Biology subjects

Jaworski, E.

Publications and source records attributed to Jaworski, E..

8 recordsLinked to original sources

bioRxiv2022

Flies adaptively control flight to compensate for added inertia

Animal locomotion is highly adaptive, displaying a large degree of flexibility, yet how this flexibility arises from the integration of mechanics, sensing and neural control remains elusive. For instance, animals require flexible strategies to maintain performance as changes in mass or inertia impact stability. Compensatory strategies to mechanical loading are especially critical for animals that rely on flight for survival. To shed light on the capacity and flexibility of flight neuromechanics to mechanical loading, we pushed the performance of fruit flies (Drosophila) near its limit and implemented a control theoretic framework to quantify how flies compensated for added inertia. Flies with added inertia were placed inside a virtual reality arena which enabled free rotation about the vertical (yaw) axis. Adding inertia increased the flys response time yet had little influence on overall gaze performance. Flies maintained stability following the addition of inertia by adaptively modulating both visuomotor gain and damping. In contrast, mathematical modeling predicted a significant decrease in flight stability and performance. Adding inertia altered saccades, however flies compensated for the added inertia by increasing yaw torque production, indicating that flies sense that they are mechanically loaded. Taken together, in response to added inertia flies trade off reaction time to maintain flight performance through adaptive neural modulation. Our work highlights the flexibility and capacity of motor control in flight.

biophysics↗

bioRxiv2022

SARS-CoV-2 Uses Nonstructural Protein 16 to Evade Restriction by IFIT1 and IFIT3

Understanding the molecular basis of innate immune evasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important consideration for designing the next wave of therapeutics. Here, we investigate the role of the nonstructural protein 16 (NSP16) of SARS-CoV-2 in infection and pathogenesis. NSP16, a ribonucleoside 2-O methyltransferase (MTase), catalyzes the transfer of a methyl group to mRNA as part of the capping process. Based on observations with other CoVs, we hypothesized that NSP16 2-O MTase function protects SARS-CoV-2 from cap-sensing host restriction. Therefore, we engineered SARS-CoV-2 with a mutation that disrupts a conserved residue in the active site of NSP16. We subsequently show that this mutant is attenuated both in vitro and in vivo, using a hamster model of SARS-CoV-2 infection. Mechanistically, we confirm that the NSP16 mutant is more sensitive to type I interferon (IFN-I) in vitro. Furthermore, silencing IFIT1 or IFIT3, IFN-stimulated genes that sense a lack of 2-O methylation, partially restores fitness to the NSP16 mutant. Finally, we demonstrate that sinefungin, a methyltransferase inhibitor that binds the catalytic site of NSP16, sensitizes wild-type SARS-CoV-2 to IFN-I treatment. Overall, our findings highlight the importance of SARS-CoV-2 NSP16 in evading host innate immunity and suggest a possible target for future antiviral therapies. ImportanceSimilar to other coronaviruses, disruption of SARS-CoV-2 NSP16 function attenuates viral replication in a type I interferon-dependent manner. In vivo, our results show reduced disease and viral replication at late times in the hamster lung, but an earlier titer deficit for the NSP16 mutant (dNSP16) in the upper airway. In addition, our results confirm a role for IFIT1, but also demonstrate the necessity of IFIT3 in mediating dNSP16 attenuation. Finally, we show that targeting NSP16 activity with a 2-O methyltransferase inhibitor in combination with type I interferon offers a novel avenue for antiviral development.

microbiology↗

bioRxiv2021

Co-variation of viral recombination with single nucleotide variants during virus evolution revealed by CoVaMa

Adaptation of viruses to their environments occurs through the acquisition of both novel Single-Nucleotide Variants (SNV) and recombination events including insertions, deletions, and duplications. The co-occurrence of SNVs in individual viral genomes during their evolution has been well-described. However, unlike covariation of SNVs, studying the correlation between recombination events with each other or with SNVs has been hampered by their inherent genetic complexity and a lack of bioinformatic tools. Here, we expanded our previously reported CoVaMa pipeline (v0.1) to measure linkage disequilibrium between recombination events and SNVs within both short-read and long-read sequencing datasets. We demonstrate this approach using long-read nanopore sequencing data acquired from Flock House virus (FHV) serially passaged in vitro. We found SNVs that were either correlated or anti-correlated with large genomic deletions generated by nonhomologous recombination that give rise to Defective-RNAs. We also analyzed NGS data from longitudinal HIV samples derived from a patient undergoing antiretroviral therapy who proceeded to virological failure. We found correlations between insertions in the p6Gag and mutations in Gag cleavage sites. This report confirms previous findings and provides insights on novel associations between SNVs and specific recombination events within the viral genome and their role in viral evolution.

