A dominant negative mutation in the conserved RNA helicase motif ‘SAT’ causes splicing factor PRP2 to stall in spliceosomes.

Abstract

To characterize sequences in the RNA helicase‐like PRP2 protein of Saccharomyces cerevisiae that are essential for its function in pre‐mRNA splicing, a pool of random PRP2 mutants was generated. A dominant negative allele was isolated which, when overexpressed in a wild‐type yeast strain, inhibited cell growth by causing a defect in pre‐mRNA splicing. This defect was partially alleviated by simultaneous co‐overexpression of wild‐type PRP2. The dominant negative PRP2 protein inhibited splicing in vitro and caused the accumulation of stalled splicing complexes. Immunoprecipitation with anti‐PRP2 antibodies confirmed that dominant negative PRP2 protein competed with its wild‐type counterpart for interaction with spliceosomes, with which the mutant protein remained associated. The PRP2‐dn1 mutation led to a single amino acid change within the conserved SAT motif that in the prototype helicase eIF‐4A is required for RNA unwinding. Purified dominant negative PRP2 protein had approximately 40% of the wild‐type level of RNA‐stimulated ATPase activity. As ATPase activity was reduced only slightly, but splicing activity was abolished, we propose that the dominant negative phenotype is due primarily to a defect in the putative RNA helicase activity of PRP2 protein.

Comprehensive Mutational Analysis of Yeast DEXD/H Box RNA Helicases Required for Small Ribosomal Subunit Synthesis

“…Such an approach has been successfully applied to precisely determine the function of many RNA helicases in energy-dependent steps of spliceosome assembly-disassembly. In almost all cases, these proteins were trapped in inactive spliceosomes (2,10,19,26,31,39,40). This strategy has also been adapted to characterize the recently discovered Q motif (44).…”

“…The first aspartic acid in motif II (DEXD/H box), which may coordinate binding of the Mg 2ϩ cofactor (3), was replaced by alanine. The first amino acid in motif III was mutated to a leucine, which, based on in vivo and in vitro studies, may uncouple ATP hydrolysis and RNA helicase activity (30,31,40). Overall, we predicted that these mutations would specifically affect ATP binding and hydrolysis and would have the lowest probability of affecting RNA or RNP substrate binding.…”

“…Previous results suggest that motif III has a role in coupling ATP hydrolysis with helicase activity but is not required for binding ATP. In the prototypical DEAD box protein eIF4-A and the DEAH box protein Prp22, substitution of the serine (SAT) in motif III abrogated RNA helicase activity but did not dramatically affect ATP hydrolysis (30,40); in the case of Prp2, it also did not interfere with ATP hydrolysis (31). We showed that the serine-to-leucine substitutions in motif III only affected the DEAH box RNA helicases Dhr1 and Dhr2; the latter mutant also conferred a strong dominant negative growth defect when overexpressed (Table 1).…”

