RNA-dependent RNA polymerase of Japanese encephalitis virus binds the initiator nucleotide GTP to form a mechanistically important pre-initiation state

Flaviviral RNA-dependent RNA polymerases (RdRps) initiate replication of the single-stranded RNA genome in the absence of a primer. The template sequence 5'-CU-3' at the 3'-end of the flaviviral genome is highly conserved. Surprisingly, flaviviral RdRps require high concentrations of the second incoming nucleotide GTP to catalyze de novo template-dependent RNA synthesis. We show that GTP stimulates de novo RNA synthesis by RdRp from Japanese encephalitis virus (jRdRp) also. Crystal structures of jRdRp complexed with GTP and ATP provide a basis for specific recognition of GTP. Comparison of the jRdRp(GTP) structure with other viral RdRp-GTP structures shows that GTP binds jRdRp in a novel conformation. Apo-jRdRp structure suggests that the conserved motif F of jRdRp occupies multiple conformations in absence of GTP. Motif F becomes ordered on GTP binding and occludes the nucleotide triphosphate entry tunnel. Mutational analysis of key residues that interact with GTP evinces that the jRdRp(GTP) structure represents a novel pre-initiation state. Also, binding studies show that GTP binding reduces affinity of RdRp for RNA, but the presence of the catalytic Mn2+ ion abolishes this inhibition. Collectively, these observations suggest that the observed pre-initiation state may serve as a check-point to prevent erroneous template-independent RNA synthesis by jRdRp during initiation.

Analysis of the Genome and Transcriptome of Cryptococcus neoformans var. grubii Reveals Complex RNA Expression and Microevolution Leading to Virulence Attenuation

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.

3 `-Amino-5 `-carboxymethy1-3 `,5 `-dideoxy nucleosides for the synthesis of fully amide-linked RNA mimics

A convenient protocol is developed for the synthesis of 3 `-N-(fluorenylmethoxycarbonyl)-amino]-5 `-carboxymethyl derivatives of all four natural ribonucleosides from cheap chiral pool compound glucose. Synthesis of fully amide-linked RNA analogues of small oligonucleotides containing, for the first time, all four nucleoside amino acids using standard solid phase Fmoc-chemistry is described. (C) 2014 Elsevier Ltd. All rights reserved.

Mycobacterium tuberculosis Rho Is an NTPase with Distinct Kinetic Properties and a Novel RNA-Binding Subdomain

Two mechanisms - factor independent and dependent termination - ensure the completion of RNA synthesis in eubacteria. Factor-dependent mechanism relies on the Rho protein to terminate transcription by interacting with RNA polymerase. Although well studied in Escherichia coli, the properties of the Rho homologs from most bacteria are not known. The rho gene is unusually large in genus Mycobacterium and other members of actinobacteria, having,150 additional residues towards the amino terminal end. We describe the distinct properties of Rho from Mycobacterium tuberculosis. It is an NTPase with a preference for purine nucleoside triphosphates with kinetic properties different from E. coli homolog and an ability to use various RNA substrates. The N-terminal subdomain of MtbRho can bind to RNA by itself, and appears to contribute to the interaction of the termination factor with RNAs. Furthermore, the interaction with RNA induces changes in conformation and oligomerization of MtbRho.

Re-analysis of cryoEM data on HCV IRES bound to 40S subunit of human ribosome integrated with recent structural information suggests new contact regions between ribosomal proteins and HCV RNA

In this study, we combine available high resolution structural information on eukaryotic ribosomes with low resolution cryo-EM data on the Hepatitis C Viral RNA (IRES) human ribosome complex. Aided further by the prediction of RNA-protein interactions and restrained docking studies, we gain insights on their interaction at the residue level. We identified the components involved at the major and minor contact regions, and propose that there are energetically favorable local interactions between 40S ribosomal proteins and IRES domains. Domain II of the IRES interacts with ribosomal proteins S5 and S25 while the pseudoknot and the downstream domain IV region bind to ribosomal proteins S26, S28 and S5. We also provide support using UV cross-linking studies to validate our proposition of interaction between the S5 and IRES domains II and IV. We found that domain IIIe makes contact with the ribosomal protein S3a (S1e). Our model also suggests that the ribosomal protein S27 interacts with domain IIIc while S7 has a weak contact with a single base RNA bulge between junction IIIabc and IIId. The interacting residues are highly conserved among mammalian homologs while IRES RNA bases involved in contact do not show strict conservation. IRES RNA binding sites for S25 and S3a show the best conservation among related viral IRESs. The new contacts identified between ribosomal proteins and RNA are consistent with previous independent studies on RNA-binding properties of ribosomal proteins reported in literature, though information at the residue level is not available in previous studies.

