Star-PAP RNA Binding Landscape Reveals Novel Role of Star-PAP in mRNA Metabolism That Requires RBM10-RNA Association.

Star-PAP is a non-canonical poly(A) polymerase that selects mRNA targets for polyadenylation. Yet, genome-wide direct Star-PAP targets or the mechanism of specific mRNA recognition is still vague. Here, we employ HITS-CLIP to map the cellular Star-PAP binding landscape and the mechanism of global Star-PAP mRNA association. We show a transcriptome-wide association of Star-PAP that is diminished on Star-PAP depletion. Consistent with its role in the 3'-UTR processing, we observed a high association of Star-PAP at the 3'-UTR region. Strikingly, there is an enrichment of Star-PAP at the coding region exons (CDS) in 42% of target mRNAs. We demonstrate that Star-PAP binding de-stabilises these mRNAs indicating a new role of Star-PAP in mRNA metabolism. Comparison with earlier microarray data reveals that while UTR-associated transcripts are down-regulated, CDS-associated mRNAs are largely up-regulated on Star-PAP depletion. Strikingly, the knockdown of a Star-PAP coregulator RBM10 resulted in a global loss of Star-PAP association on target mRNAs. Consistently, RBM10 depletion compromises 3'-end processing of a set of Star-PAP target mRNAs, while regulating stability/turnover of a different set of mRNAs. Our results establish a global profile of Star-PAP mRNA association and a novel role of Star-PAP in the mRNA metabolism that requires RBM10-mRNA association in the cell.

Binding of bis-ANS to Bacillus subtilis lipase: a combined computational and experimental investigation.

8-Anilino-1-naphthalene sulfonate (ANS) and its covalent dimer bis-ANS are widely used for titrating hydrophobic surfaces of proteins. Interest to understand the nature of interaction of these dyes with proteins was seriously pursued. However as the techniques used in these studies varied, they often provided varied information regarding stoichiometry, binding affinity, actual binding sites etc. In the present study, we used combination of computation methods (docking and MD simulation) and experimental methods (mutations, steady-state and time-resolved fluorescence) to investigate bis-ANS interaction with Bacillus subtilis lipase. We identified seven binding sites for bis-ANS on lipase using computational docking and MD simulation and verified these data using a set of single amino acid substituted mutants. Docking and MD simulation studies indicated that the binding sites were various indentations and grooves on protein surface with hydrophobic characteristics. Both hydrophobic and ionic interactions were involved in each of these binding events. We further examine the fluorescence properties of bis-ANS bound to mutant lipases that either gained or lost a binding site. Our results indicated that neither gain nor loss of single binding site caused any change in fluorescence lifetimes (and their relative amplitudes) of mutant lipase-bound bis-ANS in comparison to that bound to wild type; hence, it suggested that nature of bis-ANS binding to each of the sites in lipase was very similar.

The parasite specific substitution matrices improve the annotation of apicomplexan proteins.

BACKGROUND: A number of apicomplexan genomes have been sequenced successfully in recent years and this would help in understanding the biology of apicomplexan parasites. The members of the phylum Apicomplexa are important protozoan parasites (Plasmodium, Toxoplasma and Cryptosporidium etc) that cause some of the deadly diseases in humans and animals. In our earlier studies, we have shown that the standard BLOSUM matrices are not suitable for compositionally biased apicomplexan proteins. So we developed a novel series (SMAT and PfFSmat60) of substitution matrices which performed better in comparison to standard BLOSUM matrices and developed ApicoAlign, a sequence search and alignment tool for apicomplexan proteins. In this study, we demonstrate the higher specificity of these matrices and make an attempt to improve the annotation of apicomplexan kinases and proteases. RESULTS: The ROC curves proved that SMAT80 performs best for apicomplexan proteins followed by compositionally adjusted BLOSUM62 (PSI-BLAST searches), BLOSUM90 and BLOSUM62 matrices in terms of detecting true positives. The poor E-values and/or bit scores given by SMAT80 matrix for the experimentally identified coccidia-specific oocyst wall proteins against hematozoan (non-coccidian) parasites further supported the higher specificity of the same. SMAT80 uniquely detected (missed by BLOSUM) orthologs for 1374 apicomplexan hypothetical proteins against SwissProt database and predicted 70 kinases and 17 proteases. Further analysis confirmed the conservation of functional residues of kinase domain in one of the SMAT80 detected kinases. Similarly, one of the SMAT80 detected proteases was predicted to be a rhomboid protease. CONCLUSIONS: The parasite specific substitution matrices have higher specificity for apicomplexan proteins and are helpful in detecting the orthologs missed by BLOSUM matrices and thereby improve the annotation of apicomplexan proteins which are hypothetical or with unknown function.

ApicoAlign: an alignment and sequence search tool for apicomplexan proteins.

