The carboxy terminal WD domain of the pre-mRNA splicing factor Prp17p is critical for function

In Saccharomyces cerevisiae, Prp17p is required for the efficient completion of the second step of pre-mRNA splicing. The function and interacting factors for this protein have not been elucidated. We have performed a mutational analysis of yPrp17p to identify protein domains critical for function. A series of deletions were made throughout the region spanning the N-terminal 158 amino acids of the protein, which do not contain any identified structural motifs. The C-terminal portion (amino acids 160–455) contains a WD domain containing seven WD repeats. We determined that a minimal functional Prp17p consists of the WD domain and 40 amino acids N-terminal to it. We generated a three-dimensional model of the WD repeats in Prp17p based on the crystal structure of the [beta]-transducin WD domain. This model was used to identify potentially important amino acids for in vivo functional characterization. Through analysis of mutations in four different loops of Prp17p that lie between [beta] strands in the WD repeats, we have identified four amino acids, 235TETG238, that are critical for function. These amino acids are predicted to be surface exposed and may be involved in interactions that are important for splicing. Temperature-sensitive prp17 alleles with mutations of these four amino acids are defective for the second step of splicing and are synthetically lethal with a U5 snRNA loop I mutation, which is also required for the second step of splicing. These data reinforce the functional significance of this region within the WD domain of Prp17p in the second step of splicing.

Trans-saccadic processing of visual and motor planning during sequential eye movements

How the brain maintains perceptual continuity across eye movements that yield discontinuous snapshots of the world is still poorly understood. In this study, we adapted a framework from the dual-task paradigm, well suited to reveal bottlenecks in mental processing, to study how information is processed across sequential saccades. The pattern of RTs allowed us to distinguish among three forms of trans-saccadic processing (no trans-saccadic processing, trans-saccadic visual processing and trans-saccadic visual processing and saccade planning models). Using a cued double-step saccade task, we show that even though saccade execution is a processing bottleneck, limiting access to incoming visual information, partial visual and motor processing that occur prior to saccade execution is used to guide the next eye movement. These results provide insights into how the oculomotor system is designed to process information across multiple fixations that occur during natural scanning.

Saccharomyces cerevisiae NineTeen Complex (NTC)-associated Factor Bud31/Ycr063w Assembles on Precatalytic Spliceosomes and Improves First and Second Step Pre-mRNA Splicing Efficiency

Pre-mRNA splicing occurs in spliceosomes whose assembly and activation are critical for splice site selection and catalysis. The highly conserved NineTeen complex protein complex stabilizes various snRNA and protein interactions early in the spliceosome assembly pathway. Among several NineTeen complex-associated proteins is the nonessential protein Bud31/Ycr063w, which is also a component of the Cef1p subcomplex. A role for Bud31 in pre-mRNA splicing is implicated by virtue of its association with splicing factors, but its specific functions and spliceosome interactions are uncharacterized. Here, using in vitro splicing assays with extracts from a strain lacking Bud31, we illustrate its role in efficient progression to the first catalytic step and its requirement for the second catalytic step in reactions at higher temperatures. Immunoprecipitation of functional epitope-tagged Bud31 from in vitro reactions showed that its earliest association is with precatalytic B complex and that the interaction continues in catalytically active complexes with stably bound U2, U5, and U6 small nuclear ribonucleoproteins. In complementary experiments, wherein precatalytic spliceosomes are selected from splicing reactions, we detect the occurrence of Bud31. Cross-linking of proteins to pre-mRNAs with a site-specific 4-thio uridine residue at the -3 position of exon 1 was tested in reactions with WT and bud31 null extracts. The data suggest an altered interaction between a similar to 25-kDa protein and this exonic residue of pre-mRNAs in the arrested bud31 null spliceosomes. These results demonstrate the early spliceosomal association of Bud31 and provide plausible functions for this factor in stabilizing protein interactions with the pre-mRNA.

