ATP-dependent interaction of yeast U5 snRNA loop 1 with the 5′ splice site

Abstract

Pre-messenger RNA splicing is a two-step process by which introns are removed and exons joined together. In yeast, the U5 snRNA loop 1 interacts with the 59 exon before the first step of splicing and with the 59 and 39 exons before the second step. In vitro studies revealed that yeast U5 loop 1 is not required for the first step of splicing but is essential for holding the 59 and 39 exons for ligation during the second step. It is critical, therefore, that loop 1 contacts the 59 exon before the first step of splicing to hold this exon following cleavage from the pre-mRNA. At present it is not known how U5 loop 1 is positioned on the 59 exon prior to the first step of splicing. To address this question, we have used site-specific photoactivated crosslinking in yeast spliceosomes to investigate the interaction of U5 loop 1 with the pre-mRNA prior to the first step of splicing. We have found that the highly conserved uridines in loop 1 make ATP-dependent contacts with an approximately 8-nt region at the 59 splice site that includes the invariant GU. These interactions are dependent on functional U2 and U6 snRNAs. Our results support a model where U5 snRNA loop 1 interacts with the 59 exon in two steps during its targeting to the 59 splice site.

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

“…Results from the S. cerevisiae splicing system indicate that, although base pairing with the U1 snRNA may not be a major determinant in defining the 59 splice site cleavage site, the U6 snRNA appears to be involved in the definition of the exact scissile bond (Séraphin et al+, 1988;Siliciano & Guthrie, 1988;Séraphin & Rosbash, 1990;Kandels-Lewis & Séraphin, 1993;Lesser & Guthrie, 1993)+ In agreement with this, there appears to be flexibility with respect to the position of U1 snRNA binding in the mammalian system+ For instance, U1 snRNAs with an altered 59 splice site recognition sequence (U1 snRNA nt 3-11), designed to base pair with sequences either upstream or downstream of the 59 splice site, can activate splicing of otherwise defective 59 splice sites mutated at positions ϩ3 to ϩ6 (Cohen et al+, 1994)+ Other mechanisms for early 59 splice site definition probably exist, as the U1 snRNP is not always required for splicing+ Inhibition of splicing upon depletion or inactivation of the U1 snRNP can be abrogated by the addition of high amounts of SR proteins, which appear to replace the U1 snRNP function and restore the splicing efficiency of some but not all pre-mRNA substrates (Crispino et al+, 1994;Tarn & Steitz, 1994)+ U5 snRNA, which enters the spliceosome as a component of the U4/U6+U5 tri-snRNP to form complex B, also contacts the 59 splice site (Fig+ 1)+ Phylogenetic comparisons show that the U5 snRNA contains an invariant U-rich sequence of 9 nt displayed in an 11-nt loop structure (Frank et al+, 1994)+ Data obtained from genetic experiments suggests that this sequence interacts with exon nucleotides immediately upstream of the 59 splice site in the pre-mRNA (Newman & Norman, 1991 and this is supported by the results from crosslinking experiments (Wassarman & Steitz, 1992;Wyatt et al+, 1992;Sontheimer & Steitz, 1993)+ In addition, the U5 loop sequence contacts the first 2 nt of the intron (Alvi et al+, 2001)+ Because the 59 exon region is not very well conserved, the interaction is believed to involve nonconventional base pairs+ The U5 snRNP may play a direct role in splice site selection based on the observation that, when normal 59 splice site definition is inhibited by mutation of the first 2 nt of the intron, the U5 loop sequence can influence the choice of the cleavage site (Cortes et al+, 1993)+ A potential protein factor involved in 59 splice site recognition is the U5 snRNP component Prp8, which has been shown to contact nucleotides on both sites of the 59 splice site scissile bond, including the first 2 nt of the intron, suggesting that the U1 and U5 snRNPs functionally collaborate in the recognition of the 59 splice site (Wyatt et al+, 1992;Teigelkamp et al+, 1995;…”

Prp8 protein: At the heart of the spliceosome

“…Both U5 snRNA loop 1 and Prp8p can be cross-linked to the pre-mRNA 5Ј and 3Ј splice sites in HeLa and yeast extracts, and these contacts occur at similar stages in the splicing reaction Wyatt et al 1992;Sontheimer and Steitz 1993;Newman et al 1995;Teigelkamp et al 1995a;Umen and Guthrie 1995a;O'Keefe et al 1996;Reyes et al 1996;Dix et al 1998;Segault et al 1999;Alvi et al 2001;McConnell and Steitz 2001;O'Keefe 2002). U5 snRNA was proposed to anchor and align the ends of the exons in the catalytic center of the spliceosome; however, U5 snRNA loop 1 is not essential for splicing in HeLa nuclear extract (Segault et al 1999), and is only required for step two in yeast extracts (O'Keefe et al 1996; O'Keefe and Newman 1998).…”

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

“…When Y2 and Y4 from the spliceosome formed in NTCdepleted extracts were digested with RNase H, each cross-linkcontaining fragment appeared as a smeared band (lanes [25][26][27][28][29][30][31][32][33][34][35][36], indicating that there might be multiple cross-linking sites on pre-mRNA and/or U5. Since cross-linking on the pre-mRNA was mapped near the 5Ј-end of the pre-mRNA, it is possible that the removal of half of U5 after RNase H digestion resulted FIG.…”