Differential chemical probing of a group II self-splicing intron identifies bases involved in tertiary interactions and supports an alternative secondary structure model of domain V

Abstract

Dimethyl sulfate modification was used to probe for tertiary structural elements in the group II intron Pl.LSU/2 from the mitochondrial pre-ribosomal RNA of the brown alga Pylaiella littoralis. Modification of the lariat form of the intron under conditions that allow both native folding and conformational homogeneity is found to be generally consistent with secondary and tertiary structural features identified previously for group II ribozymes. A comparison of chemical probing at temperatures just below and above the first melting transition illustrates the cooperative unfolding of tertiary structure and identifies novel candidates for tertiary interactions in addition to defining elements of secondary structure. Substitution of the GAAA terminal loop of domain V is shown to be compatible with retention of conformational homogeneity (despite the loss of an important tertiary interaction), but produces a concise methylation footprint in domain I at the site previously shown to harbor the receptor for that loop. The analysis also identified two nucleotide positions in domain V with novel secondary and potential tertiary structural roles. The proposed refinement of domain V secondary structure is supported by an expanded comparative analysis of group II sequences and bears increased resemblance to U2:U6 snRNA pairing in the spliceosome.

A group II intron encodes a functional LAGLIDADG homing endonuclease and self-splices under moderate temperature and ionic conditions

“…The 59 and 39 splice sites are marked by black arrowheads and the bulged adenosine residue in DVI is indicated by the asterisk. EBS and IBS nucleotides are shown, as well as nucleotides/segments involved in tertiary interactions (a-a9, g-g9, d-d9, e-e9, z-z9, k-k9, and u-u9). d-d9, e-e9, z-z9, k-k9, and u-u9) that are involved in ribozyme folding, and the bulged adenosine residue in DVI (involved in the branch reaction) were identified using previously published structural models (Costa et al 1997(Costa et al , 1998(Costa et al , 2000Pyle et al 2007). …”

RNA recognition by the DNA end-binding Ku heterodimer

“…The structure of this bulge motif cannot be predicted solely from the modification pattern. Some possibilities for the protection of A292, but not U293, include the formation of a base-triple including A292, intercalation of bases into the helical stack, ion-coordination, or water-mediated hydrogen bonding (Costa et al 1998;Leontis and Westhof 1998). Further investigation will be necessary to distinguish between these possibilities.…”

“…The modification data suggest that this portion of the KBS may fold into a specific structural motif. Varying chemical reactivity has been observed in the 2-nt bulge of domain V in the group II intron (Costa et al 1998), which can likely be explained by the formation of a base-triple including one of the nucleotides in the bulge (Keating et al 2010). Factors such as ion-binding sites or water-mediated hydrogen bonds may contribute to the protection of single-stranded nucleotides as well (Leontis and Westhof 1998).…”

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

“…In spite of the high conservation of the structure of this element and the apparent conservation of its function as shown by these studies, we do not have a clear idea of what its function might be in the spliceosome+ Its location near the sites of splicing chemistry have led to speculation that it plays a role in the active site of the spliceosome (reviewed in Nilsen, 1998;Collins & Guthrie, 2000)+ Several groups have suggested parallels with features of various ribozymes including the hairpin ribozyme (Tani & Ohshima, 1991;Sun & Manley, 1995) and domain 5 of group II self-splicing introns (see discussions in Sun & Manley, 1997;Costa et al+, 1998;Nilsen, 1998)+ The group II domain 5 comparison is particularly interesting+ A recent revision of the proposed structure of the domain 5 stem-loop has emphasized the similarity between it and the U6 (or U6atac) intramolecular stem-loop (Costa et al+, 1998)+ A phosphorothioate modification-interference study of domain 5 in self-splicing identified a phosphate group in the bulge region as important for splicing catalysis (Chanfreau & Jacquier, 1994)+ This phosphate is located in a very similar position to one identified as important for U6 snRNA function in in vitro pre-mRNA splicing in both yeast (Fabrizio & Abelson, 1992) and nematodes (Yu et al+, 1995)+ Recent investigations of both phosphorothioate diastereomers at this position in yeast U6 snRNA have revealed a metal ion specificity switch for the first step of splicing (Yean et al+, 2000)+ This suggests that U6 snRNA participates in the catalysis of splicing through metal ion coordination and places this stem-loop element at or very near the catalytic center of the spliceosome+ Pyle's group has also suggested that the bulge and lower stem regions of domain 5 serve to position a critical metal ion for group II splicing (Konforti et al+, 1998)+ Such a function would mainly involve the positioning of phosphate groups to coordinate the metal and would be compatible with many but perhaps not all base paired sequences within the stem region+ Basespecific interactions would be limited to residues in unpaired regions and functional groups in the major and minor groves of the helical regions+ Such a function would fit with the apparent tolerance of the stem regions of U6atac snRNA for many but not all substitutions that maintain base pairing+…”