Biology subjects

Singh, J.

Publications and source records attributed to Singh, J..

At least 109 records · Page 6Linked to original sources

bioRxiv2021

A DCL3 dicing code within Pol IV-RDR2 transcripts diversifies the siRNA pool guiding RNA-directed DNA methylation

In plants, selfish genetic elements including retrotransposons and DNA viruses are transcriptionally silenced by RNA-directed DNA methylation. Guiding the process are short interfering RNAs (siRNAs) cut by DICER-LIKE 3 (DCL3) from double-stranded precursors of [~]30 bp synthesized by NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). We show that Pol IV initiating nucleotide choice, RDR2 initiation 1-2 nt internal to Pol IV transcript ends and RDR2 terminal transferase activity collectively yield a code that influences which end of the precursor is diced and whether 24 or 23 nt siRNAs are generated from the Pol IV or RDR2-transcribed strands. By diversifying the size, sequence, and strand polarity of siRNAs derived from a given precursor, alternative patterns of DCL3 dicing allow maximal siRNA coverage at methylated target loci.

molecular biology↗

bioRxiv2021

Structure and RNA template requirements of Arabidopsis RNA-DEPENDENT RNA POLYMERASE 2

RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In Arabidopsis, RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest or termination, involving the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The dsRNAs are then released from the Pol IV-RDR2 complex and diced into siRNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 [A], determined by cryo-electron microscopy. The N-terminal region contains an RNA-recognition motif (RRM) adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1-2 nucleotides (nt) internal to the 3 ends of its templates and can transcribe the RNA of an RNA-DNA hybrid provided that 9 or more nucleotides at the RNAs 3 end is unpaired. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3 end occurs as the DNA template and non-template strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3 end as the DNA strands reanneal, allowing RDR2 to engage the RNA and transcribe the second strand. SignificanceRDR2 is critical for siRNA-directed DNA methylation in Arabidopsis, functioning in physical association with DNA-dependent Pol IV to synthesize the second strands of double-stranded siRNA precursors. Basepairing between the DNA template strand transcribed by Pol IV and the nontemplate DNA strand is known to induce Pol IV arrest and Pol IV-RDR2 transcriptional coupling, but how this occurs is unknown. We report the structure of RDR2 and experimental evidence for how RDR2 engages its RNA templates and initiates transcription. RDR2 engages the ends of RNAs displaced from RNA-DNA hybrids, suggesting a model in which Pol IV arrest and backtracking, accompanied by DNA strand reannealing, extrudes the 3 end of the Pol IV transcript, allowing RNA engagement and second-strand synthesis.

molecular biology↗

bioRxiv2021

Notch-dependent Abl signaling regulates cell motility during ommatidial rotation in Drosophila

A collective cell motility event that occurs during Drosophila eye development, ommatidial rotation (OR), serves as a paradigm for signaling pathway-regulated directed movement of cell clusters. OR is instructed by several signaling events, including the EGFR and Notch pathways, and planar cell polarity (PCP) signaling, all of which are associated with photoreceptor R3 and R4 specification and differentiation. Here, we show that Abl kinase negatively regulates ommatidial rotation through its activity in the R3/R4 pair. Interestingly in wild-type, Abl is localized to apical junctional regions in R4 but not in R3 during OR, and this apical enrichment requires Notch signaling. We further demonstrate that Abl and Notch genetically interact during OR, and Abl co-immunoprecipitates in complexes with Notch in the developing eye disc. Perturbations of Abl interfere with adherens junction dynamics of the ommatidial preclusters, which are critical for the OR process. Taken together, our data suggest a model in which Abl kinase acts directly downstream of the Notch receptor in R4 to fine-tune OR via its input into adherens junction complexes.

developmental biology↗

bioRxiv2021

SPOT-Contact-Single: Improving Single-Sequence-Based Prediction of Protein Contact Map using a Transformer Language Model, Large Training Set and Ensembled Deep Learning