Diffusion ordered spectroscopy for resolution of double bonded cis, trans-isomers

NMR spectroscopic separation of double bonded cis- and trans-isomers, that have different molecular shapes but identical mass have been carried out using Diffusion Ordered Spectroscopy (DOSY). The mixtures of fumaric acid and maleic acid, that have similar hydrodynamic radii, have resolved been on the basis of their diffusion coefficients arising due to their different tendencies to associate with micelles or reverse micelles. Sodium dodecyl sulfate (SDS) and Dioctyl sulfosuccinate sodium salt (AOT) have been used as the media to mimic the chromatographic conditions, modify the average mobility and to achieve differential diffusion rates. The best separation of the components has been achieved by Dioctyl sulfosuccinate sodium salt (AOT) in D2O solution. (C) 2012 Elsevier B.V. All rights reserved.

Cis-trans peptide variations in structurally similar proteins

The presence of energetically less favourable cis peptides in protein structures has been observed to be strongly associated with its structural integrity and function. Inter-conversion between the cis and trans conformations also has an important role in the folding process. In this study, we analyse the extent of conservation of cis peptides among similar folds. We look at both the amino acid preferences and local structural changes associated with such variations. Nearly 34% of the Xaa-Proline cis bonds are not conserved in structural relatives; Proline also has a high tendency to get replaced by another amino acid in the trans conformer. At both positions bounding the peptide bond, Glycine has a higher tendency to lose the cis conformation. The cis conformation of more than 30% of beta turns of type VIb and IV are not found to be conserved in similar structures. A different view using Protein Block-based description of backbone conformation, suggests that many of the local conformational changes are highly different from the general local structural variations observed among structurally similar proteins. Changes between cis and trans conformations are found to be associated with the evolution of new functions facilitated by local structural changes. This is most frequent in enzymes where new catalytic activity emerges with local changes in the active site. Cis-trans changes are also seen to facilitate inter-domain and inter-protein interactions. As in the case of folding, cis-trans conversions have been used as an important driving factor in evolution.

Context dependent splicing functions of Bud31/Ycr063w define its role in budding and cell cycle progression

The yeast Bud31 protein, a Prp19 complex (NTC) member, aids spliceosome assembly and thus promotes efficient pre-mRNA splicing. The bud31 null cells show mild budding abnormalities at optimal growth temperatures and, at higher temperatures, have growth defects with aberrant budding. Here we have assessed cell cycle transitions which require Bud31. We find Bud31 facilitates passage through G1-S regulatory point (Start) but is not needed for G2-M transition or for exit from mitosis. To co-relate Bud31 functions in cell division with splicing, we studied the splicing status of transcripts that encode proteins involved in budding. We find Bud31 promotes efficient splicing of only some of these pre-mRNAs, for example, ARP2 and SRC1. Wild type cells have a long and a short isoform of SRC1 mRNA and protein, out of which the shorter mRNA splice variant is predominant. bud31 Delta cells show inefficient SRC1 splicing and entirely lack the shorter SRC1 spliced mRNA isoform. Yeast PRP17, another NTC sub-complex member, is also required for G1-S and G2-M cell cycle transitions. We examined genetic interactions between BUD31 and PRP17. While both factors were needed for efficient cell cycle dependent gene expression, our data indicate that distinct pre-mRNAs depend on each of these non-essential splicing factors.

Plasmodium falciparum Prp16 homologue and its role in splicing

Large numbers of Plasmodium genes have been predicted to have introns. However, little information exists on the splicing mechanisms in this organism. Here, we describe the DExD/DExH-box containing Pre-mRNA processing proteins (Prps), PfPrp2p, PfPrp5p, PfPrp16p, PfPrp22p, PfPrp28p, PfPrp43p and PfBrr2p, present in the Plasmodium falciparum genome and characterized the role of one of these factors, PfPrp16p. It is a member of DEAH-box protein family with nine collinear sequence motifs, a characteristic of helicase proteins. Experiments with the recombinantly expressed and purified PfPrp16 helicase domain revealed binding to RNA, hydrolysis of ATP as well as catalytic helicase activities. Expression of helicase domain with the C-terminal helicase-associated domain (HA2) reduced these activities considerably, indicating that the helicase-associated domain may regulate the PfPrp16 function. Localization studies with the PfPrp16 GFP transgenic lines suggested a role of its N-terminal domain (1-80 amino acids) in nuclear targeting. Immunodepletion of PfPrp16p, from nuclear extracts of parasite cultures, blocked the second catalytic step of an in vitro constituted splicing reaction suggesting a role for PfPrp16p in splicing catalysis. Further we show by complementation assay in yeast that a chimeric yeast-Plasmodium Prp16 protein, not the full length PfPrp16, can rescue the yeast prp16 temperature-sensitive mutant. These results suggest that although the role of Prp16p in catalytic step II is highly conserved among Plasmodium, human and yeast, subtle differences exist with regards to its associated factors or its assembly with spliceosomes. (C) 2012 Elsevier B.V. All rights reserved.