Local Structural and Environmental Factors Define the Efficiency of an RNA Pseudoknot Involved in Programmed Ribosomal Frameshift Process

In programmed -1 ribosomal frameshift, an RNA pseudoknot stalls the ribosome at specific sequence and restarts translation in a new reading frame. A precise understanding of structural characteristics of these pseudoknots and their PRF inducing ability has not been clear to date. To investigate this phenomenon, we have studied various structural aspects of a -1 PRF inducing RNA pseudoknot from BWYV using extensive molecular dynamics simulations. A set of functional and poorly functional forms, for which previous mutational data were available, were chosen for analysis. These structures differ from each other by either single base substitutions or base-pair replacements from the native structure. We have rationalized how certain mutations in RNA pseudoknot affect its function; e.g., a specific base substitution in loop 2 stabilizes the junction geometry by forming multiple noncanonical hydrogen bonds, leading to a highly rigid structure that could effectively resist ribosome-induced unfolding, thereby increasing efficiency. While, a CG to AU pair substitution in stem 1 leads to loss of noncanonical hydrogen bonds between stems and loop, resulting in a less stable structure and reduced PRF inducing ability, inversion of a pair in stem 2 alters specific base-pair geometry that might be required in ribosomal recognition of nucleobase groups, negatively affecting pseudoknot functioning. These observations illustrate that the ability of an RNA pseudoknot to induce -1 PRF with an optimal rate depends on several independent factors that contribute to either the local conformational variability or geometry

Interaction of Sesbania mosaic virus (SeMV) RNA-dependent RNA polymerase (RdRp) with the p10 domain of polyprotein 2a and its implications in SeMV replication

Identification of viral encoded proteins that interact with RNA-dependent RNA polymerase (RdRp) is an important step towards unraveling the mechanism of replication. Sesbania mosaic virus (SeMV) RdRp was shown to interact strongly with p10 domain of polyprotein 2a and moderately with the protease domain. Mutational analysis suggested that the C-terminal disordered domain of RdRp is involved in the interaction with p10. Coexpression of full length RdRp and p10 resulted in formation of RdRp-p10 complex which showed significantly higher polymerase activity than RdRp alone. Interestingly, C Delta 43 RdRp also showed a similar increase in activity. Thus, p10 acts as a positive regulator of RdRp by interacting with the C-terminal disordered domain of RdRp. (C) 2014 The Authors. Published by Elsevier B.V.

Stacking Interactions in RNA and DNA: Roll-Slide Energy Hyperspace for Ten Unique Dinucleotide Steps

Understanding dinucleotide sequence directed structures of nuleic acids and their variability from experimental observation remained ineffective due to unavailability of statistically meaningful data. We have attempted to understand this from energy scan along twist, roll, and slide degrees of freedom which are mostly dependent on dinucleotide sequence using ab initio density functional theory. We have carried out stacking energy analysis in these dinucleotide parameter phase space for all ten unique dinucleotide steps in DNA and RNA using DFT-D by B97X-D/6-31G(2d,2p), which appears to satisfactorily explain conformational preferences for AU/AU step in our recent study. We show that values of roll, slide, and twist of most of the dinucleotide sequences in crystal structures fall in the low energy region. The minimum energy regions with large twist values are associated with the roll and slide values of B-DNA, whereas, smaller twist values correspond to higher stability to RNA and A-DNA like conformations. Incorporation of solvent effect by CPCM method could explain the preference shown by some sequences to occur in B-DNA or A-DNA conformations. Conformational preference of BII sub-state in B-DNA is preferentially displayed mainly by pyrimidine-purine steps and partly by purine-purine steps. The purine-pyrimidine steps show largest effect of 5-methyl group of thymine in stacking energy and the introduction of solvent reduces this effect significantly. These predicted structures and variabilities can explain the effect of sequence on DNA and RNA functionality. (c) 2014 Wiley Periodicals, Inc. Biopolymers 103: 134-147, 2015.