MotivationAccurate prediction of protein contact-map is essential for accurate protein structure and function prediction. As a result, many methods have been developed for protein contact map prediction. However, most methods rely on protein-sequence-evolutionary information, which may not exist for many proteins due to lack of naturally occurring homologous sequences. Moreover, generating evolutionary profiles is computationally intensive. Here, we developed a contact-map predictor utilizing the output of a pre-trained language model ESM-1b as an input along with a large training set and an ensemble of residual neural networks. ResultsWe showed that the proposed method makes a significant improvement over a single-sequence-based predictor SSCpred with 15% improvement in the F1-score for the independent CASP14-FM test set. It also outperforms evolutionary-profile-based methods TrRosetta and SPOT-Contact with 48.7% and 48.5% respective improvement in the F1-score on the proteins without homologs (Neff=1) in the independent SPOT-2018 set. The new method provides a much faster and reasonably accurate alternative to evolution-based methods, useful for large-scale prediction. AvailabilityStand-alone-version of SPOT-Contact-Single is available at https://github.com/jas-preet/SPOT-Contact-Single. Direct prediction can also be made at https://sparks-lab.org/server/spot-contact-single. The datasets used in this research can also be downloaded from the GitHub. Contactjaspreetsingh2@griffithuni.edu.au, k.paliwal@griffith.edu.au, and zhouyq@szbl.ac.cn Supplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2021

Epitome: Predicting epigenetic events in novel cell types with multi-cell deep ensemble learning

The accumulation of large epigenomics data consortiums provides us with the opportunity to extrapolate existing knowledge to new cell types and conditions. We propose Epitome, a deep neural network that learns similarities of chromatin accessibility between well characterized reference cell types and a query cellular context, and copies over signal of transcription factor binding and modification of histones from reference cell types when chromatin profiles are similar to the query. Epitome achieves state-of-the-art accuracy when predicting transcription factor binding sites on novel cellular contexts, and can further improve predictions as more epigenetic signals are collected from both reference cell types and the query cellular context of interest.

bioinformatics↗

bioRxiv2021

In-vitro Reconstitution of Membrane Contact Sites between the Endoplasmic Reticulum and Lipid Droplets

Coordinated cell function requires inter-organelle communication across Membrane Contact Sites (MCS). Here we deposit ER-enriched microsomes purified from rat liver or from cultured cells on a coverslip in the form of a continuous planar membrane. We visualize real-time protein and lipid exchanges across MCS that form between this ER-mimicking membrane and lipid droplets purified from rat liver. An Optical trap is used to demonstrate physical tethering of individual lipid droplets to the ER-mimicking membrane at MCS, and to directly measure the strength of this tether. In-vitro MCS formation changes dramatically in response to metabolic state and immune activation in the animal. Surprisingly, we find that the Rab18 GTPase and Phosphatidic acid are common molecular factors to control both of these pathways. This assay could possibly be adapted to interrogate MCS formation between other membranes (e.g. mitochondria, peroxisomes, endosomes etc.), and abnormalities therein that cause neurological, metabolic and pathogenic diseases.

cell biology↗

bioRxiv2021

Possible link between higher transmissibility of B.1.617 and B.1.1.7 variants of SARS-CoV-2 and increased structural stability of its spike protein and hACE2 affinity

The Severe Acute syndrome corona Virus 2 (SARS-CoV-2) outbreak in December 2019 has caused a global pandemic. The rapid mutation rate in the virus has caused alarming situations worldwide and is being attributed to the false negativity in RT-PCR tests, which also might lead to inefficacy of the available drugs. It has also increased the chances of reinfection and immune escape. We have performed Molecular Dynamic simulations of three different Spike-ACE2 complexes, namely Wildtype (WT), B.1.1.7 variant (N501Y Spike mutant) and B.1.617 variant (L452R, E484Q Spike mutant) and compared their dynamics, binding energy and molecular interactions. Our result shows that mutation has caused the increase in the binding energy between the Spike and hACE2. In the case of B.1.617 variant, the mutations at L452R and E484Q increased the stability and intra-chain interactions in the Spike protein, which may change the interaction ability of human antibodies to this Spike variant. Further, we found that the B.1.1.7 variant had increased hydrogen interaction with LYS353 of hACE2 and more binding affinity in comparison to WT. The current study provides the biophysical basis for understanding the molecular mechanism and rationale behind the increase in the transmissivity and infectivity of the mutants compared to wild-type SARS-CoV-2.