genetics↗

bioRxiv2021

Tau modulates mRNA transcription, alternative polyadenylation profiles of hnRNPs, chromatin remodeling and spliceosome complexes

Tau protein is a known contributor in several neurodegenerative diseases, including Alzheimer s disease (AD) and frontotemporal dementia (FTD). It is well-established that tau forms pathological aggregates and fibrils in these diseases. Tau has been observed within the nuclei of neurons, but there is a gap in understanding regarding the mechanism by which tau modulates transcription. We are interested in the P301L mutation of tau, which has been associated with FTD and increased tau aggregation. Our study utilized tau-inducible HEK (iHEK) cells to reveal that WT and P301L tau distinctively alter the transcription and alternative polyadenylation (APA) profiles of numerous nuclear precursors mRNAs, which then translate to form proteins involved in chromatin remodeling and splicing. We isolated total mRNA before and after over-expressing tau and then performed Poly(A)-ClickSeq (PAC-Seq) to characterize mRNA expression and APA profiles. We characterized changes in Gene Ontology (GO) pathways using EnrichR and Gene Set Enrichment Analysis (GSEA). We observed that P301L tau up-regulates genes associated with reactive oxygen species responsiveness as well as genes involved in dendrite, microtubule, and nuclear body/speckle formation. The number of genes regulated by WT tau is greater than the mutant form, which indicates that the P301L mutation causes loss-of-function at the transcriptional level. WT tau up-regulates genes contributing to cytoskeleton-dependent intracellular transport, microglial activation, microtubule and nuclear chromatin organization, formation of nuclear bodies and speckles. Interestingly, both WT and P301L tau commonly down-regulate genes responsible for ubiquitin-proteosome system. In addition, WT tau significantly down-regulates several genes implicated in chromatin remodeling and nucleosome organization. Although there are limitations inherent to the model systems used, this study will improve understanding regarding the nuclear impact of tau at the transcriptional and post-transcriptional level. This study also illustrates the potential impact of P301L tau on the human brain genome during early phases of pathogenesis. Author summaryWhile tau biology has been extensively studied and closely linked to several neurodegenerative diseases, our current understanding of taus functions in the nucleus is limited. Given the role of tau in disease progression and pathogenesis, elucidating the function of tau activity in transcription and its nuclear accumulation may reveal novel therapeutic targets; therefore, helping identify new upstream pathways that have yet to be investigated. In this study, we used tau-inducible cell lines to uncover new molecular mechanisms by which tau functions in the nucleus. This study systematically investigates the changes in transcriptomic and alternative polyadenylation profiles modulated by WT and mutant P301L tau protein. In this manuscript, we report following new findings (i) tau modulates gene expression of transcripts associated with chromatin remodeling and splicing complexes; (ii) WT and mutant P301L tau regulate, differentially, transcription and alternative polyadenylation (APA) profiles; and (iii) P301L mutation affects the transcription mediated by tau protein. The potential role of tau in mediating transcription and alternative polyadenylation processes is not well studied, representing a novelty in the field. Therefore, this research establishes a new direction for investigating tau nuclear function in both human and mouse brains.

neuroscience↗

bioRxiv2021

Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for Next-Generation Sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called Tiled-ClickSeq, which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

molecular biology↗

bioRxiv2020

RBFOX2 Is Critical For Maintaining Alternative Polyadenylation And Mitochondrial Health In Myoblasts

RBFOX2, which has a well-established role in alternative splicing, is linked to heart diseases. However, it is unclear whether RBFOX2 has other roles in RNA processing that can influence gene expression/function in muscle cells, contributing to disease pathology. Here, we employed both 3-end and nanopore cDNA sequencing to reveal a previously unrecognized role for RBFOX2 in maintaining alternative polyadenylation (APA) signatures in myoblasts. We found that RBFOX2-mediated APA modulates both mRNA levels and isoform expression of a collection of genes including contractile and mitochondrial genes. We identified the key muscle-specific contractile gene, Tropomyosin 1 and essential mitochondrial gene, Slc25a4 as APA targets of RBFOX2. Unexpectedly, depletion of RBFOX2 adversely affected mitochondrial health in myoblasts that is in part mediated by disrupted APA of mitochondrial gene Slc25a4. Mechanistically, we found that RBFOX2 regulation of Slc25a4 APA is mediated through consensus RBFOX2 binding motifs near the distal polyadenylation site enforcing the use of the proximal polyadenylation site. In sum, our results unveiled a new role for RBFOX2 in fine tuning expression levels of mitochondrial and contractile genes via APA in myoblasts relevant to heart diseases.

