“…Splicing of pre-mRNA is carried out by the spliceosome that consists of five small nuclear ribonucleoprotein particles (snRNPs), U1, U2, U4, U5, and U6, and a number of non-snRNP protein factors+ The snRNPs consist of several protein components and a unique small nuclear RNA (snRNA)+ The sequences of mammalian 59 splice sites vary considerably but generally conform to the consensus A Ϫ2 G Ϫ1 /G 1 U 2 R 3 A 4 G 5 U 6 (R ϭ purine, nucleotides in the pre-mRNA are numbered relative to the exon/intron junction throughout this paper; Mount, 1982;Padgett et al+, 1986;Shapiro & Senapathy, 1987)+ Three of the five spliceosomal snRNAs interact with the 59 splice site during the course of the splicing reaction (Fig+ 1)+ U1 snRNP associates with the 59 splice site in an ATP-independent step and forms the earliest detectable splicing complex, the commitment complex (CC), thereby protecting a region spanning positions Ϫ3 to ϩ12 relative to the 59 splice site from RNase digestion (Mount et al+, 1983)+ The 59 splice site is complementary to the 59 end of the U1 snRNA and recognition of the 59 splice site involves base pairing with the U1 snRNA (Zhuang & Weiner, 1986;Séraphin et al+, 1988;Siliciano & Guthrie, 1988)+ The interaction between the pre-mRNA and U1 snRNP is stabilized by other interactions, as in both the Saccharomyces cerevisiae and mammalian splicing systems a U1 snRNP particle lacking the 59 end of the U1 snRNA is still able to interact specifically with an RNA oligonucleotide containing a consensus 59 splice site, albeit with lower stability (Rossi et al+, 1996;Du & Rosbash, 2001)+ Moreover, in S. cerevisiae, several U1 snRNP protein components can be crosslinked to the 59 splice site region (Zhang & Rosbash, 1999;see Discussion), implying that both U1 snRNP proteins and snRNA associate with the pre-mRNA+ In addition to U1 snRNP-associated proteins, other splicing factors may facilitate binding of the U1 snRNP to the 59 splice site+ The best studied example is the splicing factor ASF/SF2, which, upon binding to nearby purine-rich sequences, enhances the interaction of the U1 snRNP with the 5 splice site, prob-ably through an interaction between the RS domains of ASF/SF2 and the U1 snRNP component U1-70K (Wu & Maniatis, 1993;Kohtz et al+, 1994)+ U6 snRNA also interacts with the 59 splice site during splicing+ Crosslinking experiments in both mammalian and yeast systems place positions ϩ2 to ϩ6 at the 59 splice site in close proximity of the conserved A 41 CAGAG 46 sequence in U6 snRNA (Wassarman & Steitz, 1992;Kim & Abelson, 1996; numbers refer to the human U6 snRNA; Fig+ 1)+ A site-specific crosslink to position ϩ2 in the intron was only observed in lariat species, suggesting that this base pair only forms during the second step of splicing (Sontheimer & Steitz, 19...…”