biophysics↗

bioRxiv2021

Whole-cell segmentation of tissue images with human-level performance using large-scale data annotation and deep learning

Understanding the spatial organization of tissues is of critical importance for both basic and translational research. While recent advances in tissue imaging are opening an exciting new window into the biology of human tissues, interpreting the data that they create is a significant computational challenge. Cell segmentation, the task of uniquely identifying each cell in an image, remains a substantial barrier for tissue imaging, as existing approaches are inaccurate or require a substantial amount of manual curation to yield useful results. Here, we addressed the problem of cell segmentation in tissue imaging data through large-scale data annotation and deep learning. We constructed TissueNet, an image dataset containing >1 million paired whole-cell and nuclear annotations for tissue images from nine organs and six imaging platforms. We created Mesmer, a deep learning-enabled segmentation algorithm trained on TissueNet that performs nuclear and whole-cell segmentation in tissue imaging data. We demonstrated that Mesmer has better speed and accuracy than previous methods, generalizes to the full diversity of tissue types and imaging platforms in TissueNet, and achieves human-level performance for whole-cell segmentation. Mesmer enabled the automated extraction of key cellular features, such as subcellular localization of protein signal, which was challenging with previous approaches. We further showed that Mesmer could be adapted to harness cell lineage information present in highly multiplexed datasets. We used this enhanced version to quantify cell morphology changes during human gestation. All underlying code and models are released with permissive licenses as a community resource.

bioinformatics↗

bioRxiv2021

CRISPR-Cas9 gene editing and rapid detection of gene-edited mutants using high-resolution melting in the apple scab fungus, Venturia inaequalis

BackgroundScab, or black spot, caused by the filamentous fungal pathogen Venturia inaequalis, is the most economically important disease of apple (Malus x domestica) worldwide. To develop durable control strategies against this disease, a better understanding of the genetic mechanisms underlying the growth, reproduction, virulence and pathogenicity of V. inaequalis is required. A major bottleneck for the genetic characterization of V. inaequalis is the inability to easily delete or disrupt genes of interest using homologous recombination. Indeed, no gene deletions or disruptions in V. inaequalis have yet been published. Recently, CRISPR-Cas9 has emerged as an efficient tool for gene editing in filamentous fungi. With this in mind, we set out to establish CRISPR-Cas9 as a gene editing tool in V. inaequalis. ResultsWe showed that CRISPR-Cas9 can be used for gene inactivation in the apple scab fungus. As a proof of concept, we targeted the melanin biosynthesis pathway gene trihydroxynaphthalene reductase (THN), which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference. Using one of two CRISPR-Cas9 single guide RNAs (sgRNAs) targeted to the THN gene, delivered by a single autonomously replicating Golden Gate-compatible plasmid, we were able to identify six of 36 stable transformants with a light-brown phenotype, indicating an ~16.7% gene inactivation efficiency. Notably, of these six THN mutants, five had an independent mutation. As part of our pipeline, we also report a high-resolution melting (HRM) curve protocol for the rapid detection of CRISPR-Cas9 gene-edited mutants of V. inaequalis. This protocol identified a single base pair deletion mutation in a sample containing only 5% mutant genomic DNA, indicating high sensitivity for mutant screening. ConclusionsIn establishing CRISPR-Cas9 as a tool for gene editing in V. inaequalis, we have provided a strong starting point for studies aiming to decipher the function of genes associated with the growth, reproduction, virulence and pathogenicity of this fungus. The associated HRM curve protocol will enable CRISPR-Cas9 transformants to be screened for gene inactivation in a high-throughput and low-cost manner, which will be particularly powerful in cases where the CRISPR-Cas9-mediated gene inactivation efficiency is low.

microbiology↗

bioRxiv2021

Coordinated overexpression of OsSUT1, OsSWEET11 and OsSWEET14 in rice impairs carbohydrate metabolism that has implications in plant growth, yield and susceptibility to Xanthomonas oryzae pv oryzae (Xoo)