Annexin A2 and PSF proteins interact with p53 IRES and regulate translation of p53 mRNA

p53 mRNA has been shown to be translated into two isoforms, full-length p53 (FL-p53) and a truncated isoform Delta N-p53, which modulates the functions of FL-p53 and also has independent functions. Previously, we have shown that translation of p53 and Delta N-p53 can be initiated at Internal Ribosome Entry Sites (IRES). These two IRESs were shown to regulate the translation of p53 and Delta N-p53 in a distinct cell-cycle phase-dependent manner. Earlier observations from our laboratory also suggest that the structural integrity of the p53 RNA is critical for IRES function and is compromised by mutations that affect the structure as well as RNA protein interactions. In the current study, using RNA affinity approach we have identified Annexin A2 and PTB associated Splicing Factor (PSF/SFPQ) as novel ITAFs for p53 IRESs. We have showed that the purified Annexin A2 and PSF proteins specifically bind to p53 IRES elements. Interestingly, in the presence of calcium ions Annexin A2 showed increased binding with p53 IRES. Immunopulldown experiments suggest that these two proteins associate with p53 mRNA ex vivo as well. Partial knockdown of Annexin A2 and PSF showed decrease in p53 IRES activity and reduced levels of both the p53 isoforms. More importantly the interplay between Annexin A2, PSF and PTB proteins for binding to p53mRNA appears to play a crucial role in IRES function. Taken together, our observations suggest pivotal role of two new trans-acting factors in regulating the p53-IRES function, which in turn influences the synthesis of p53 isoforms.

Splicing Functions and Global Dependency on Fission Yeast Slu7 Reveal Diversity in Spliceosome Assembly

The multiple short introns in Schizosaccharomyces pombe genes with degenerate cis sequences and atypically positioned polypyrimidine tracts make an interesting model to investigate canonical and alternative roles for conserved splicing factors. Here we report functions and interactions of the S. pombe slu7(+) (spslu7(+)) gene product, known from Saccharomyces cerevisiae and human in vitro reactions to assemble into spliceosomes after the first catalytic reaction and to dictate 3' splice site choice during the second reaction. By using a missense mutant of this essential S. pombe factor, we detected a range of global splicing derangements that were validated in assays for the splicing status of diverse candidate introns. We ascribe widespread, intron-specific SpSlu7 functions and have deduced several features, including the branch nucleotide-to-3' splice site distance, intron length, and the impact of its A/U content at the 5' end on the intron's dependence on SpSlu7. The data imply dynamic substrate-splicing factor relationships in multiintron transcripts. Interestingly, the unexpected early splicing arrest in spslu7-2 revealed a role before catalysis. We detected a salt-stable association with U5 snRNP and observed genetic interactions with spprp1(+), a homolog of human U5-102k factor. These observations together point to an altered recruitment and dependence on SpSlu7, suggesting its role in facilitating transitions that promote catalysis, and highlight the diversity in spliceosome assembly.

