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

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

The electrostatic characteristics of G·U wobble base pairs

G·U wobble base pairs are the most common and highly conserved non-Watson–Crick base pairs in RNA. Previous surface maps imply uniformly negative electrostatic potential at the major groove of G·U wobble base pairs embedded in RNA helices, suitable for entrapment of cationic ligands. In this work, we have used a Poisson–Boltzmann approach to gain a more detailed and accurate characterization of the electrostatic profile. We found that the major groove edge of an isolated G·U wobble displays distinctly enhanced negativity compared with standard GC or AU base pairs; however, in the context of different helical motifs, the electrostatic pattern varies. G·U wobbles with distinct widening have similar major groove electrostatic potentials to their canonical counterparts, whereas those with minimal widening exhibit significantly enhanced electronegativity, ranging from 0.8 to 2.5 kT/e, depending upon structural features. We propose that the negativity at the major groove of G·U wobble base pairs is determined by the combined effect of the base atoms and the sugar-phosphate backbone, which is impacted by stacking pattern and groove width as a result of base sequence. These findings are significant in that they provide predictive power with respect to which G·U sites in RNA are most likely to bind cationic ligands

4. The School Develops

Between 1947 and 1953, when M.P. Catherwood left the deanship to become New York’s industrial commissioner, the ILR School developed into a full fledged enterprise. These pages attempt to capture some of the excitement of this period of the school’s history, which was characterized by vigor, growth, and innovation. Includes: Alumni Recall Their Lives as Students; The Faculty Were Giants; Alice Cook: Lifelong Scholar, Consummate Teacher; Frances Perkins; Visits and Visitors; Tenth Anniversary: Reflection and Change; The Emergence of Departments at ILR; Development of International Programs and Outreach

3. Launching the New Enterprise

As the academic year of 1945-46 approached, the intensity of activity in preparation for actually opening the school in the fall term became overwhelming. Incredible though it may seem, Ives and Day were able in a period of a few weeks to assemble the nucleus of a faculty, several of whom formed a continuing source of counsel and advice both during the school’s formative years and thereafter. Includes: The First Dean and the School’s Dedication; A Participant’s View of the Early Years; Ives Moves On; Several Views of Martin P. Catherwood; The Founders

A High Incidence of Meiotic Silencing of Unsynapsed Chromatin Is Not Associated with Substantial Pachytene Loss in Heterozygous Male Mice Carrying Multiple Simple Robertsonian Translocations

Meiosis is a complex type of cell division that involves homologous chromosome pairing, synapsis, recombination, and segregation. When any of these processes is altered, cellular checkpoints arrest meiosis progression and induce cell elimination. Meiotic impairment is particularly frequent in organisms bearing chromosomal translocations. When chromosomal translocations appear in heterozygosis, the chromosomes involved may not correctly complete synapsis, recombination, and/or segregation, thus promoting the activation of checkpoints that lead to the death of the meiocytes. In mammals and other organisms, the unsynapsed chromosomal regions are subject to a process called meiotic silencing of unsynapsed chromatin (MSUC). Different degrees of asynapsis could contribute to disturb the normal loading of MSUC proteins, interfering with autosome and sex chromosome gene expression and triggering a massive pachytene cell death. We report that in mice that are heterozygous for eight multiple simple Robertsonian translocations, most pachytene spermatocytes bear trivalents with unsynapsed regions that incorporate, in a stage-dependent manner, proteins involved in MSUC (e.g., γH2AX, ATR, ubiquitinated-H2A, SUMO-1, and XMR). These spermatocytes have a correct MSUC response and are not eliminated during pachytene and most of them proceed into diplotene. However, we found a high incidence of apoptotic spermatocytes at the metaphase stage. These results suggest that in Robertsonian heterozygous mice synapsis defects on most pachytene cells do not trigger a prophase-I checkpoint. Instead, meiotic impairment seems to mainly rely on the action of a checkpoint acting at the metaphase stage. We propose that a low stringency of the pachytene checkpoint could help to increase the chances that spermatocytes with synaptic defects will complete meiotic divisions and differentiate into viable gametes. This scenario, despite a reduction of fertility, allows the spreading of Robertsonian translocations, explaining the multitude of natural Robertsonian populations described in the mouse

Surface electrostatic potential of () uBP, () ψBP major groove () ψBP minor groove in which only a −0

<p><b>Copyright information:</b></p><p>Taken from "Recognition of the spliceosomal branch site RNA helix on the basis of surface and electrostatic features"</p><p>Nucleic Acids Research 2005;33(4):1154-1161.</p><p>Published online 23 Feb 2005</p><p>PMCID:PMC549433.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p>5 charge was placed on each nonbridging phosphate oxygen. The atomic radii were taken from the AMBER94 force field. Here, the electrostatic potential is negative over the whole molecular surface with yellow (−3 kcal/mol/), red, white, blue and green (−1 kcal/mol/) going from most negative to least negative potential. The calculation was carried out using the hybrid PBE approach as described in the Methods

A schematic view of uBP () and ψBP () from structures solved by Newby and Greenbaum (; uBP PDB:1LMV; ψBP PDB: 1LPW

<p><b>Copyright information:</b></p><p>Taken from "Recognition of the spliceosomal branch site RNA helix on the basis of surface and electrostatic features"</p><p>Nucleic Acids Research 2005;33(4):1154-1161.</p><p>Published online 23 Feb 2005</p><p>PMCID:PMC549433.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Model #1 from both duplexes was used). RNA helices were rendered using the DINO () visualization program. The color schemes are as follows: the backbone is green; the sugar is yellow; base A is cyan; base C is red; base G is orange and base U and ψ are blue. Note that uBP adopts a typical A-helical pattern, whereas ψBP is characterized by an extrahelical branch site adenosine