An evolutionarily conserved U 5 snRNP-specific protein is a GTP-binding factor closely related to the ribosomal translocase EF2 a large number of transacting factors that assemble in

Institut für Molekularbiologie und Tumorforschung, Philippsincorporated into the pre-mRNA during splicing, ATP is Universität Marburg, Emil-Mannkopff-Strasse 2, D-35037 Marburg, an essential cofactor (Hardy et al., 1984; Frendewey and Germany and 2Harvard Microchemistry Facility, 16 Divinity Avenue, Keller, 1985) and has been shown to be involved in Cambridge, MA 02138, USA several steps from spliceosome assembly to product release 1Present address: Qiagen GmbH, Max-Volmer-Strasse 4, 40724 Hilden, (reviewed in Guthrie, 1991; Moore et al., 1993). Germany Two classes of splicing factors are distinguished cur3Corresponding author rently. The first class comprises four evolutionarily cone-mail: luehrmann@imt.uni-marburg.de served small nuclear ribonucleoprotein (snRNP) particles, U1, U2, U4/U6 and U5, that contain either one (U1, U2, The driving forces behind the many RNA conformU5) or two (U4/U6) snRNA components (for review, see ational changes occurring in the spliceosome are not Green, 1991; Guthrie, 1991; Rymond and Rosbash, 1992; well understood. Here we characterize an evoluMoore et al., 1993); the second class consists of an as yet tionarily conserved human U5 small nuclear ribounknown number of proteins that are not tightly bound to nucleoprotein (snRNP) protein (U5-116kD) that is snRNPs and are therefore termed non-snRNP splicing strikingly homologous to the ribosomal elongation factors (see Lamm and Lamond, 1993; Beggs, 1995; factor EF-2 (ribosomal translocase). A 114 kDa protein Krämer, 1995). (Snu114p) homologous to U5-116kD was identified in The composition of the U snRNPs has been studied Saccharomyces cerevisiae and was shown to be essential most extensively in HeLa cells (Will et al., 1995). At low for yeast cell viability. Genetic depletion of Snu114p salt concentrations (up to 100 mM), where HeLa nuclear results in accumulation of unspliced pre-mRNA, extracts support pre-mRNA splicing in vitro, a 12S U1 indicating that Snu114p is essential for splicing in vivo. snRNP, 17S U2 snRNP and a 25S [U4/U6·U5] tri-snRNP Antibodies specific for U5-116kD inhibit pre-mRNA complex are found. At high salt concentrations (350– splicing in a HeLa nuclear extract in vitro. In HeLa 450 mM), the tri-snRNP complex dissociates into a 20S cells, U5-116kD is located in the nucleus and coU5 and a 12S U4/U6 particle. In the U4/U6 snRNP, the localizes with snRNP-containing subnuclear structures U4 and U6 snRNAs interact through extensive sequence referred to as speckles. The G domain of U5-116kD/ complementarity (Bringmann et al., 1984; Hashimoto and Snu114p contains the consensus sequence elements Steitz, 1984; Rinke et al., 1985; Brow and Guthrie, 1988). G1–G5 important for binding and hydrolyzing GTP. The proteins of the snRNPs fall into two groups, the Consistent with this, U5-116kD can be cross-linked common proteins (B/B , D1, D2, D3, E, F and G), specifically to GTP by UV irradiation of U5 snRNPs. which are present in each snRNP, and the particle-specific Moreover, a single amino acid substitution in the G1 proteins. While U1 and U2 snRNPs contain three (70K, sequence motif of Snu114p, expected to abolish GTPA and C) and 11 specific proteins respectively, the tribinding activity, is lethal, suggesting that GTP binding snRNP has an even more complex protein composition. and probably GTP hydrolysis is important for the The 20S U5 snRNP component contains nine specific function of U5-116kD/Snu114p. This is to date the first proteins with apparent mol. wts of 15, 40, 52, 100, 102, evidence that a G domain-containing protein plays an 110, 116, 200 and 220 kDa (Behrens and Lührmann, essential role in the pre-mRNA splicing process. 1991), while two proteins with apparent mol. wts of 60