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- Materials and methods
The driving forces behind the many RNA conformational changes occurring in the spliceosome are not well understood. Here we characterize an evolutionarily conserved human U5 small nuclear ribonucleoprotein (snRNP) protein (U5-116kD) that is strikingly homologous to the ribosomal elongation factor EF-2 (ribosomal translocase). A 114 kDa protein (Snu114p) homologous to U5-116kD was identified in Saccharomyces cerevisiae and was shown to be essential for yeast cell viability. Genetic depletion of Snu114p results in accumulation of unspliced pre-mRNA, indicating that Snu114p is essential for splicing in vivo. Antibodies specific for U5-116kD inhibit pre-mRNA splicing in a HeLa nuclear extract in vitro. In HeLa cells, U5-116kD is located in the nucleus and co-localizes with snRNP-containing subnuclear structures referred to as speckles. The G domain of U5-116kD/Snu114p contains the consensus sequence elements G1–G5 important for binding and hydrolyzing GTP. Consistent with this, U5-116kD can be cross-linked specifically to GTP by UV irradiation of U5 snRNPs. Moreover, a single amino acid substitution in the G1 sequence motif of Snu114p, expected to abolish GTP-binding activity, is lethal, suggesting that GTP binding and probably GTP hydrolysis is important for the function of U5-116kD/Snu114p. This is to date the first evidence that a G domain-containing protein plays an essential role in the pre-mRNA splicing process.
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- Materials and methods
Splicing of nuclear mRNA precursors (pre-mRNA) proceeds via two consecutive transesterification steps. In the first step, the 2′ hydroxyl group of the branch-point adenosine attacks the 5′ splice site, generating the splicing intermediates, exon 1 and lariat-exon 2. The second step involves nucleophilic attack by the 3′ hydroxyl group of exon 1 at the 3′ splice site, producing ligated exons 1 and 2 and excised intron in the form of a lariat. Splicing requires a large number of trans-acting factors that assemble in an orderly manner on the pre-mRNA, thereby forming the catalytic splicing machinery known as the spliceosome. Despite the fact that exogenous phosphates are not incorporated into the pre-mRNA during splicing, ATP is an essential cofactor (Hardy et al., 1984; Frendewey and Keller, 1985) and has been shown to be involved in several steps from spliceosome assembly to product release (reviewed in Guthrie, 1991; Moore et al., 1993).
Two classes of splicing factors are distinguished currently. The first class comprises four evolutionarily conserved small nuclear ribonucleoprotein (snRNP) particles, U1, U2, U4/U6 and U5, that contain either one (U1, U2, U5) or two (U4/U6) snRNA components (for review, see Green, 1991; Guthrie, 1991; Rymond and Rosbash, 1992; Moore et al., 1993); the second class consists of an as yet unknown number of proteins that are not tightly bound to snRNPs and are therefore termed non-snRNP splicing factors (see Lamm and Lamond, 1993; Beggs, 1995; Krämer, 1995).
The composition of the U snRNPs has been studied most extensively in HeLa cells (Will et al., 1995). At low salt concentrations (up to 100 mM), where HeLa nuclear extracts support pre-mRNA splicing in vitro, a 12S U1 snRNP, 17S U2 snRNP and a 25S [U4/U6.U5] tri-snRNP complex are found. At high salt concentrations (350–450 mM), the tri-snRNP complex dissociates into a 20S U5 and a 12S U4/U6 particle. In the U4/U6 snRNP, the U4 and U6 snRNAs interact through extensive sequence complementarity (Bringmann et al., 1984; Hashimoto and Steitz, 1984; Rinke et al., 1985; Brow and Guthrie, 1988).
The proteins of the snRNPs fall into two groups, the common proteins (B/B′, D1, D2, D3, E, F and G), which are present in each snRNP, and the particle-specific proteins. While U1 and U2 snRNPs contain three (70K, A and C) and 11 specific proteins respectively, the tri-snRNP has an even more complex protein composition. The 20S U5 snRNP component contains nine specific proteins with apparent mol. wts of 15, 40, 52, 100, 102, 110, 116, 200 and 220 kDa (Behrens and Lührmann, 1991), while two proteins with apparent mol. wts of 60 and 90 kDa are associated with the 12S U4/U6 snRNP (Gozani et al., 1994; Lauber et al., in preparation). The 25S [U4/U6.U5] tri-snRNP complex contains five additional proteins with apparent mol. wts of 15.5, 20, 27, 61 and 63 kDa, which are required for tri-snRNP formation (Behrens and Lührmann, 1991; Utans et al., 1992; Lauber et al., in preparation; reviewed in Will et al., 1995). The protein composition of the snRNPs of Saccharomyces cerevisiae has not yet been studied in detail, but recent genetic and biochemical results strongly indicate that not only the snRNA components, but also the snRNP proteins are evolutionarily highly conserved (Fabrizio et al., 1994; Lauber et al., 1996; Neubauer et al., 1997; for review, see Beggs, 1995; Krämer, 1995).
