Characterization of U4 and U6 interactions with the 5′ splice site using a S. cerevisiae in vitro trans-splicing system

Abstract

Spliceosome assembly has been characterized as the ordered association of the snRNP particles U1, U2, and U4/U6·U5 onto pre-mRNA. We have used an in vitro trans-splicing/cross-linking system in Saccharomyces cerevisiae nuclear extracts to examine the first step of this process, 5 splice site recognition. This trans-splicing reaction has ATP, Mg 2+, and splice-site sequence requirements similar to those of cis-splicing reactions. Using this system, we identified and characterized a novel U4-5 splice site interaction that is ATP-dependent, but does not require the branch point, the 3 splice site, or the 5 end of the U1 snRNA. Additionally, we identified several ATP-dependent U6 cross-links at the 5 splice site, indicating that different regions of U6 sample it before a U6-5 splice site interaction is stabilized that persists through the first step of splicing. This work provides evidence for ATP-dependent U4/U6 association with the 5 splice site independent of ATP-mediated U2 association with the branch point. Furthermore, it defines specific nucleotides in U4 and U6 that interact with the 5 splice site at this early stage, even in the absence of base-pairing with the U1 snRNA. Messenger RNA splicing is a critical step in the process of eukaryotic gene expression. In splicing, the introns are removed from newly synthesized RNA by the complex molecular machinery of the spliceosome. Assembly of the spliceosome is driven by a series of interactions between the pre-mRNA, five small nuclear ribonucleoprotein particles, and a host of trans-acting, non-snRNP proteins (Will and Lührmann 1997;Burge et al. 1999;Stevens and Abelson 1999). Extensive biochemical and genetic analyses have been carried out to identify the steps in assembly as well as the specific RNA-RNA and RNA-protein contacts that are involved.The first step of splicing is the accurate recognition of the pre-mRNA at the correct splice sites. These events are thought to be initiated when, in the absence of ATP, the U1-snRNP particle associates with the 5Ј splice site via base-pairing with the 5Ј end of the U1 snRNA (Mount 1982;Zhuang and Weiner 1986;Seraphin et al. 1988;Siliciano and Guthrie 1988) and association of U1-snRNP proteins with sequences around the 5Ј splice site (Liu et al. 1998;Zhang and Rosbash 1999). In what has been thought to be the first energy-requiring step, U2 snRNA base-pairs with the pre-mRNA branch-point sequence (Cheng and Abelson 1987;Parker et al. 1987;Krämer 1988;Wu and Manley 1989;Zhuang and Weiner 1989;Pruzan et al. 1990;Michaud and Reed 1991;Liao et al. 1992). Once U1 and U2 are bound to the pre-mRNA, U4/U6·U5 associates with the 5Ј splice site region of the pre-mRNA (Ruby and Abelson 1988;Liao et al. 1992), and the U5 snRNP is positioned such that the invariant loop 1 is aligned with exon sequences near the 5Ј and 3Ј splice sites (Wyatt et al. 1992;Sontheimer and Steitz 1993;Newman et al. 1995). In turn, U6 associates with the intron nucleotides at the 5Ј splice site, replacing U1 interactions. Although neither U1 nor U4 appears...

A conserved Lsm-interaction motif in Prp24 required for efficient U4/U6 di-snRNP formation

“…The intriguing absence of the half-TPR domain in yeast Prp24 itself (Fig+ 1A) may indicate that its function is not required in budding yeast+ A more interesting possibility, supported by the presence of a half-TPR domain in the C. albicans Prp24 homolog, is that the function of the missing N-terminal domain is provided by a separate polypeptide+ The most likely candidate, based on sequence homology, is Prp39, a U1 snRNP component consisting solely of half-TPRs (Lockhart & Rymond, 1994)+ Although a functional association of Prp24 with the U1 snRNP has not been reported to date, the proposed switch of U6 for U1 at the premRNA 59 splice site (Staley & Guthrie, 1999) is one place where such an interaction might occur+ Indeed, it has recently been reported that nucleotides in both U4 and U6 can be crosslinked to the 59 splice site in a trans-splicing assay (Johnson & Abelson, 2001)+ Consistent with this hypothesis, we have observed a weak but reproducible two-hybrid interaction between Prp24 and Prp39 (our unpubl+ data)+ Further experiments in both the yeast and mammalian systems will be required to reveal the function of the enigmatic half-TPR domain+…”

The Prp19-associated Complex Is Required for Specifying Interactions of U5 and U6 with Pre-mRNA during Spliceosome Activation

“…U4/5Ј splice site interaction has been demonstrated in the yeast spliceosome using an in vitro trans-splicing system. The ϩ2 residue of the intron can cross-link to residues 75, 78, and 82 of U4 downstream of the U4/U6 helix I independent of the U2 association with the branch point, indicating that U4 RNA also contacts the pre-mRNA during its association with the spliceosome (10). By carrying out splicing at lower concentrations of ATP in a conventional cis-splicing reaction, we have been able to isolate the preactivated spliceosome and detected multiple cross-linked products of U4 to the pre-mRNA (data not shown).…”

“…X1, appearing only in the active spliceosome, contains cross-links between the Lsm binding site of U6 and the intron sequence in a region ϳ30 bases downstream from the 5Ј splice site (21). X2a and X2b are cross-linked products between the 5Ј splice site and U6 in a region near the conserved ACAGA box (10). X2a, previously identified as dX2, accumulated in the spliceosome formed in NTC-depleted extracts (lane 2).…”

“…U6 has been demonstrated to be a key element in formation of the catalytic core of the spliceosome by simultaneously base pairing with the 5Ј splice site and with the U2 RNA (17)(18)(19)(20). Two types of U6/5Ј splice site base pairing, differed by a shift of 5 bases, were proposed based on biochemical and genetic analysis, respectively (10,19,20). Both types of interaction, as shown in Fig.…”

“…Binding of U2 to the branch site is mediated by base pairing between U2 RNA and the branch site sequence (8,9). The association of tri-snRNP with the spliceosome appears to also involve RNA-RNA interactions as demonstrated by isolation of cross-linked products of U4 small nuclear RNA to the second residue of the 5Ј splice site using a Saccharomyces cerevisiae in vitro trans-splicing system (10).…”