Enhancing carbohydrate export to sink tissues is considered as a feasible approach for improving photosynthetic efficiency and crop yield. In Oryza sativa Sucrose Transporter OsSUT1 located in companion cells and Sugars Will Eventually be Exported Transporters (SWEETs); OsSWEET11 and OsSWEET14 present in phloem parenchyma mesophyll cell plasma membranes are involved in long distance sucrose transport. OsSWEET11 and OsSWEET14 also play important role in host-pathogen interaction of rice plants and Xanthomonas oryzae pv oryzae (Xoo) that causes bacterial leaf blight. Three genes, OsSUT1, OsSWEET11, and OsSWEET14 were overexpressed under the control of their native promoters in rice to modulate long distance sugar transport and disease resistance. The transgenics displayed several phenotypic aberrations such as reduced plant height and seed weight due to altered sucrose transport and metabolism. Lower sucrose transport rate in transgenics than the WT resulted in reduced sucrose, fructose and glucose and increased starch accumulation in their leaves at the end of dark period. Transcriptional analysis revealed a reduction in the expression of genes involved in sucrose synthesis pathway in transgenics. Normal growth and development of transgenic seedlings were restored in growth media supplemented with 3% sucrose demonstrating in planta sucrose limitation. Remarkably, transgenic lines had diminished susceptibility to Xoo than the WTs due to low sugar content in the leaves demonstrating that rice plants maintain an optimum level of SWEETs for proper plant growth and development, and upregulation of these SWEETs in rice mimicks Xoo attack impelling plants to reduce sugar content in the apoplasm to inhibit pathogen growth.

plant biology↗

bioRxiv2021

Structure-function investigation of a new VUI-202012/01 SARS-CoV-2 variant

The SARS-CoV-2 (Severe Acute Respiratory Syndrome-Coronavirus) has accumulated multiple mutations during its global circulation. Recently, a new strain of SARS-CoV-2 (VUI 202012/01) had been identified leading to sudden spike in COVID-19 cases in South-East England. The strain has accumulated 23 mutations which have been linked to its immune evasion and higher transmission capabilities. Here, we have highlighted structural-function impact of crucial mutations occurring in spike (S), ORF8 and nucleocapsid (N) protein of SARS-CoV-2. Some of these mutations might confer higher fitness to SARS-CoV-2. SummarySince initial outbreak of COVID-19 in Wuhan city of central China, its causative agent; SARS-CoV-2 virus has claimed more than 1.7 million lives out of 77 million populations and still counting. As a result of global research efforts involving public-private-partnerships, more than 0.2 million complete genome sequences have been made available through Global Initiative on Sharing All Influenza Data (GISAID). Similar to previously characterized coronaviruses (CoVs), the positive-sense single-stranded RNA SARS-CoV-2 genome codes for ORF1ab non-structural proteins (nsp(s)) followed by ten or more structural/nsps [1, 2]. The structural proteins include crucial spike (S), nucleocapsid (N), membrane (M), and envelope (E) proteins. The S protein mediates initial contacts with human hosts while the E and M proteins function in viral assembly and budding. In recent reports on evolution of SARS-CoV-2, three lineage defining non-synonymous mutations; namely D614G in S protein (Clade G), G251V in ORF3a (Clade V) and L84S in ORF 8 (Clade S) were observed [2-4]. The latest pioneering works by Plante et al and Hou et al have shown that compared to ancestral strain, the ubiquitous D614G variant (clade G) of SARS-CoV-2 exhibits efficient replication in upper respiratory tract epithelial cells and transmission, thereby conferring higher fitness [5, 6]. As per latest WHO reports on COVID-19, a new strain referred as SARS-CoV-2 VUI 202012/01 (Variant Under Investigation, year 2020, month 12, variant 01) had been identified as a part of virological and epidemiological analysis, due to sudden rise in COVID-19 detected cases in South-East England [7]. Preliminary reports from UK suggested higher transmissibility (increase by 40-70%) of this strain, escalating Ro (basic reproduction number) of virus to 1.5-1.7 [7, 8]. This apparent fast spreading variant inculcates 23 mutations; 13 non-synonymous, 6 synonymous and 4 amino acid deletions [7]. In the current scenario, where immunization programs have already commenced in nations highly affected by COVID-19, advent of this new strain variant has raised concerns worldwide on its possible role in disease severity and antibody responses. The mutations also could also have significant impact on diagnostic assays owing to S gene target failures.

