AN EVOLUTIONARILY CONSERVED U5 SNRNP-SPECIFIC PROTEIN IS A GTP-BINDING FACTOR CLOSELY-RELATED TO THE RIBOSOMAL TRANSLOCASE EF-2

The driving forces behind the many RNA conformational changes occurrin g in the spliceosome are not well understood. Here we characterize an evolutionarily conserved human U5 small nuclear ribonucleoprotein (snR NP) protein (U5-116kD) that is strikingly homologous to the ribosomal elongation factor EF-2 (ribosomal translocase). A 114 kDa protein (Snu 114p) homologous to U5-116kD was identified in Saccharomyces cerevisia e and was shown to be essential for yeast cell viability. Genetic depl etion of Snu114p results in accumulation of unspliced pre-mRNA, indica ting that Snu114p is essential for splicing in vivo. Antibodies specif ic 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 colocali zes with snRNP-containing subnuclear structures referred to as speckle s. The G domain of U5-116kD/Snu114p contains the consensus sequence el ements G1-G5 important for binding and hydrolyzing GTP. Consistent wit h this, U5-116kD can be cross-linked specifically to GTP by UV irradia tion of U5 snRNPs. Moreover, a single amino acid substitution in the G 1 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 f irst evidence that a G domain-containing protein plays an essential ro le in the pre-mRNA splicing process.