Recognition of the spliceosomal branch site RNA helix on the basis of surface and electrostatic features

Abstract

We have investigated electrostatic and surface features of an essential region of the catalytic core of the spliceosome, the eukaryotic precursor messenger (pre-m)RNA splicing apparatus. The nucleophile for the first of two splicing reactions is the 2′-hydroxyl (OH) of the ribose of a specific adenosine within the intron. During assembly of the spliceosome's catalytic core, this adenosine is positioned by pairing with a short region of the U2 small nuclear (sn)RNA to form the pre-mRNA branch site helix. The solution structure of the spliceosomal pre-mRNA branch site [Newby,M.I. and Greenbaum,N.L. (2002) Nature Struct. Biol., 9, 958–965] showed that a phylogenetically conserved pseudouridine (ψ) residue in the segment of U2 snRNA that pairs with the intron induces a markedly different structure compared with that of its unmodified counterpart. In order to achieve a more detailed understanding of the factors that contribute to recognition of the spliceosome's branch site helix and activation of the nucleophile for the first step of pre-mRNA splicing, we have calculated surface areas and electrostatic potentials of ψ-modified and unmodified branch site duplexes. There was no significant difference between the total accessible area or ratio of total polar:nonpolar groups between modified and unmodified duplexes. However, there was substantially greater exposure of nonpolar area of the adenine base, and less exposure of the 2′-OH, in the ψ-modified structure. Electrostatic potentials computed using a hybrid boundary element and finite difference nonlinear Poisson–Boltzmann approach [Boschitsch, A.H. and Fenley, M.O. (2004) J. Comput. Chem., 25, 935–955] revealed a region of exceptionally negative potential in the major groove surrounding the 2′-OH of the branch site adenosine. These surface and electrostatic features may contribute to the overall recognition of the pre-mRNA branch site region by other components of the splicing reaction.

Different Mechanisms for Pseudouridine Formation in Yeast 5S and 5.8S rRNAs

“…These observed associations suggest the possibility that Pus7 activity is connected to the nucleolus and, more importantly, point to the possibility that the modification status provides a signal allowing the coregulation of the production and turnover of rRNAs, spliceosomes, and tRNAs. These observations also suggest a link to the production of ribosomal proteins, since the Pus7-catalyzed ⌿35 in U2 snRNA is important for the high-efficiency splicing of S. cerevisiae introns (79,80,111), which are preferentially located in ribosomal protein genes (32). Indeed, the interdependencies are consistent with the concept that the production of RNAP II and RNAP III transcripts is regulated in response to prerRNA synthesis via an extensive network of interactions (18,56,74,95,96).…”

A Fast and Robust Poisson–Boltzmann Solver Based on Adaptive Cartesian Grids

“…Here high resolution surface potential maps capturing these features are produced. As portrayed in Figure 8a the G-stretch of the major groove side of the DNA structure and its locally narrowed minor groove have a deep negative potential 67 . The extensive region of negative potential along the G-stretch of the major groove is mostly due to the deformation of the DNA structure.…”

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

“…Among them, electrostatic features of the c-modified branch site duplex (Xu et al 2005) may impact on recognition and function of the branch site. Smith et al (2009) have recently shown that the branching activity in yeast spliceosomes is also affected by the distance of the branch site A from the U2-U6 helix Ia, suggesting that long range interactions with remote sections of the U2-U6 snRNA complex may play a role in recognition and/or positioning of the branch site adenosine.…”

“…A duplex that included the conserved pseudouridine (c) residue in position 35 of the U2 snRNA strand was characterized by a kinked backbone of the intron strand and an extrahelical conformation of the branch site A, with its 29OH exposed in the widened major groove. Calculation of electrostatic surface potentials by a nonlinear Poisson-Boltzmann approach identified a region of significant negative potential in the major groove surrounding the 29OH of the branch site A (Xu et al 2005) that may contribute to the recognition of the branch site A by other spliceosomal components or its activity in the first step of splicing. In contrast, the branch site 4 Present address: LECOM-Bradenton, Bradenton,FL 34211,USA 5 A in the unmodified counterpart (U in place of the naturally occurring c in the U2 snRNA strand) adopted an intrahelical conformation, and both strands had A-type helical parameters throughout.…”