molecular biology↗

bioRxiv2020

Differential alphavirus defective RNA diversity between intracellular and encapsidated compartments is driven by subgenomic recombination events

Alphaviruses are positive-sense RNA arboviruses that can cause either a chronic arthritis or a potentially lethal encephalitis. Like other RNA viruses, alphaviruses produce truncated, defective genomes featuring large deletions during replication. Defective RNAs (D-RNAs) have primarily been isolated from virions after high-multiplicity of infection passaging. Here, we aimed to characterize both intracellular and packaged viral D-RNA populations during early passage infections under the hypothesis that D-RNAs arise de novo intracellularly that may not be packaged and thus have remained undetected. To this end, we generated NGS libraries using RNA derived from passage 1 (P1) stock chikungunya virus (CHIKV) 181/clone 25, intracellular virus, and encapsidated P2 virus and analyzed samples for D-RNA expression, followed by diversity and differential expression analyses. We found that the diversity of D-RNA species is significantly higher for intracellular D-RNA populations than encapsidated and specific populations of D-RNAs are differentially expressed between intracellular and encapsidated compartments. Importantly, these trends were likewise observed in a murine model of CHIKV 15561 infection, as well as in vitro studies using related Mayaro, Sindbis, and Aura viruses. Additionally, we identified a novel subtype of subgenomic D-RNA that are conserved across arthritogenic alphaviruses. D-RNAs specific to intracellular populations were defined by recombination events specifically in the subgenomic region, which was confirmed by direct RNA nanopore sequencing of intracellular CHIKV RNAs. Together, these studies show that only a portion of D-RNAs generated intracellularly are packaged and D-RNAs readily arise de novo in the absence of transmitted template. IMPORTANCEOur understanding of viral defective RNAs (D-RNAs), or truncated viral genomes, comes largely from passaging studies in tissue culture under artificial conditions and/or packaged viral RNAs. Here, we show that specific populations of alphavirus D-RNAs arise de novo and that they are not packaged into virions, thus imposing a transmission bottleneck and impeding their prior detection. This raises important questions about the roles of D-RNAs, both in nature and in tissue culture, during viral infection and whether their influence is constrained by packaging requirements. Further, during the course of these studies, we found a novel type of alphavirus D-RNA that is enriched intracellularly; dubbed subgenomic D-RNAs (sgD-RNAs), they are defined by deletion boundaries between capsid/E3 and E1/3UTR regions and are common to chikungunya, Mayaro, Sindbis, and Aura viruses. These sgD-RNAs are enriched intracellularly and do not appear to be selectively packaged, and additionally may exist as subgenome-derived transcripts.

molecular biology↗

bioRxiv2017

Poly(A)-ClickSeq: click-chemistry for next-generation 3´-end sequencing without RNA enrichment or fragmentation

The recent emergence of alternative polyadenylation (APA) as an engine driving transcriptomic diversity has stimulated the development of sequencing methodologies designed to assess genome-wide polyadenylation events. The goal of these approaches is to enrich, partition, capture, and ultimately sequence poly(A) site junctions. However, these methods often require poly(A) enrichment, 3{acute} linker ligation steps, and RNA fragmentation, which can necessitate higher levels of starting RNA, increase experimental error, and potentially introduce bias. We recently reported a click-chemistry based method for generating RNAseq libraries called \"ClickSeq\". Here, we adapt this method to direct the cDNA synthesis specifically toward the 3{acute} UTR/poly(A) tail junction of cellular RNA. With this novel approach, we demonstrate sensitive and specific enrichment for poly(A) site junctions without the need for complex sample preparation, fragmentation or purification. Poly(A)-ClickSeq (PAC-seq) is therefore a simple procedure that generates high-quality RNA-seq poly(A) libraries. As a proof-of-principle, we utilized PAC-seq to explore the poly(A) landscape of both human and Drosophila cells in culture and observed outstanding overlap with existing poly(A) databases and also identified previously unannotated poly(A) sites. Moreover, we utilize PAC-seq to quantify and analyze APA events regulated by CFIm25 illustrating how this technology can be harnessed to identify alternatively polyadenylated RNA.

molecular biology↗