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Using a strategy of compensatory nucleotide changes between yeast U1 and a 5' splice site, we have analyzed the contribution of base-pairing to the efficiency and fidelity of pre-mRNA splicing in vivo. Watson-Crick base-pairing interactions with U1 can be demonstrated at intron positions 1 and 5 but not at position 4. Moreover, restoration of the ability to pair with U1 is not sufficient to restore activity in the second step of splicing to intron position 1 mutants. Finally, in contrast to recent observations in mammalian systems, we find that the precise position of 5' splice site cleavage is not determined solely by the base-pairing interaction with U1. Rather, the presence of a G residue at position 5 is required for the correct localization of the nucleolytic event. Taken together, these results indicate that the demands for 5' splice site selection and utilization are more complex than a simple maximization of Watson-Crick interactions with U1.[Key Words: Splicing; 5'splice site; U1 snRNA] Received June 22, 19881 revised version accepted August 18, 1988. Understanding the specificity of splice site selection remains one of the most central problems in pre-mRNA splicing. Since the potential complementarity between the conserved 5' end of the U1 snRNA and the consensus sequence spanning the 5' splice site ICAG/GUPuAGU} was first noted {Lemer et al. 1980~ Rogers and Wall 1980}, specific hypotheses have focused on the role of base-pairing between conserved intron signals and the snRNAs. Numerous biochemical experiments indicated that U1 is required for splicing [Kramer et al. 1984;Black et al. 1985;Krainer and Maniatis 1985}, that it binds to the 5' splice site (Mount et al. 19831, and that this binding depends on the 5' end of U1 .Zhuang and Weiner {19861 first demonstrated specific base-pairing interactions between U1 and the 5' splice site by achieving in vivo suppression of 5' splice site mutants via compensatory changes in U1. Splicing of adenovirus EIA transcripts carrying a G--~ A transition at the fifth position of the intron {herein designated by the convention IVS-AS} was efficiently restored by expression of a mutant U1 with a compensatory change at position 4 {U1-4U). By the same assay, however, suppression of mutants at intron position 3 was found to be very inefficient, at best {Zhuang and Weiner 1986}. Other experiments (in which only the intron was changed} showed that mutation of the SV40 large T antigen 5' splice site to improve its match to the consensus results in an increased usage of this site, relative to the small t 5' splice site, both in vivo and in vitro (Zhuang et al. 1987}. In combination with other studies (see below}, the view has emerged that the net strength of basepairing interactions is a crucial determinant of the efficiency of 5' splice site utilization, particularly when several potential sites are competing in cis.Moreover, the results of a systematic analysis of mutations at intron positions 1 and 2 suggest that the precise site of 5' cleavage is not determined by the...

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Nuclear pre-messenger RNA splicing requires the action of five small nuclear (sn) RNAs, U1, U2, U4, U5 and U6, and more than 50 proteins. The mechanistic similarity of nuclear pre-mRNA splicing and group II self-splicing suggests that many of the central processes of nuclear pre-mRNA splicing are based on RNA-RNA interaction. To understand the mechanism of pre-mRNA splicing, the interactions, and their temporal relationships, that occur between the snRNAs and the pre-mRNA during splicing must be identified. Several snRNA-snRNA and snRNA-intron interactions have been demonstrated but the putative RNA-based interactions that recognize the AG dinucleotide at the 3' splice site during 3' cleavage and exon ligation are unknown. We report here the reciprocal suppression between 5' and 3' splice site mutations in the yeast actin intron, and propose that the 3' splice site is positioned for 3' cleavage and exon ligation, at least in part, through a non-Watson-Crick interaction between the guanosines at the 5' and 3' splice sites.

Aminoacyl-tRNA synthetases catalyze the attachment of cognate amino acids to specific tRNA molecules. To prevent potential errors in protein synthesis caused by misactivation of noncognate amino acids, some synthetases have evolved editing mechanisms to hydrolyze misactivated amino acids (pre-transfer editing) or misacylated tRNAs (post-transfer editing). In the case of post-transfer editing, synthetases employ a separate editing domain that is distinct from the site of amino acid activation, and the mechanism is believed to involve shuttling of the flexible CCA-3 end of the tRNA from the synthetic active site to the site of hydrolysis. The mechanism of pre-transfer editing is less well understood, and in most cases, the exact site of pre-transfer editing has not been conclusively identified. Here, we probe the pre-transfer editing activity of class II prolyl-tRNA synthetases from five species representing all three kingdoms of life. To locate the site of pre-transfer editing, truncation mutants were constructed by deleting the insertion domain characteristic of bacterial prolyl-tRNA synthetase species, which is the site of posttransfer editing, or the N-or C-terminal extension domains of eukaryotic and archaeal enzymes. In addition, the pre-transfer editing mechanism of Escherichia coli prolyl-tRNA synthetase was probed in detail. These studies show that a separate editing domain is not required for pre-transfer editing by prolyl-tRNA synthetase. The aminoacylation active site plays a significant role in preserving the fidelity of translation by acting as a filter that selectively releases non-cognate adenylates into solution, while protecting the cognate adenylate from hydrolysis.