Steric and Electronic Interactions Controlling the cis/trans Isomer Equilibrium at X-Pro Tertiary Amide Motifs in Solution

A systematic understanding of the noncovalent interactions that influence the structures of the cis conformers and the equilibrium between the cis and the trans conformers, of the X-Pro tertiary amide motifs, is presented based on analyses of H-1-, C-13-NMR and FTIR absorption spectra of two sets of homologous peptides, X-Pro-Aib-OMe and X-Pro-NH-Me (where X is acetyl, propionyl, isobutyryl and pivaloyl), in solvents of varying polarities. First, this work shows that the cis conformers of any X-Pro tertiary amide motif, including Piv-Pro, are accessible in the new motifs X-Pro-Aib-OMe, in solution. These conformers are uniquely observable by FTIR spectroscopy at ambient temperatures and by NMR spectroscopy from temperatures as high as 273 K. This is made possible by the persistent presence of n(i-1i)* interactions at Aib, which also influence the disappearance of steric effects at these cis X-Pro rotamers. Second, contrary to conventional understanding, the energy contribution of steric effects to the cis/trans equilibrium at the X-Pro motifs is found to be nonvariant (0.54 +/- 0.02 kcal/mol) with increase in steric bulk on the X group. Third, the current studies provide direct evidence for the weak intramolecular interactions namely the n(i-1i)*, the N-Pro center dot center dot center dot Hi+1 (C(5)a), and the C-7 hydrogen bond that operate and influence the structures, stabilities, and dynamics between different conformational states of X-Pro tertiary amide motifs. NMR and IR spectral data suggest that the cis conformers of X-Pro motifs are ensembles of short-lived rotamers about the C-X-N-Pro bond. (c) 2013 Wiley Periodicals, Inc. Biopolymers 101: 66-77, 2014.

Conformational Diversity in Contryphans from Conus Venom: cis-trans Isomerisation and Aromatic/Proline Interactions in the 23-Membered Ring of a 7-Residue Peptide Disulfide Loop

Conformational diversity or shapeshifting in cyclic peptide natural products can, in principle, confer a single molecular entity with the property of binding to multiple receptors. Conformational equilibria have been probed in the contryphans, which are peptides derived from Conus venom possessing a 23-membered cyclic disulfide moiety. The natural sequences derived from Conus inscriptus, GCV(D)LYPWC* (In936) and Conus loroisii, GCP(D)WDPWC* (Lo959) differ in the number of proline residues within the macrocyclic ring. Structural characterisation of distinct conformational states arising from cis-trans equilibria about Xxx-Pro bonds is reported. Isomerisation about the C2-P3 bond is observed in the case of Lo959 and about the Y5-P6 bond in In936. Evidence is presented for as many as four distinct species in the case of the synthetic analogue V3P In936. The Tyr-Pro-Trp segment in In936 is characterised by distinct sidechain orientations as a consequence of aromatic/proline interactions as evidenced by specific sidechain-sidechain nuclear Overhauser effects and ring current shifted proton chemical shifts. Molecular dynamics simulations suggest that Tyr5 and Trp7 sidechain conformations are correlated and depend on the geometry of the Xxx-Pro bond. Thermodynamic parameters are derived for the cis trans equilibrium for In936. Studies on synthetic analogues provide insights into the role of sequence effects in modulating isomerisation about Xxx-Pro bonds.

A Thiol- ene Clickable Hyperbranched Polyester via a Simple Single- Step Melt Trans- Esterification Process

Self-condensation of AB(2) type monomers (containing one A-type and two B-type functional groups) generates hyperbranched (HB) polymers that carry numerous B-type end-groups at their molecular periphery; thus, development of synthetic methods that directly provide quantitatively transformable peripheral B groups would be of immense value as this would provide easy access to multiply functionalized HB systems. A readily accessible AB(2) monomer, namely diallyl, 5-(4-hydroxybutoxy)isophthalate was synthesized, which on polymerization under standard melt-transesterfication conditions yielded a peripherally clickable HB polyester in a single step; the allyl groups were quantitatively reacted with a variety of thiols using the facile photoinitiated thiol-ene reaction to generate a wide range of derivatives, with varying solubility and thermal properties. Furthermore, it is shown that the peripheral allyl double bonds can also be readily epoxidized using meta-chloroperoxybenzoic acid to yield interesting HB systems, which could potentially serve as a multifunctional cross-linking agent in epoxy formulations. (c) 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40248.