Evidence for a base-pairing interaction between U6 small nuclear RNA and 5' splice site during the splicing reaction in yeast.

Abstract

U6 small nuclear RNA (snRNA) is an essential factor in mRNA splicing. On the basis of the high conservation of its sequence, it has been proposed that U6 suRNA may function catalytically during the splicing reaction. If this is the case, it is likely that U6 snRNA interacts with the splice sites in the spliceosome to catalyze the reaction. We have used UV crosslinking to analyze the interactions of U6 snRNA with the splicing substrates during the yeast splicing reaction.Crosslinked products in which the central region of U6 snRNA was joined to the 5' splice site region of mRNA precursor and lariat intermediate were identified. The crosslinking sites were precisely located in one of these products. The results suggest a possible base-pairing interaction between U6 snRNA and the 5' splice site of the mRNA precursor.In nuclear pre-mRNA splicing, a number of factors including five small nuclear ribonucleoproteins (snRNPs) assemble on the mRNA precursor (pre-mRNA) in an ordered pathway to form a large RNP complex called the spliceosome (1)(2)(3). In this complex, the intron is excised in a two-step reaction. The first step involves the cleavage at the 5' splice site and the formation of a lariat structure. In the second step, the 3' splice site is cleaved and concomitantly the 5' exon is ligated to the 3' exon. This two-step reaction is fundamentally similar to the self-splicing pathway ofgroup II introns, which is known to be catalyzed by the intron itself (4,5). This has led to the hypothesis that the mechanisms of pre-mRNA splicing and self-splicing are related and that pre-mRNA splicing is . Although no conserved secondary structures have been reported in pre-mRNA introns, small nuclear RNAs (snRNAs) are known to participate in the splicing reaction as trans-acting components. Therefore, it has been argued that snRNAs might interact with each other and/or with pre-mRNA to form the structure that catalyzes the splicing reaction.Several RNA-RNA interactions are known to be involved in mRNA splicing. U1 snRNA interacts with the 5' splice site by base pairing at an early stage in the spliceosome assembly pathway (9-11). This interaction is apparently destabilized after spliceosome formation (12)(13)(14). Recently U1 snRNA was also shown to base pair with the 3' splice site in Schizosaccharomyces pombe (15). U2 snRNA base pairs with the branch point (16)(17)(18). A study of cryptic splice site activation in Saccharomyces cerevisiae suggests that U5 snRNA interacts with the exon sequences at the 5' and 3' splice sites (19). U4 and U6 snRNAs base pair with each other in the U4/U6 snRNP (20)(21)(22). This base-pairing interaction is destabilized after the entry of this snRNP into the spliceosome prior to the first-step reaction (23-25). In the mammalian system, base pairing between the 5'-end region of U2 snRNA and the 3'-end region of U6 snRNA has also been demonstrated (26,27 for a base-pairing interaction between U6 snRNA and a conserved sequence near the 5' splice site of the intron. The formation of such a s...

Functional selection of splicing enhancers that stimulate trans-splicing in vitro