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

“…Important information concerning the 59SS:hPrp28p interaction was obtained from the mapping of the site of crosslink within the protein+ Because of the remarkable specificity of the BP-mediated crosslinking, the resulting product is virtually homogenous, representing a single contact between the 59SS RNA and hPrp28p+ The crosslink site maps to a short segment in the helicase domain of hPrp28p (amino acid positions 601-609), spanning the conserved TAT motif (motif III, positions 606-608; Lüking et al+, 1998)+ Mutational studies suggested that motif III (which in many other DExD/Hbox proteins contains an SAT rather than TAT sequence) is involved in coupling ATP hydrolysis with duplex unwinding (Schwer & Guthrie, 1992;Plumpton et al+, 1994;Gross & Shuman, 1998)+ In fact, in several DExH-box proteins, mutations of this motif result in inhibition of splicing (Plumpton et al+, 1994; B+ Schwer, pers+ comm+)+ Based on the structural similarity of several helicases, including PcrA and Rep DNA helicases (Subramanya et al+, 1996;Korolev et al+, 1997) and HCV RNA helicase (Yao et al+, 1997), a unifying mechanism for this class of proteins has been suggested (Bird et al+, 1998)+ Helicases coordinate a cycle of ATP binding and hydrolysis to an ordered series of conformational changes that allow the protein to move along its substrate+ Because of its localization in a hinge region connecting the two protein domains, motif III is thought to play a pivotal role in these conformational changes+ The current models predict that, upon binding of ATP and the single stranded portion of the substrate, the protein assumes a closed conformation+ Subsequent ATP hydrolysis yields an opened conformation of the protein, resulting in a stretched motion that pulls the single strand of RNA (or DNA) and as a result, unwinds the substrate duplex+ The structural organization of helicases requires that during catalysis, the single-stranded substrate is positioned in proximity of the conserved motif III hinge connecting the two protein domains that undergo the ATP-dependent conformational changes (Bird et al+, 1998;Velankar et al+, 1999)+ Although the structure of hPrp28p is not currently known, its TAT motif most likely also connects the two conformationally changing domains of the protein+ If the position of the single-stranded RNA relative to motif III is similar in hPrp28p and HCV NS3 RNA helicase, the observed BP-mediated crosslink (;15 Å linker) between the 59SS RNA and the TAT motif of hPrp28p is fully consistent with the predicted structure, strongly suggesting that the 59SS represents a true substrate for hPrp28p+ Although it is formally possible that the observed 59SS:hPrp28p crosslink reflects only a close proximity, and not the direct contact between the 59SS and the helicase, the simplest interpretation is that the crosslink results from the proper positioning of the 59SS in the opened conformation of hPrp28p+ In fact, the present study offers the first biochemical evidence of the direct, specific contact between an RNA substrate and its putative RNA helicase+…”

The first ATPase domain of the yeast 246-kDa protein is required for in vivo unwinding of the U4/U6 duplex

“…The DEAD/DEXH-box proteins are involved in diverse cellular functions including pre-mRNA splicing (Wassarman & Steitz, 1991;Ripmaster et al+, 1992;Schmid & Linder, 1992;Czaplinski et al+, 1995;Liang et al+, 1996;Margossian et al+, 1996;Ohno & Shimura, 1996;Py et al+, 1996;Chuang et al+, 1997)+ This family is characterized by several highly conserved ATPase motifs including the DEAD or DExH sequence (Chang et al+, 1990;Koonin, 1991;Bork & Koonin, 1993)+ Functional assignments for several of the motifs have been made through analyses of various RNA helicases, including the extensively studied eIF4A (Pause & Sonenberg, 1992;Pause et al+, 1993Pause et al+, , 1994+ Mutations in ATPase A motif (GKT, motif I) affect ATP binding, whereas mutations in ATPase motif B (DEAD or DExH, motif II) affect ATP hydrolysis+ Motif VI (GRAGR) is involved in ATP hydrolysis, and the SAT sequence element (motif III) is essential for RNA unwinding+ Mutational alterations in these motifs result in specific functional impairment of each helicase thus far studied (Ohno & Simura, 1996;Burgess & Guthrie, 1993;Plumpton et al+, 1994)+ Analysis of dominant negative mutations has been proposed as a tool for identifying the function of a gene product by observing the functional and biochemical consequences of overexpressing mutant proteins in the presence of the wild-type protein (Herskowitz, 1987)+ It has been suggested that the generation of dominant negative mutations that affect the ATPase domains of DEAD or DExH proteins could provide a powerful strategy for elucidating the diverse roles of this gene/ protein family (Schwer & Guthrie, 1992b)+ Several dominant negative mutants in ATPase domains have been created to investigate the function of DEAD/Hbox proteins (Schwer & Guthrie, 1992b;Burgess & Guthrie, 1993;Plumpton et al+, 1994;Ohno & Simura, 1996;Kim & Lin, 1996)+ Studies of mutations in pre-mRNA splicing factors PRP2 and PRP16 have shown that mutant proteins with reduced ATPase activity remain tightly bound to their spliceosomal substrate (Schwer & Guthrie, 1992b;…”