimulating its helicase activity. We identify missense mutations in Prp43 and Ntr1 that disrupt protein-protein interaction and impair Ntr1 enhancement of Prp43 RNA unwinding. Our results demonstrate for the first time that regulating the motor activity of a DEAH-box protein by an accessory factor is critical for mRNA splicing.[Keywords: Pre-mRNA splicing; DEAH-box helicase; Prp43; Ntr1; spliceosome] Supplemental material is available at http:/www.genesdev.org. Nuclear pre-mRNA splicing proceeds via two successive transesterification reactions (Burge et al. 1999). In the first step, the 2Ј-OH of the branch site adenosine within the intron attacks the phosphodiester at the 5Ј exonintron boundary to form a lariat-intermediate. In the second step, the 3Ј-OH of the 5Ј exon attacks the phosphodiester at the intron-3Ј exon junction to form mature mRNA and lariat-intron. Pre-mRNA splicing requires five snRNAs and >80 proteins (Will and Lührmann 2006). The small nuclear RNAs (snRNAs), packaged as snRNPs, assemble together with non-snRNP proteins onto the precursor RNA to form the spliceosome. The snRNPs are involved in identifying the splice junctions and positioning the reactive nucleotides for catalysis (Valadkhan 2005). ATP-dependent remodeling of RNA-RNA and RNA-protein interactions is essential for faithful spliceosome assembly, for the catalytic activation of the splice sites, and for spliceosome disassembly (Staley and Guthrie 1998).The remodeling events in the spliceosome are catalyzed by DExD/H NTPases that act at discrete stages of the splicing pathway (Staley and Guthrie 1998). Splicing factors Prp2, Prp16, Prp22, and Prp43 are members of the DEAH family of RNA-dependent NTPases (Burgess et al. 1990;Chen and Lin 1990;Company et al. 1991;Arenas and Abelson 1997). They are composed of a central NTPase/helicase domain, characteristic of all DExH proteins, plus an ∼300-amino-acid C-terminal domain unique to the Prp2/Prp16/Prp22/Prp43 clade (Tanner and Linder 2001;Silverman et al. 2003). The N-terminal segments are distinctive in each DEAH-box splicing factor. Understanding how the DEAH proteins carry out their specific functions during the splicing cycle requires knowledge about how they are recruited to the splicing complex, what their biochemical activities are, and whether they are regulated.Prp43 catalyzes the final step of ...