Domains of U4 and U6 snRNAs required for snRNP assembly and splicing complementation in Xenopus oocytes.

Abstract

Structure‐function relationships in the vertebrate U4‐U6 snRNP have been analysed by assaying the ability of mutant RNAs to form U4‐U6 snRNPs and to function in splicing complementation in Xenopus oocytes. The mutants define three categories of domain within the RNAs. First, domains which are not essential for splicing. These include regions of U6 which have previously been implicated in the capping and transport to the nucleus of U6 RNA as well as, less surprisingly, regions of U4 and U6 which have been poorly conserved in evolution. Second, domains whose mutation reduces U4‐U6 snRNP assembly or stability. This group includes mutations in both the proposed U4‐U6 interaction domain, and also, in the case of U6, in a highly conserve sequence flanking stem I of the interaction domain. These mutants are all defective in splicing. Third, regions not required for U4‐U6 assembly, but required for splicing complementation. This category defines domains which are likely to be required for specific contacts with other components of the splicing machinery. Combinations of mutants in the U4 and U6 interaction domain are used to show that there are not only requirements for base complementarity but also for specific sequences in these regions.

Splicing function of mammalian U6 small nuclear RNA: conserved positions in central domain and helix I are essential during the first and second step of pre-mRNA splicing.

“…In the current singular U6 structure (9) (23)(24)(25), revealed no dramatic phenotypes. This result is consistent with previous mutant studies in yeast (15) and Xenopus (27). Such a base-pairing interaction, however, may exist and contribute to the splicing efficiency of specific pre-mRNAs but may not be essential for the general splicing mechanism.…”

In vitro reconstitution of yeast splicing with U4 snRNA reveals multiple roles for the 3′ stem–loop

“…Here, we instead targeted the 59 end of U4 (Fig. 1A), as it has been shown to be essential for interaction with U6 and, consequently, for splicing (Vankan et al 1990;Bindereif 1990, 1992).…”

“…Given that stems I and II and the 59 stem-loop are all required for efficient spliceosome assembly and splicing in human extract and Xenopus oocytes (Vankan et al 1990;Bindereif 1990, 1992), we tested a mutant containing these three structural features, which consisted of nt 1-68. This mutant, U4 1-68, lacks the Sm binding site, the 39 stem-loop, and most of the central domain (Fig.…”

Structure Probing of U4 snRNA: Investigations into the Mechanism of U4/U6 di-snRNP Formation.

“…Notably this is in agreement with literature regarding the necessity of stem II for di-snRNP formation in yeast, humans, and Xenopus. In humans, deletion of all nucleotides that form stem II, or half of these (nucleotides 1 -8) resulted in complete abolishment of disnRNP formation in vitro (Wersig and Bindereif 1990), and in Xenopus deletion of stem II was found to inhibit U4/U6 di-snRNP assembly (Vankan et al 1990, Vankan et al 1992). …”

“…In an in vivo study using Xenopus oocytes substitution of the proposed kissing loop nucleotides in the U4 snRNA blocked di-snRNP formation, while substitution of surrounding nucleotides allowed formation of the di-snRNP to continue (Vankan et al 1990). The results demonstrate strong support for an initial interaction between U4 and U6 that leads to propagation of stem II, though it is unclear if this region in U4 forms a stem-loop structure.…”