BACKGROUND: Over the recent years, a number of genomes have been successfully sequenced and this was followed by genome annotation projects to help understand the biological capabilities of newly sequenced genomes. To improve the annotation of Plasmodium falciparum proteins, we earlier developed parasite specific matrices (PfSSM) and demonstrated their (Smat80 and PfFSmat60) better performance over standard matrices (BLOSUM and PAM). Here we extend that study to nine apicomplexan species other than P. falciparum and develop a web application ApicoAlign for improving the annotation of apicomplexan proteins. RESULTS: The SMAT80 and PfFSmat60 matrices perform better for apicomplexan proteins compared to BLOSUM in detecting the orthologs and improving the alignment of these proteins with their potential orthologs respectively. Database searches against non-redundant (nr) database have shown that SMAT80 gives superior performance compared to BLOSUM series in terms of E-values, bit scores, percent identity, alignment length and mismatches for most of the apicomplexan proteins studied here. Using these matrices, we were able to find orthologs for rhomboid proteases of P. berghei, P. falciparum & P. vivax and large subunit of U2 snRNP auxiliary factor of Cryptosporidium parvum in Arabidopsis thaliana. We also show improved pairwise alignments of proteins from Apicomplexa viz. Cryptosporidium parvum and P. falciparum with their orthologs from other species using the PfFSmat60 matrix. CONCLUSIONS: The SMAT80 and PfFSmat60 substitution matrices perform better for apicomplexan proteins compared to BLOSUM series. Since they can be helpful in improving the annotation of apicomplexan genomes and their functional characterization, we have developed a web server ApicoAlign for finding orthologs and aligning apicomplexan proteins.

Characterization of Lipid Binding Properties of Plasmodium falciparum Acyl-Coenzyme A Binding Proteins and Their Competitive Inhibition by Mefloquine.

Malaria remains a worldwide concern in terms of morbidity and mortality. Limited understanding of the Plasmodium proteome makes it challenging to control malaria. Understanding of the expression and functions of different Plasmodium proteins will help in knowing this organism's virulence properties, besides facilitating the drug development process. In this study, we characterize the lipid binding and biophysical properties of the putative Plasmodium falciparum acyl-CoA binding proteins (PfACBPs), which may have intriguing functions in different stages of P. falciparum life cycle. While the PfACBPs can bind to long-chain fatty acyl-CoAs with high affinity, their affinity for short-chain fatty acyl-CoAs is weak. Base-stacking, electrostatic, and hydrophobic interactions between the aromatic rings, charged groups or residues, and hydrophobic chains or residues are responsible for acyl-CoA binding to PfACBPs. PfACBPs can also bind to phospholipids. PfACBPs cannot bind to the fatty acids and unphosphorylated fatty acid esters. PfACBPs are globular-helical proteins that contain a conserved acyl-CoA binding region. They exist in folded or unfolded conformations without attaining any intermediate state. In a systematic high-throughput in silico screening, mefloquine is identified as a potential ligand of PfACBPs. Binding affinities of mefloquine are much higher than those of fatty acyl-CoAs for all PfACBPs. Mefloquine binds to the acyl-CoA binding pocket of PfACBPs, thereby engaging many of the critical residues. Thus, mefloquine acts as a competitive inhibitor against fatty acyl-CoA binding to PfACBPs, leading to the prevention of P. falciparum growth and proliferation. Taken together, our study characterizes the functions of annotated PfACBPs and highlights the mechanistic details of their inactivation by mefloquine.

Sequencing of the genus Arabidopsis identifies a complex history of nonbifurcating speciation and abundant trans-specific polymorphism.

The notion of species as reproductively isolated units related through a bifurcating tree implies that gene trees should generally agree with the species tree and that sister taxa should not share polymorphisms unless they diverged recently and should be equally closely related to outgroups. It is now possible to evaluate this model systematically. We sequenced multiple individuals from 27 described taxa representing the entire Arabidopsis genus. Cluster analysis identified seven groups, corresponding to described species that capture the structure of the genus. However, at the level of gene trees, only the separation of Arabidopsis thaliana from the remaining species was universally supported, and, overall, the amount of shared polymorphism demonstrated that reproductive isolation was considerably more recent than the estimated divergence times. We uncovered multiple cases of past gene flow that contradict a bifurcating species tree. Finally, we showed that the pattern of divergence differs between gene ontologies, suggesting a role for selection.

Temperature controlled ionic liquid-based dispersive micro-extraction using two ligands, for determination of aluminium in scalp hair samples of Alzheimer's patients: a multivariate study.

A green and sensitive temperature controlled dispersive liquid-liquid microextraction (TIL-DLLME) methodology based on the application of ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate, [C4mim][PF6], as an extractant solvent was proposed for the preconcentration of trace levels of aluminium (Al(3+)) in scalp hair samples of Alzheimer's (AD) patients, prior to analyzing by flame atomic absorption spectrometry (FAAS). The Al(3+) was complexed with 8-hydrooxyquinoline (oxine) (L1) and 3,5,7,2'-4' pentahydroxy flavone (morin) (L2) separately and then extracted by IL at temperature (50±2.0°C). Some effective factors that influence the TIL-DLLME efficiency such as pH, ligands concentrations, volume of IL, ionic strength, and incubation time were investigated and optimized by multivariate analysis. In the optimum experimental conditions, the limit of detection (3s) and enhancement factor were 0.56 µg L(-1), 0.64 µg L(-1) and 85, 73 for both ligands, respectively. The relative standard deviation (RSD) for six replicate determinations of 100 µg L(-1) Al(3+) complexed with oxine and morin were found to be 3.88% and 4.74%, respectively. The developed method was validated by the analysis of certified reference material of human hair (NCSZC81002).and applied satisfactorily to the determination of Al(3+) in acid digested scalp hair samples of AD patients and healthy controls. The resulted data shows significant higher level in scalp hair samples of AD male patients with related to referents of same age and socioeconomic status.