bioinformatics↗

bioRxiv2020

Evolution of the SARS-CoV-2 proteome in three dimensions (3D) during the first six months of the COVID-19 pandemic

Three-dimensional structures of SARS-CoV-2 and other coronaviral proteins archived in the Protein Data Bank were used to analyze viral proteome evolution during the first six months of the COVID-19 pandemic. Analyses of spatial locations, chemical properties, and structural and energetic impacts of the observed amino acid changes in >48,000 viral proteome sequences showed how each one of the 29 viral study proteins have undergone amino acid changes. Structural models computed for every unique sequence variant revealed that most substitutions map to protein surfaces and boundary layers with a minority affecting hydrophobic cores. Conservative changes were observed more frequently in cores versus boundary layers/surfaces. Active sites and protein-protein interfaces showed modest numbers of substitutions. Energetics calculations showed that the impact of substitutions on the thermodynamic stability of the proteome follows a universal bi-Gaussian distribution. Detailed results are presented for six drug discovery targets and four structural proteins comprising the virion, highlighting substitutions with the potential to impact protein structure, enzyme activity, and functional interfaces. Characterizing the evolution of the virus in three dimensions provides testable insights into viral protein function and should aid in structure-based drug discovery efforts as well as the prospective identification of amino acid substitutions with potential for drug resistance.

molecular biology↗

bioRxiv2020

Swi6/HP1 binding to RNA-DNA hybrids initiates heterochromatin assembly at the centromeric dg-dh repeats in Fission Yeast

Canonically, heterochromatin formation in fission yeast and metazoans is initiated by di/trimethylation of histone H3 at lysine 9 position by the histone methyltransferase Suv39H1/Clr4, followed by binding of Swi6/HP1 to H3-K9-me2/me3 via its chromodomain. Subsequent self-association of Swi6/HP1 on adjacent nucleosomes or a cooperative interaction between Clr4 and Swi6/HP1 leads to folded heterochromatin structure. HP1 binding to RNA is shown to facilitate its localization at and assembly of heterochromatin in metazoans. Likewise, recruitment of Swi6/HP1 to centromere depends on the RNAi pathway in fission yeast; paradoxically, Swi6/HP1 is also thought to play a role in RNA turnover. Here we provide evidence in support of RNAi-independent recruitment of Swi6. We show that, apart from the low affinity binding to RNAs through its hinge domain, as already reported, Swi6/HP1 displays a hierarchy of increasing binding affinity through its chromodomain to the siRNAs corresponding to specific dg-dh repeats and even stronger binding to the cognate siRNA-DNA hybrids than to the siRNA precursors or general RNAs. Our results support a mechanism of recruitment of Swi6, which is dependent on its specific and high affinity binding to siRNA-DNA hybrid at the dg-dh repeats. This binding, which is independent of, albeit augmented by binding to H3-K9-Me2, leads to heterochromatin formation and silencing. We suggest that the net role of Swi6 in RNA physiology may be regulated by a balance between abundance and affinity of Swi6 towards heterochromatic and euchromatic RNAs and siRNAs.

genetics↗

bioRxiv2020

Heterochromatin Replication: Direct Interaction of DNA replication machinery with heterochromatin code writer Clr4/Suv39 and reader Swi6/HP1 in S. pombe

