The U1 small nuclear RNA-protein complex selectively binds a 5′ splice site in vitro

Defining a 5??? splice site by functional selection in the presence and absence of U1 snRNA 5??? end

“…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...…”

Identification and purification of a 62,000-dalton protein that binds specifically to the polypyrimidine tract of introns.

“…It has long been recognized that the formation of spe cific complexes containing Ul and U2 snRNPs, respec tively, bound to the 5' splice site, and the branch site is an early step in the splicing of pre-mRNAs (Mount et al 1983;Black et al 1985;Konarska and Sharp 1986;Ruby and Abelson 1988). Depletion of nuclear extracts of Ul snRNP abolishes the activity of the 5' splice site in pro moting the U2 snRNP/pre-mRNA association (Ruby and Abelson 1988;Seraphin et al 1988).…”

Synthesis of U1 RNA in isolated nuclei from sea urchin embryos: U1 RNA is initiated at the first nucleotide of the RNA.

“…The sequence of this RNA has been highly conserved in evolution, with only a 25 to 30% difference between Ul RNA from such diverse organisms as rats, drosophila, and sea urchins (3,5,28). Much evidence is accumulating that Ul RNA functions in the biosynthesis of mRNA at the level of RNA splicing (17,27,31). However, little is known about the biosynthesis of Ul RNA.…”