Genome wide chromatin occupancy of mrhl RNA and its role in gene regulation in mouse spermatogonial cells

Mrhl RNA is a nuclear lncRNA encoded in the mouse genome and negatively regulates Wnt signaling in spermatogonial cells through p68/Ddx5 RNA helicase. Mrhl RNA is present in the chromatin fraction of mouse spermatogonial Gc1-Spg cells and genome wide chromatin occupancy of mrhl RNA by ChOP (Chromatin oligo affinity precipitation) technique identified 1370 statistically significant genomic loci. Among these, genes at 37 genomic loci also showed altered expression pattern upon mrhl RNA down regulation which are referred to as GRPAM (Genes Regulated by Physical Association of Mrhl RNA). p68 interacted with mrhl RNA in chromatin at these GRPAM loci. p68 silencing drastically reduced mrhl RNA occupancy at 27 GRPAM loci and also perturbed the expression of GRPAM suggesting a role for p68 mediated mrhl RNA occupancy in regulating GRPAM expression. Wnt3a ligand treatment of Gc1-Spg cells down regulated mrhl RNA expression and also perturbed expression of these 27 GRPAM genes that included genes regulating Wnt signaling pathway and spermatogenesis, one of them being Sox8, a developmentally important transcription factor. We also identified interacting proteins of mrhl RNA associated chromatin fraction which included Pc4, a chromatin organizer protein and hnRNP A/B and hnRNP A2/B1 which have been shown to be associated with lincRNA-Cox2 function in gene regulation. Our findings in the Gc1-Spg cell line also correlate with the results from analysis of mouse testicular tissue which further highlights the in vivo physiological significance of mrhl RNA in the context of gene regulation during mammalian spermatogenesis.

Demonstration of helicase activity in the nonstructural protein, NSs, of the negative-sense RNA virus, Groundnut bud necrosis virus

The nonstructural protein NSs, encoded by the S RNA of groundnut bud necrosis virus (GBNV) (genus Tospovirus, family Bunyaviridae) has earlier been shown to possess nucleic-acid-stimulated NTPase and 50 a phosphatase activity. ATP hydrolysis is an essential function of a true helicase. Therefore, NSs was tested for DNA helicase activity. The results demonstrated that GBNV NSs possesses bidirectional DNA helicase activity. An alanine mutation in the Walker A motif (K189A rNSs) decreased DNA helicase activity substantially, whereas a mutation in the Walker B motif resulted in a marginal decrease in this activity. The parallel loss of the helicase and ATPase activity in the K189A mutant confirms that NSs acts as a non-canonical DNA helicase. Furthermore, both the wild-type and K189A NSs could function as RNA silencing suppressors, demonstrating that the suppressor activity of NSs is independent of its helicase or ATPase activity. This is the first report of a true helicase from a negative-sense RNA virus.

RNA nucleosides as chiral sensing agents in NMR spectroscopy

The study reports chiral sensing properties of RNA nucleosides. Adenosine, guanosine, uridine and cytidine are used as chiral derivatizing agents to differentiate chiral 1 degrees-amines. A three component protocol has been adopted for complexation of nucleosides and amines. The chiral differentiating ability of nucleosides is examined for different amines based on the H-1 NMR chemical shift differences of diastereomers (Delta delta(R,S)). Enantiomeric differentiation has been observed at multiple chemically distinct proton sites. Adenosine and guanosine exhibit large chiral differentiation (Delta delta(R,S)) due to the presence of a purine ring. The diastereomeric excess (de) measured by using adenosine is in good agreement with the gravimetric values.

A carboxy terminal domain of the L protein of rinderpest virus possesses RNA triphosphatase activity - The first enzyme in the viral mRNA capping pathway

The large protein L of negative-sense RNA viruses is a multifunctional protein involved in transcription and replication of genomic RNA. It also possesses enzymatic activities involved in capping and methylation of viral mRNAs. The pathway for mRNA capping followed by the L protein of the viruses in the Morbillivirus genus has not been established, although it has been speculated that these viruses may follow the unconventional capping pathway as has been shown for some viruses of Rhabdoviridae family. We had earlier shown that the large protein L of Rinderpest virus expressed as recombinant L-P complex in insect cells as well as the ribonucleoprotein complex from purified virus possesses RNA triphosphatase (RTPase) and guanylyltransferase activities, in addition to RNA dependent RNA polymerase activity. In the present work, we demonstrate that RTPase as well as nucleoside triphosphatase (NTPase) activities are exhibited by a subdomain of the L protein in the C terminal region (a.a. 1640 1840). The RTPase activity depends absolutely on a divalent cation, either magnesium or manganese. Both the RTPase and NTPase activities of the protein show dual metal specificity. Two mutant proteins having alanine mutations in the glutamic acid residues in motif-A of the RTPase domain did not show RTPase activity, while exhibiting reduced NTPase activity suggesting overlapping active sites for the two enzymatic functions. The RTPase and NTPase activities of the L subdomain resemble those of the Vaccinia capping enzyme D1 and the baculovirus LEF4 proteins. (C) 2015 Elsevier Inc. All rights reserved.