The establishment of heterochromatin in fission yeast involves methyltransferase Clr4-mediated H3-Lys9 methylation, which is bound specifically by Swi6/HP1. However, the mechanism of propagation of heterochromatin through multiple cell divisions is not known. A role of DNA replication in propagating the heterochromatin is envisaged. Studies in S. pombe have indicated a direct interaction between DNA Pol and Swi6/HP1 and between DNA Pol{varepsilon} and Rik1-Dos2 complex, suggesting a coupling between DNA replication and heterochromatin assembly. Here, we show that like DNA Pol, Pol{delta}, which plays a role in both leading and lagging strand replication, also plays a role in silencing at mating type and centromere. We show that both the polymerases and {delta} interact directly with both Clr4 and Swi6/HP1. Mutations in both the polymerases lead to decrease in H3-Lys9 methylation and Swi6 at the mating type and left outer repeats of centromeres I and II, with a reciprocal increase in their level at the central element, cnt, at all the three centromeres. These mutations also cause defects in chromosome segregation, recruitment of Cohesin and chromosome dynamics during mitosis and meiosis. Thus, our results indicate that a tight coordination between DNA replication machinery and propagation of the heterochromatin-specific epigenetic mark.

genetics↗

bioRxiv2020

Genomic Similarity of Nucleotides in SARS CoronaVirus using K-Means Unsupervised Learning Algorithm

The drastic increase in the number of coronaviruses discovered and coronavirus genomes being sequenced have given us a great opportunity to perform genomics and bioinformatics analysis on this family of viruses. Coronaviruses possess the largest genomes (26.4 to 31.7 kb) among all known RNA viruses, with G + C contents varying from 32% to 43%. Phylogenetically, three genera, Alphacoronavirus, Betacoronavirus and Gammacoronavirus, with Betacoronavirus consisting of subgroups A, B, C were known to exist but now a new genus D also exists,namely the Deltacoronavirus. In such a situation, it becomes highly important for efficient classification of all virus data so that it helps us in suitable planning,containment and treatment. The objective of this paper is to classify SARS corona-virus nucleotide sequences based on parameters such as sequence length,percentage similarity between the sequence information,open and closed gaps in the sequence due to multiple mutations and many others.By doing this,we will be able to predict accurately the similarity of SARS CoV-2 virus with respect to other corona-viruses like the Wuhan corona-virus,the bat corona-virus and the pneumonia virus and would help us better understand about the taxonomy of the corona-virus family. SUMMARYIn addition to the guidelines provided in the abstract above,the following points summarizes the article below: O_LIThe article discusses an application of Machine Learning in the field of virology. C_LIO_LIIt aims to classify the SARS CoV2 virus as per the already known sequences of the bat-coronavirus, the Wuhan Sea Food Market pneumonia virus and the Wuhan coronavirus. C_LIO_LITo solve and predict the similarity of the SARS CoV2 coronavirus w.r.t other viruses discussed above,K-Means Unsupervised Learning Algorithm has been chosen. C_LIO_LIThe data-set used is MN997409.1-4NY0T82X016-Alignment-HitTable.csv found on www.kaggle.com.(Complete link shared in the references section).[17] C_LIO_LIThe results have been validated by using a simple data-correlation technique namely Spearmans Rank Correlation Coeffecient. C_LIO_LII have also discussed my future work using Deep Neural Nets that can help predict new virus sequences and effectively find similarity if any with already discovered viruses. C_LI

molecular biology↗

bioRxiv2020

Mutational signatures in countries affected by SARS-CoV-2: Implications in host-pathogen interactome

We are in the midst of the third severe coronavirus outbreak caused by SARS-CoV-2 with unprecedented health and socio-economic consequences due to the COVID-19. Globally, the major thrust of scientific efforts has shifted to the design of potent vaccine and anti-viral candidates. Earlier genome analyses have shown global dominance of some mutations purportedly indicative of similar infectivity and transmissibility of SARS-CoV-2 worldwide. Using high-quality large dataset of 25k whole-genome sequences, we show emergence of new cluster of mutations as result of geographic evolution of SARS-CoV-2 in local population ([≥]10%) of different nations. Using statistical analysis, we observe that these mutations have either significantly co-occurred in globally dominant strains or have shown mutual exclusivity in other cases. These mutations potentially modulate structural stability of proteins, some of which forms part of SARS-CoV-2-human interactome. The high confidence druggable host proteins are also up-regulated during SARS-CoV-2 infection. Mutations occurring in potential hot-spot regions within likely T-cell and B-cell epitopes or in proteins as part of host-viral interactome, could hamper vaccine or drug efficacy in local population. Overall, our study provides comprehensive view of emerging geo-clonal mutations which would aid researchers to understand and develop effective countermeasures in the current crisis. SignificanceOur comparative analysis of globally dominant mutations and region-specific mutations in 25k SARS-CoV-2 genomes elucidates its geo-clonal evolution. We observe locally dominant mutations (co-occurring or mutually exclusive) in nations with contrasting COVID-19 mortalities per million of population) besides globally dominant ones namely, P314L (ORF1b) and D164G (S) type. We also see exclusive dominant mutations such as in Brazil (I33T in ORF6 and I292T in N protein), England (G251V in ORF3a), India (T2016K and L3606F in ORF1a) and in Spain (L84S in ORF8). The emergence of these local mutations in ORFs within SARS-CoV-2 genome could have interventional implications and also points towards their potential in modulating infectivity of SARS-CoV-2 in regional population.

