An Early Hierarchic Role of U1 Small Nuclear Ribonucleoprotein in Spliceosome Assembly

Abstract

Splicing of nuclear precursor messenger RNA (pre-mRNA) occurs on a large ribonucleoprotein complex, the spliceosome. Several small nuclear ribonucleoproteins (snRNP's) are subunits of this complex that assembles on the pre-mRNA. Although the U1 snRNP is known to recognize the 5' splice site, its roles in spliceosome formation and splice site alignment have been unclear. A new affinity purification method for the spliceosome is described which has provided insight into the very early stages of spliceosome formation in a yeast in vitro splicing system. Surprisingly, the U1 snRNP initially recognizes sequences at or near both splice junctions in the intron. This interaction must occur before the other snRNP's (U2, U4, U5, and U6) can join the complex. The results suggest that interaction of the two splice site regions occurs at an early stage of spliceosome formation and is probably mediated by U1 snRNP and perhaps other factors.

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).…”

“…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). Recognition of both sites is important in the formation of each of these snRNA/pre-mRNA complexes.…”

“…For example, mutational changes of sequences at the 5' splice site reduce the rate of formation of the U2 snRNP complex at the branch site (Lamond et al 1987). Similarly, mutational changes at the branch site region reduce the rate of association of Ul snRNP at the 5' splice site (LeGrain et al 1988;Ruby and Abelson 1988). Depletion of nuclear extracts of U2 snRNP, however, does not affect the activity of the branch site in promoting the Ul snRNP association with the 5' spHce site (LeGrain et al 1988;Ruby and Abelson 1988).…”

“…Similarly, mutational changes at the branch site region reduce the rate of association of Ul snRNP at the 5' splice site (LeGrain et al 1988;Ruby and Abelson 1988). Depletion of nuclear extracts of U2 snRNP, however, does not affect the activity of the branch site in promoting the Ul snRNP association with the 5' spHce site (LeGrain et al 1988;Ruby and Abelson 1988). Thus, it is likely that RNA sequence-spe cific binding proteins are important in recognition of the branch site at early stages in splicing and in promoting formation of complexes containing Ul and U2 snRNPs (Gerke and Steitz 1986;Mayeda et al 1986;Steitz 1986;Tazi et al 1986;Ruskin et al 1988).…”

Sequences upstream of the branch site are required to form helix II between U2 and U6 snRNA in a trans-splicing reaction

“…Splicing of major class U2-dependent introns involves sequential and coordinated binding of five snRNPs (U1, U2, U4, U5 and U6) to the mRNA precursor. In the conventional view, U1 first binds across the exon/intron boundary at the 5′-end of introns [5′ splice site (SS); 7-9], leading to formation of an ATP-independent complex committed to splicing (complex E in mammalian extract and complexes CC1 and CC2 in yeast extract; [10][11][12][13]. Upon addition of ATP, complex E assembles into complex A, U2 stably associates with the branch site (BS) and U1 dissociates from the 5′ SS (14)(15)(16)(17)(18).…”