Evidence that U 5 snRNP recognizes the 3 9 splice site for catalytic step II in mammals sites in pre-mRNA very near or at the catalytic center

sites in pre-mRNA very near or at the catalytic center Maria Dolores Chiara1, Leon Palandjian, of the spliceosome, and these interactions are detected Rebecca Feld Kramer and Robin Reed2 immediately prior to the two steps of splicing (Parker Department of Cell Biology, Harvard Medical School, Boston, et al., 1987; Wu and Manley, 1989; Zhuang and Weiner, MA 02115, USA 1989; Newman and Norman, 1991, 1992; Sawa and 1Present address: Departamento de Fisiologia Medica y Biofisica, Abelson, 1992; Sawa and Shimura, 1992; Wassarman and Facultad de Medicina, Universidad de Sevilla, E-41009, Sevilla, Steitz, 1992; Wyattet al., 1992; Corteset al., 1993; Spain Kandels-Lewis and Seraphin, 1993; Lesser and Guthrie, 2Corresponding author 1993; Sontheimer and Steitz, 1993; Newman et al., 1995; O’Keefe et al., 1996). One or more of these snRNAs are M.D.Chiara and L.Palandjian contributed equally to this work believed to be the catalytic moieties of the spliceosome (for reviews, see Madhani and Guthrie, 1994; Newman, The first AG dinucleotide downstream from the 1994; Nilsen, 1994; Kramer, 1996). In contrast to other branchpoint sequence (BPS) is chosen as the 3 9 splice site during catalytic step II of the splicing reaction. examples of RNA-mediated catalysis, proteins play crucial The mechanism and factors involved in selection of this roles in splicing. However, the technical difficulty of AG are not known. Early in mammalian spliceosome pinpointing specific binding sites of proteins on RNA has assembly, U2AF65binds to the pyrimidine tract between hampered progress in identifying RNA–protein interthe BPS and AG. Here we show that U2AF65 crosslinkactions at the catalytic center of the spliceosome. ing is replaced by crosslinking of three proteins of 110, One step for which little information is available is 116 and 220 kDa prior to catalytic step II, and we selection of the 3 9 splice site during catalytic step II. In provide evidence that all three proteins are components both yeast and metazoans, the 3 9 splice site consists of of U5 snRNP. These proteins interact with pre-mRNA YAG (Y 5 pyrimidine), and the pre-mRNA is cleaved in the region spanning from immediately downstream after the G residue during catalytic step II. The YAG is of U2 snRNP’s binding site at the BPS to just beyond preceded by a pyrimidine tract, with this element being the 39 splice site. We also demonstrate that there are less conserved in yeast. In most introns, the first AG strict constraints on both the sequence and the distance downstream of the branchpoint sequence (BPS) serves as between the BPS and AG for catalytic step II. Together, the 39 splice site, and this AG is typically located within these observations suggest that U5 snRNP is positioned 20–40 nt of the BPS. Although the mechanism and factors on the 39 splice site by an interaction (direct or indirect) involved in selecting the correct AG are not understood, with U2 snRNP bound at the BPS and by a direct considerable progress has been made in the yeast system interaction with the pyrimidine tract. The functional (for review, see Umen and Guthrie, 1995a). Analysis of AG for catalytic step II may be specified, in turn, by mutant pre-mRNAs has revealed that a uridine-rich tract its location with respect to the U5 snRNP binding site. adjacent to the AG enhances the efficiency of step II