The spliceosome is formed by the ordered, stepwise assembly of both snRNPs and other splicing factors with the pre-mRNA. A striking feature of the spliceosome assembly pathway is the formation of a dynamic RNA network which not only involves interactions between the pre-mRNA and the snRNAs but also among the snRNAs themselves (Moore et al., 1993; Madhani and Guthrie, 1994; Nilsen, 1994). In the early phase of spliceosome formation, U1 snRNA base-pairs with the 5′ splice site, while U2 snRNA interacts with the branch site. At this stage, several non-snRNP splicing factors, such as SF2/ASF, U2AF, SC35 and SF1, cooperate with U1 and U2 to form the mammalian pre-spliceosome (Krämer and Utans, 1991; Fu and Maniatis, 1992; Krämer, 1992; Zamore et al., 1992; Eperon et al., 1993; Zuo and Manley, 1994; reviewed in Hodges and Beggs, 1994; Reed, 1996). In the final step of spliceosome assembly, the 25S [U4/U6.U5] tri-snRNP complex and an as yet unknown number of non-snRNP splicing factors interact with the pre-spliceosome to form the mature spliceosome (reviewed in Moore et al., 1993). Before (or concomitantly with) the first step of splicing, the two helices of the U4/U6 interaction domain dissociate, and a new base-pairing interaction is formed between U2 and U6 (Datta and Weiner, 1991; Wu and Manley, 1991; Madhani and Guthrie, 1992; Sun and Manley, 1995). In addition, the U1 snRNA dissociates from the 5′ splice site which is then recognized by the conserved ACAGAG sequence of the U6 snRNA (Fabrizio and Abelson, 1990; Sawa and Abelson, 1992; Kandels-Lewis and Séraphin, 1993; Lesser and Guthrie, 1993; Sontheimer and Steitz, 1993). The conserved loop I of U5 snRNA also contacts exon sequences at the 5′ and 3′ splice sites, probably in a sequential manner, while the splicing reaction proceeds from step I to step II (Newman and Norman, 1991; Wyatt et al., 1992; Cortes et al., 1993; Sontheimer and Steitz, 1993). Upon completion of the splicing reaction, the spliceosome dissociates, after which the snRNPs are thought to undergo a recycling process. The interaction between the U2 and U6 snRNA and that between U6 snRNA and the pre-mRNA must revert to the earlier U4/U6 snRNA interaction, a process that is not yet well understood (reviewed in Moore et al., 1993).
A major goal of current spliceosome research is to understand the driving forces behind the many conformational changes occurring within the spliceosome and during the recycling of the spliceosomal subunits. A key to understanding these processes may lie in the protein moiety of the spliceosome. In yeast, several non-snRNP proteins have been shown to be essential for splicing; their sequences designate them as putative ATP-dependent RNA helicases of the DEAD-box family or its DEAH subgroup. These include Prp5p (Dalbadie-McFarland and Abelson, 1990) and Prp28p (Strauss and Guthrie, 1991), which belong to the DEAD-box family, and Prp2p (Chen and Lin, 1990; King and Beggs, 1990), Prp16p (Burgess et al., 1990) and Prp22p (Company et al., 1991), which are members of the DEAH-box subgroup (Wassarman and Steitz, 1991; Schmid and Linder, 1992). The participation of these essential proteins also explains, at least in part, the requirement for ATP in the splicing process. We recently have identified a novel putative RNA helicase in purified human 20S U5 snRNPs, the first intrinsic snRNP protein in this category (Lauber et al., 1996). Interestingly, an evolutionarily conserved homolog of this protein has also been found in yeast. The yeast homolog Snu246p is essential for cell viability and is also an integral component of yeast [U4/U6.U5] tri-snRNPs (Lauber et al., 1996; Lin and Rossi, 1996; Noble and Guthrie, 1996; Xu et al., 1996).
The helicases described above are thought to affect the conformation of the spliceosome through a direct interaction with RNA. Although ATP-dependent RNA helicases appear to play a major role in the structural rearrangements of the spliceosome, structural changes in other ribonucleoprotein complexes, such as the ribosome, are mediated by a different class of enzymes. In particular, conformational changes within the ribosome are dependent upon the GTPases EF-1α and EF-2 (EF-Tu and EF-G in procaryotes) rather than ATP-dependent helicases. These proteins interact transiently with the ribosome: their GTPase activity induces dissociation from the ribosome before a new cycle of translation can begin. EF-G becomes activated upon association with the ribosome and induces a transition of the ribosome from a pre- to a post-translocational state (reviewed in Noller et al., 1990; Nierhaus et al., 1993; Abel and Jurnack, 1996). The other ribosomal GTPase, EF-Tu, is thought to play a critical role in the fidelity of the translation process (reviewed in Noller et al., 1990; Nierhaus et al., 1993; Abel and Jurnack, 1996). As pre-mRNA splicing also requires a precise chemistry, it is conceivable that the spliceosome utilizes similar mechanisms to provide fidelity (Burgess and Guthrie, 1993).
In search of additional snRNP proteins that might play a role in regulating spliceosomal conformational changes, we initially microsequenced proteins present in the U5 and [U4/U6.U5] snRNPs. Strikingly, we discovered that the 116 kDa protein of human U5 snRNPs (U5-116kD) is closely related to the eukaryotic ribosomal translocase (elongation factor EF-2). U5-116kD contains the consensus sequence elements typical of the GTPase superfamily of proteins that bind and hydrolyze GTP. We demonstrate that this protein indeed binds GTP specifically. Homologs of U5-116kD were also identified in yeast (Snu114p) and nematodes and mouse. In yeast, Snu114p is essential for cell viability and splicing in vivo. In addition, a point mutation in the P-loop of the putative GTP-binding site of Snu114p is lethal. Taken together, the data presented here suggest that the GTP-binding domain of the U5-116kD protein plays an important role in either the splicing process itself or the recycling of spliceosomal snRNPs.