A tertiary interaction that links active-site domains to the 5′ splice site of a group II intron

Abstract

Group II introns are self-splicing RNAs that are commonly found in the genes of plants, fungi, yeast and bacteria. Little is known about the tertiary structure of group II introns, which are among the largest natural ribozymes. The most conserved region of the intron is domain 5 (D5), which, together with domain 1 (D1), is required for all reactions catalysed by the intron. Despite the importance of D5, its spatial relationship and tertiary contacts to other active-site constituents have remained obscure. Furthermore, D5 has never been placed directly at a site of catalysis by the intron. Here we show that a set of tertiary interactions (lambda-lambda') links catalytically essential regions of D5 and D1, creating the framework for an active-site and anchoring it at the 5' splice site. Highly conserved elements similar to components of the lambda-lambda' interaction are found in the eukaryotic spliceosome.

The intramolecular stem-loop structure of U6 snRNA can functionally replace the U6atac snRNA stem-loop

“…More recently, a tertiary interaction has been described between the 59 splice site region and two C-G or G-C base pairs in the upper helix of domain 5 in a group II intron (Boudvillain et al+, 2000)+ These authors suggest that a similar interaction could take place between the 59 splice site of spliceosomal introns and conserved C-G base pairs in the upper helix of U6 snRNA+ These two C-G base pairs are present in both human and yeast U6 snRNA as well as in human U6atac snRNA (Fig+ 2)+ However, the upper C-G base pair is replaced by a U-A base pair in the plant U6atac snRNA+ Furthermore, as shown by mutant VII in Figure 7, mutation of this entire helix in human U6atac snRNA does not abolish in vivo splicing activity+ It is possible that both C-G and G-C base pairs could participate in the tertiary interaction proposed by Boudvillain et al+ (2000)+ Additional mutations at these positions of U6atac will be necessary to test this proposed interaction in the U12-dependent spliceosome+ Interestingly, mutations in the two C-G base pairs in U6 snRNA were not examined in the extensive study of U6 snRNA by Sun and Manley (1997)+ When the results of this work are compared to the similar study of human U6 snRNA by Sun and Manley (1997), some interesting similarities and differences appear+ In both studies, single mutations of residues within the base-paired portions of the snRNAs were inactive for splicing in vivo+ However, in the case of U6atac, mutation of residues in the 39 portion of the lower stem showed no defect in splicing, although they could compensate for splicing defective mutations in the other side of the stem+ In both studies, splicing defective mutants in the stem regions could be rescued by compensatory mutations restoring the base pairing pattern in the stems+ An interesting difference between these two studies is seen in the effects of mutation or deletion of the U residue in the bulge regions of the two snRNAs+ Mutation or deletion of this residue (U74) in U6 abolished function (Sun & Manley, 1997) whereas mutation of U46 in U6atac to G or C (in the plant sequence) as well as deletion had no major effect on function+ In the absence of information on the actual conformation of these structures in the spliceosome and on the nature of any base-specific interactions, it is difficult to judge the significance of this difference+ Another significant difference between the two studies is the apparent lack of the need to compensate for the U6 mutations by altering U4 snRNA+ In our U6atac studies, most mutants, including some with single base changes, required the coexpression of compensatory U4atac snRNAs mutants+ The mechanistic foundation of this difference is unclear+ It might reflect a lower stability of the U4atac/U6atac interaction compared with the U4/U6 interaction+ Perhaps the 100-fold greater abundance of U4 and U6 snRNAs compared to U4atac and U6atac snRNAs (Yu et al+, 1999) more strongly favors the di-snRNP configuration+ Alternatively, the U4atac/U6atac di-snRNP may be a poorer substrate for factors that anneal the two snRNAs or a better substrate for factors that unwind the snRNA duplex+ In the yeast system, factors have been identified that appear to catalyze the formation of the U4/U6 di-snRNP and catalyze the unwinding of the duplex (Raghunathan & Guthrie, 1998a…”

Complex Formation of Cadmium with Sugar Residues, Nucleobases, Phosphates, Nucleotides, and Nucleic Acids

“…Such a rescue is also part of so-called nucleotide analogue interference mapping/suppression (NAIM/NAIS) experiments, which are used to identify catalytically crucial atoms within a RNA and/or tertiary contacts [253][254][255]. This technique has been widely applied to study metal ion binding sites and their influence on catalytic RNAs, i.e., ribozymes [256][257][258][259][260][261][262]. Major limitations of this method are the fact that (i) T7 RNA polymerase, which is generally used for the transcription of defined RNA sequences, inserts only S p thionucleotides under inversion of their conformation to R p .…”

A dynamic bulge in the U6 RNA internal stem–loop functions in spliceosome assembly and activation

“…Both in vitro and in vivo evidence from mutational analyses of the human U6 ISL suggest that both base-pairing potential and sequence identity are important for spliceosome assembly and later processes (Wolff and Bindereif 1993;Sun and Manley 1997), and hydroxyl radical probing experiments have placed the human ISL bulge in the vicinity of the U6-59splice site interaction in activated spliceosomes (Rhode et al 2006). Mutation of the D5 bulge region of group II self-splicing intron aI5g was previously found to inhibit either docking of Domain 5 within the intron, splicing catalysis, or both (Schmidt et al 1996), and a region adjacent to the bulge was shown to interact with the 59 splice site (Boudvillain et al 2000). Consistent with these findings, structural studies showed that the D5 bulge from a Pylaiella littoralis group II intron ribozyme construct undergoes dramatic conformational changes upon docking to Domains 1-3 (Gumbs et al 2006).…”