Structural Basis for a Lethal Mutation in U6 RNA^,

Abstract

U6 RNA is essential for nuclear pre-mRNA splicing and has been implicated directly in catalysis of intron removal. The U80G mutation at the essential magnesium binding site of the U6 3′ intramolecular stem-loop region (ISL) is lethal in yeast. To further understand the structure and function of the U6 ISL, we have investigated the structural basis for the lethal U80G mutation by NMR and optical spectroscopy. The NMR structure reveals that the U80G mutation causes a structural rearrangement within the ISL resulting in the formation of a new Watson-Crick base pair (C67·G80), and disrupts a protonated C67·A79 wobble pair that forms in the wild-type structure. Despite the structural change, the accessibility of the metal binding site is unperturbed, and cadmium titration produces similar phosphorus chemical shift changes for both the U80G mutant and wild-type RNAs. The thermodynamic stability of the U80G mutant is significantly increased (ΔΔG fold = −3.6 ± 1.9 kcal/mol), consistent with formation of the Watson-Crick pair. Our structural and thermodynamic data, in combination with previous genetic data, suggest that the lethal basis for the U80G mutation is stem-loop hyperstabilization. This hyperstabilization may prevent the U6 ISL melting and rearrangement necessary for association with U4.Proteomic diversity in eukaryotes is generated by alternative splicing of exons from nuclear premessenger RNA (pre-mRNA) by the spliceosome. The spliceosome is a large ribonucleoprotein complex, made up of five small nuclear RNAs (snRNAs), U1, U2, and U4, U5, U6, and more than 70 proteins (1-4). The spliceosome catalyzes a two-step transesterification reaction, speculated to be RNA-catalyzed by analogy to mitochondrial group II self-splicing ribozymes (2,3,5). Two snRNAs (U2 and U6) likely comprise part of the spliceosome active site, and U6 is essential for catalysis (2,3,5). In the active spliceosome, U2 and U6 form a complex by base pairing to each other, and have also been shown to base pair to pre-mRNA at the first step of splicing. Moreover, mutagenesis data have shown that certain regions of U6 must be intact for the assembled spliceosome to catalyze the first or second step of splicing (6). Other atomic substitution studies of U6 RNA have revealed several phosphate oxygens that are essential for splicing (7-9), which are potential sites of magnesium coordination required by the spliceosome. Recently, the U2-U6 complex was shown to catalyze a reaction similar to the first step of splicing in the † This work was supported by NIH Grant GM65166. S.R.V. absence of protein, lending more evidence to the hypothesis of an RNA active site in the spliceosome (10).The formation of the U2-U6 complex is initiated by a large conformational change in U6 RNA (1, 11). During spliceosome assembly, U6 extensively base pairs with U4 RNA, forming a helical secondary structure. After the spliceosome is assembled completely, base pairing between U4 and U6 is disrupted and U6 undergoes a large conformational change, in which an intramolec...

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

“…3; Madhani et al 1990). Furthermore, formation of the C67/G80 pair to the exclusion of the normal C67/A79 pair has been observed in the isolated ISL by NMR (Sashital et al 2003).…”

“…The cold sensitivity of the A62G and A79G mutations has been postulated to result from ISL stabilization due to the replacement of A-C by G-C base pairs (Fortner et al 1994). Similarly, the lethal U80G mutation stabilizes the ISL and blocks U4/U6 complex assembly, presumably by inhibiting ISL melting (Madhani et al 1990;Ryan and Abelson 2002;Sashital et al 2003).…”

Structure of the yeast U2/U6 snRNA complex

“…Sugar pucker conformations for residues with weak H19-H29 couplings were left unrestricted. Initial structures were calculated using CNS 1.1 (Brunger et al 1998) as described (Sashital et al 2003). The 10 lowest-energy out of 50 structures were refined using XPLOR-NIH (Schwieters et al 2003) with 42 RDC restraints.…”

The N- and C-terminal RNA recognition motifs of splicing factor Prp24 have distinct functions in U6 RNA binding

“…Given that both the U6 ISL and the U4/U6 intermolecular stems are very stable, with melting temperatures above 50°C at physiological salt concentration (Brow and Guthrie 1988;Sashital et al 2003), it is likely that their interconversion is assisted by proteins and may involve metastable intermediate structures. Indeed, dissociation of the human U4/U6 RNA complex is facilitated by formation of a longrange intramolecular helix in U6 RNA that extends the ISL (Brow and Vidaver 1995).…”