Evidence for N-7 guanine methyl transferase activity encoded within the modular domain of RNA-dependent RNA polymerase L of a Morbillivirus

Post-transcriptional modification of viral mRNA is essential for the translation of viral proteins by cellular translation machinery. Due to the cytoplasmic replication of Paramyxoviruses, the viral-encoded RNA-dependent RNA polymerase (RdRP) is thought to possess all activities required for mRNA capping and methylation. In the present work, using partially purified recombinant RNA polymerase complex of rinderpest virus expressed in insect cells, we demonstrate the in vitro methylation of capped mRNA. Further, we show that a recombinant C-terminal fragment (1717-2183 aa) of L protein is capable of methylating capped mRNA, suggesting that the various post-transcriptional activities of the L protein are located in independently folding domains.

Comparative sequence analyses of genome and transcriptome reveal novel transcripts and variants in the Asian elephant Elephas maximus

The Asian elephant Elephas maximus and the African elephant Loxodonta africana that diverged 5-7 million years ago exhibit differences in their physiology, behaviour and morphology. A comparative genomics approach would be useful and necessary for evolutionary and functional genetic studies of elephants. We performed sequencing of E. maximus and map to L. africana at similar to 15X coverage. Through comparative sequence analyses, we have identified Asian elephant specific homozygous, non-synonymous single nucleotide variants (SNVs) that map to 1514 protein coding genes, many of which are involved in olfaction. We also present the first report of a high-coverage transcriptome sequence in E. maximus from peripheral blood lymphocytes. We have identified 103 novel protein coding transcripts and 66-long non-coding (lnc)RNAs. We also report the presence of 181 protein domains unique to elephants when compared to other Afrotheria species. Each of these findings can be further investigated to gain a better understanding of functional differences unique to elephant species, as well as those unique to elephantids in comparison with other mammals. This work therefore provides a valuable resource to explore the immense research potential of comparative analyses of transcriptome and genome sequences in the Asian elephant.

Differential binding of ppGpp and pppGpp to E-coli RNA polymerase: photo-labeling and mass spectral studies

(p) ppGpp, a secondary messenger, is induced under stress and shows pleiotropic response. It binds to RNA polymerase and regulates transcription in Escherichia coli. More than 25 years have passed since the first discovery was made on the direct interaction of ppGpp with E. coli RNA polymerase. Several lines of evidence suggest different modes of ppGpp binding to the enzyme. Earlier cross-linking experiments suggested that the beta-subunit of RNA polymerase is the preferred site for ppGpp, whereas recent crystallographic studies pinpoint the interface of beta'/omega-subunits as the site of action. With an aim to validate the binding domain and to follow whether tetra-and pentaphosphate guanosines have different location on RNA polymerase, this work was initiated. RNA polymerase was photo-labeled with 8-azido-ppGpp/8-azido-pppGpp, and the product was digested with trypsin and subjected to mass spectrometry analysis. We observed three new peptides in the trypsin digest of the RNA polymerase labeled with 8-azido-ppGpp, of which two peptides correspond to the same pocket on beta'-subunit as predicted by X-ray structural analysis, whereas the third peptide was mapped on the beta-subunit. In the case of 8-azido-pppGpp-labeled RNA polymerase, we have found only one cross-linked peptide from the beta'-subunit. However, we were unable to identify any binding site of pppGpp on the beta-subunit. Interestingly, we observed that pppGpp at high concentration competes out ppGpp bound to RNA polymerase more efficiently, whereas ppGpp cannot titrate out pppGpp. The competition between tetraphosphate guanosine and pentaphosphate guanosine for E. coli RNA polymerase was followed by gel-based assay as well as by a new method known as DRaCALA assay.