“…Trans-splicing reactions employing progressive rounds of selected exonic sequences+ A: Trans-splicing of the wild-type starting construct compared to rounds 0-7 of the successive selected sequences+ An authentic trans-splicing substrate is also shown for comparison+ Splicing reactions were incubated for the indicated time in minutes+ Shaded boxes represent trans-spliced exons, lines indicate intronic regions, and striped boxes correspond to the transspliced leader exon (SL)+ B: Graph of splicing efficiency plotted for each of the selected rounds+ The average trans-splicing efficiency for three independent sets of splicing reactions was graphed+ From this analysis, it is clear that precursor turnover leveled off after the fourth round+ C: Splicing time course comparing the initial randomized pool with the final round+ Again, an authentic trans-splicing substrate is included for comparison+ Trans-splicing reactions proceeded for the indicated time+ Substrates and products are indicated as above+ 7+1 (3XSGACS) sequences acts as an efficient competitor for both self-competition (Fig+ 4A, lanes 8-11) as well as on the splicing of clone 7+2 (59ss; Fig+ 4B, lanes 8-11)+ Thus, because the SGACS-containing competitor acts as an effective inhibitor of the 59 splicecontaining substrate (7+2), it presumably titrates out a factor(s) required for general trans-splicing and not just a factor(s) necessary for the specific trans-splicing of substrates containing the SGACS motif+ A similar profile was observed when the exon from clone 7+2 (59ss) FIGURE 3. Isolation of individual sequences from the mixed pools+ A: Discrete sequences are grouped according to the elements that they contain+ For the SGACS, ACE-like, and RRGAGS elements, individual motifs are underlined+ S ϭ G or C; N ϭ A, C, G or U; W ϭ A or U+ Upper case letters indicate bases within the 18n randomized region, lower case letters indicate surrounding sequences+ Outlined letters (clone 2+3) indicate a change in the sequence of the surrounding sequence+ Sequences that fall into the 59 splice site-like category are drawn in a proposed base paired interaction with the 59 end of A. lumbricoides U1 snRNA (Shambaugh et al+, 1994) B: Transsplicing of individual selected sequences versus the wild-type starting construct+ Clone 7+1 contains three copies of the SGACS motif (3XSGACS) and clone 7+2 contains a 9 out of 9 complementarity to the 59 end of U1 (59 ss)+ Splicing reactions were incubated for the indicated times+ Precursors and products are indicated as in the legend to Figure 2+ was examined+ This competitor led to a substantial reduction in the trans-splicing efficiency of both the 7+1 (3XSGACS) (Fig+ 4A, lanes 12-15) and self 7+2 (59ss) substrates (Fig+ 4B, lanes 12-15)+ This result is intriguing because it demonstrates that a 59 splice sitecontaining RNA is able to compete for trans-splicing, even though the reaction does not require the 59 end of U1 (Hannon et al+, 1991)+ Therefore, other factors required for trans-splicing are presumably titrated away by the presence of excess 59 splice sites+ Possible candidates include p220/Prp8 (Wyatt et al+, 1992;Reyes et al+, 1996) and U6 snRNP (Sawa & Abelson, 1992;Wassarman & Steitz, 1992)+ Because the only difference between the three competitor fragments lies within the 18-nt region that was the focus of the selection procedure, the effects on trans-splicing efficiency can be directly attributed to this region+ To extend the characterization of the competitor RNAs, we investigated their effects on the splicing of authentic nematode trans-and cis-...…”

Pseudouridines in spliceosomal snRNAs

“…In the newly formed spliceosome, a specific sequence of U5 interacts with the exon sequences at the 5' and 3' splice sites (Newman and Norman, 1991;Newman and Norman, 1992;Wyatt et al, 1992;Cortes et al, 1993;Sontheimer and Steitz, 1993), and other sequences of U4 and U6 base-pair with each other. Then, before the first step of splicing occurs, the spliceosome undergoes dynamic changes, resulting in the departure of U1 and U4, and the formation of new duplexes, including those between U2 and U6, and between U6 and the 5' splice site (Hausner et al, 1990;Datta and Weiner, 1991;Wu and Manley, 1991;Yean and Lin, 1991;Madhani and Guthrie, 1992;Sawa and Abelson, 1992;Wassarman and Steitz, 1992;Lesser and Guthrie, 1993;Nilsen, 1994). The resulting conformational changes lead to the formation of the active spliceosome (complex B2), triggering the first step of splicing, where the bulged-out branch point adenosine nucleophilically attacks the phosphate at the 5' splice site.…”

Protein-free small nuclear RNAs catalyze a two-step splicing reaction

“…Furthermore, an evolutionarily invariant region in U6, the ACAGAGA box ( Fig. 1 A), is in close proximity to the splice sites during splicing catalysis (14)(15)(16)(17)(18), and mutagenesis studies have shown that this domain plays a crucial role in catalysis of the splicing reaction (7,10,11). Two other conserved regions, the AGC triad and an asymmetric bulge in the intramolecular stemloop of U6 (ISL), are also thought to play important roles in spliceosomal catalysis (11).…”