Impact of base pair identity 5′ to the spliceosomal branch site adenosine on branch site conformation

Abstract

The branch site helix from Saccharomyces cerevisiae with pseudouridine (c) incorporated in a phylogenetically conserved position of U2 snRNA features an extrahelical branch site adenosine (A) that forms a base triple interaction with the minor groove edge of a widely conserved purine U2 strand -pyrimidine intron strand (R U2 -Y intron ) base pair two positions upstream. In these studies, NMR spectra of a duplex in which 2-aminopurine (2ap), a fluorescent analog of adenine lacking the proposed hydrogen bond donor, was substituted for the branch site A, indicated that the substitution does not alter the extrahelical position of the branch site residue; thus, it appears that a hydrogen bond between the adenine amino group and the R-Y pair is not obligatory for stabilization of the extrahelical conformation. In contrast, reversal of the orientation of A U2 -U intron to U U2 -A intron resulted in an intrahelical position for the branch site A or 2ap. Fluorescence intensity of 2ap substituted for the branch site A with the original R U2 -Y intron orientation (AU or GC) was high, consistent with an extrahelical position, whereas fluorescence in helices with the reversed R-Y orientation, or with a mismatched pair (A-U / G d A or U d C), was markedly quenched, implying that the residue was stacked in the helix. The A 59 to the branch site residue was not extrahelical in any of the duplexes. These findings suggest that the R U2 -Y intron base pair orientation in the c-dependent branch site helix plays an important role in positioning the branch site A for recognition and/or function.

Alternative splicing ofU2AF1reveals a shared repression mechanism for duplicated exons

“…Extending base-pairing contacts between U2 and upstream BP sequences improved splicing of the BP-2U>C mutant (88) and bulged adenosines placed at position +1 or −1 relative to canonical position participated in the first-step splicing catalysis (89). Reversal of the orientation of a base pair switch A U2 -U intron to U U2 -A intron resulted in a stacked intrahelical position of the BP adenine and reduced splicing efficiency (90,91), consistent with the importance of nucleophile bulging for splicing (89). The flexibility in nucleophile specification observed in yeast is likely to be even higher for more relaxed mammalian BPs.…”

Structure modulation of helix 69 from Escherichia coli 23S ribosomal RNA by pseudouridylations

“…After incorporation of Ψ modifications at positions 1911, 1915 and 1917, these nucleotides appear to work synergistically in modulating the conformation and structural dynamics of the loop ( Figure 6 ), which plays a role in formation and maintenance of bridge B2a during various stages of translation. It has been reported in studies of telomerase ( 6 ), spliceosomal ( 7 ) and transfer ( 8 ) RNAs that Ψ helps pre-structure local RNA motifs for subsequent participation in downstream events; therefore, this work on H69 helps complete the spectrum of roles for Ψ in all major biologically relevant RNA species. The unique structural properties of Ψ and its ability to fine-tune the structure of functionally important RNAs make these sites attractive targets for drug design, and the structures obtained in this work can be applied in the development of novel inhibitors of bacterial ribosome function.…”

“…Among the >100 modifications identified to date ( 4 ), pseudouridine (Ψ) ( Figure 1 a) was the first reported and is also the most frequently encountered ( 5 ). Uridine ( Figure 1 b) is isomerized to Ψ ( Figure 1 a) by replacing the N-glycosidic bond with a C-glycosidic bond, and this covalent structural variation has been shown to modulate local conformation and overall activity in telomerase ( 6 ), spliceosomal ( 7 ) and transfer ( 8 ) RNAs. In the Escherichia coli ( E. coli ) ribosomal RNAs (rRNAs), 10 Ψ modifications are distributed in the functionally important regions (e.g .…”

CHSalign: A Web Server That Builds upon Junction-Explorer and RNAJAG for Pairwise Alignment of RNA Secondary Structures with Coaxial Helical Stacking

“…An RNA junction, also known as a multi-branch loop, forms when more than two helical segments are brought together [5][6][7][8][9][10]. RNA junctions exist in numerous RNA molecules; they play important roles in a wide variety of biochemical activities such as self-cleavage of the hammerhead ribozyme [11], the recognition of the binding pocket domain by purine riboswitches [12] and the translation initiation of the hepatitis C virus at the internal ribosome entry site [13].…”