bioinformatics↗

bioRxiv2020

Assembly of a double-stranded RNA synthesizing complex: RNA-DEPENDENT RNA POLYMERASE 2 docks with NUCLEAR RNA POLYMERASE IV at the clamp domain

In plants, transcription of selfish genetic elements such as transposons and DNA viruses is suppressed by RNA-directed DNA methylation. This process is guided by 24 nt short-interfering RNAs (siRNAs) whose double-stranded precursors are synthesized by DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). Pol IV and RDR2 co-immunoprecipitate, and their activities are tightly coupled, yet the basis for their association is unknown. Here, we show that RDR2 stably associates with Pol IVs largest catalytic subunit, NRPD1 at three sites, all within the clamp module. The clamp is a ubiquitous feature of DNA-dependent RNA polymerases that opens to allow DNA template entry and closes to encase the DNA-RNA hybrid adjacent to the RNA exit channel. The clamp also provides binding sites for polymerase-specific subunits or regulatory proteins, thus RDR2 binding to the Pol IV clamp is consistent with this theme. Within RDR2, the site of interaction with NRPD1 is very near the catalytic center. The locations of the NRPD1-RDR2 contact sites suggest a model in which transcripts emanating from Pol IVs RNA exit channel align with the template cleft of RDR2, facilitating rapid conversion of terminated Pol IV transcripts into double-stranded RNAs. Significance StatementShort interfering RNAs (siRNAs) play important roles in gene regulation by inhibiting mRNA translation into proteins or by guiding chromatin modifications that inhibit gene transcription. In plants, transcriptional gene silencing is guided by siRNAs derived from double-stranded (ds) RNAs generated by coupling the activities of DNA-dependent NUCLEAR RNA POLYMERASE IV and RNA-DEPENDENT RNA POLYMERASE 2. We show that the physical basis for Pol IV-RDR2 coupling is RDR2 binding to the clamp domain of Pol IVs largest subunit. The positions of the protein docking sites suggest that nascent Pol IV transcripts are generated in close proximity to RDR2s catalytic site, enabling rapid conversion of Pol IV transcripts into dsRNAs.

plant biology↗

bioRxiv2020

PhenoImage: an open-source GUI for plant image analysis

High-throughput genotyping coupled with molecular breeding approaches has dramatically accelerated crop improvement programs. More recently, improved plant phenotyping methods have led to a shift from manual measurements to automated platforms with increased scalability and resolution. Considerable effort has also gone into the development of large-scale downstream processing of the imaging datasets derived from high-throughput phenotyping (HTP) platforms. However, most available tools require some programing skills. We developed PhenoImage - an open-source GUI based cross-platform solution for HTP image processing with the aim to make image analysis accessible to users with either little or no programming skills. The open-source nature provides the possibility to extend its usability to meet user-specific requirements. The availability of multiple functions and filtering parameters provides flexibility to analyze images from a wide variety of plant species and platforms. PhenoImage can be run on a personal computer as well as on high-performance computing clusters. To test the efficacy of the application, we analyzed the LemnaTec Imaging system derived RGB and fluorescence shoot images from two plant species: sorghum and wheat differing in their physical attributes. In the study, we discuss the development, implementation, and working of the PhenoImage. HighlightPhenoImage is an open-source application designed for analyzing images derived from high-throughput phenotyping.

plant biology↗