Biology subjects

Srivastava, M.

Publications and source records attributed to Srivastava, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Identification and developmental profiling of microRNAs in the acoel worm Hofstenia miamia

The acoel worm Hofstenia miamia (H. miamia) has recently emerged as a model organism for studying whole-body regeneration and embryonic development, providing an opportunity to probe post-transcriptional mechanisms in both processes in the same organism. Here, we establish a resource for studying H. miamia microRNA-mediated gene regulation, a major aspect of post-transcriptional control in animals. We generated a PacBio long-read sequencing-based genome assembly. We developed a stringent microRNA annotation framework for H. miamia using small RNA-sequencing samples spanning key developmental stages. Our analysis uncovered 545 microRNA loci, including 154 highest-confidence loci based on structural features, expression levels, and prediction quality metrics. Comparison of microRNA seed sequences with those in other bilaterian species revealed that H. miamia encodes many known conserved bilaterian microRNA families and that several microRNA families previously reported only in protostomes or deuterostomes likely have ancient bilaterian origins. We profiled and characterized the expression dynamics and strand preference of microRNAs in H. miamia embryonic and post-embryonic development. An intron that is spliced in the primary transcript of co-transcribed let-7 and mir-125 microRNAs. To generate hypotheses for microRNA function, we annotated the 3 UTRs of H. miamia protein-coding genes and performed microRNA target site predictions. Focusing on genes that are known to function in the wound response, posterior patterning, and neural differentiation in H. miamia, we found that these processes may be under substantial microRNA regulation. Notably, we found that microRNAs in MIR-7 and MIR-9 families, which have target sites in the posterior genes fz-1, wnt-3, and sp5 are indeed expressed in the anterior of the animal, consistent with an anterior-biased repressive effect on their corresponding target genes. Our annotation provides a resource for future studies of post-transcriptional regulation of gene expression during development and regeneration.

genomics↗

bioRxiv2024

Reproductive life history of an acoel worm

Acoel worms belong to an enigmatic and understudied animal lineage in the phylum Xenacoelomorpha. Sparse taxonomic and histological work suggests that these worms exhibit a diversity of reproductive anatomies and likely a corresponding diversity in reproductive behavior. Here, we study the reproductive life history of the three-banded panther worm Hofstenia miamia, an acoel that is emerging as a lab-tractable model system. Using confocal microscopy and histology, we describe H. miamias reproductive organs, identifying structures previously unknown in acoels. Following a cohort of worms from zygote to adulthood, we quantify the developmental dynamics of their reproductive organs, and find that these organs emerge in a stereotyped sequence as a function of increasing body size. Studying the dynamics of organ growth and de-growth during regeneration and in starvation, we show that reproductive organs follow similar growth rules in these contexts, suggesting that they are regulated by a size-associated program in all growth contexts. Finally, we study egg-laying behavior, finding that H. miamia lay their eggs through their mouths after loading them into their pharynges. Worms lay eggs for multiple months after a single mating, suggesting long-term sperm storage despite lacking a storage organ; we also find that worms can lay viable eggs without mating, indicating a capacity for self-fertilization. Further, we show that worms assess their environment to make decisions about when and where to lay their eggs, and sometimes lay eggs in communal clutches. Together, our work establishes foundational knowledge to enable the experimental study of reproductive anatomy, physiology, and behavior in acoels.

zoology↗

bioRxiv2024

Regeneration recapitulates many embryonic processes, including reuse of developmental regulatory regions

The wide distribution of regenerative capacity across the animal tree of life raises the question of how regeneration has evolved in distantly-related animals. Given that whole-body regeneration shares the same end-point - formation of a functional body plan - as embryonic development, it has been proposed that regeneration likely recapitulates developmental processes to some extent. Therefore, understanding how developmental processes are reactivated during regeneration is important for uncovering the evolutionary history of regeneration. Comparative transcriptomic studies in some species have revealed shared gene expression between development and regeneration, but it is not known whether these shared expression profiles correspond to shared functions, and which mechanisms activate expression of developmental genes during regeneration. We sought to address these questions using the acoel Hofstenia miamia, which is amenable to studies of both embryonic development and whole-body regeneration. By examining functionally validated regeneration processes during development at single-cell resolution, we found that whereas patterning and cellular differentiation are largely similar, wound response programs have distinct dynamics between development and regeneration. Chromatin accessibility analyses revealed that regardless of playing concordant or divergent roles during regeneration and development, genes expressed in both processes are frequently controlled by the same regulatory regions, potentially via utilization of distinct transcription factor binding sites. This study extends the known correspondence of development and regeneration from broad transcriptomic similarity to include patterning and differentiation processes. Further, our work provides a catalog of regulatory regions and binding sites that potentially regulate developmental genes during regeneration, fueling comparative studies of regeneration.

developmental biology↗

bioRxiv2024

Neuronal toxicity and recovery from early bortezomib-induced neuropathy: targeting the blood nerve barrier but not the dorsal root ganglion

The use of the first in class proteasome inhibitor Bortezomib (BTZ) is highly effective in the treatment of multiple myeloma. However, its long-term use is limited by the fact, that most treated patients develop dose limiting painful polyneuropathy. In some of the treated patients, pain resolves after variable timeframes, in others it persists, despite the discontinuation of treatment, with the underlying mechanisms poorly understood. One condition of neural toxicity is the ability to penetrate the blood nerve barrier. Here we present pathways involved in early bortezomib-induced polyneuropathy (BIPN) development and its resolution, in rats and in myeloma patients. One cycle of BTZ elicited transient mechanical hyperalgesia and cold allodynia in rats. Transcriptomic signature and network analysis revealed regulation of circadian, extracellular matrix, and immune genes within the nerve and modest changes in the dorsal root ganglia. Recovery processes resealed the small molecule leakiness of the perineurial barrier, reversed axonal swelling, and normalized small fiber density in the skin. Expression of the microtubule-associated cytoskeletal protein cortactin matched this process in the perineurium. Netrin-1 (Ntn1) as a known barrier sealer was also upregulated in pain resolution in nerve and skin. In patients with painful BIPN skin NTN1 was independent of axonal damage. In summary, our data demonstrate that early BTZ toxicity targets mainly the nerve and indicates that pain resolution could be supported by protective growth factors like Ntn1 for remodeling of the extracellular matrix and neuronal barriers. SummaryBortezomib leads to dose-limiting painful polyneuropathy. Already in the first cycle, BTZ toxicity weakens the blood nerve barrier which reseals upon upregulation of netrin-1.

neuroscience↗

bioRxiv2023

Chromatin profiling data indicate regulatory mechanisms for differentiation during development in the acoel Hofstenia miamia

Chromatin profiling data can corroborate and generate hypotheses for regulatory events that underlie the control of gene expression in any biological process. Here, we applied the Assay for Transposase Accessible Chromatin (ATAC) sequencing to build a catalog of putative regulatory DNA during the process of embryonic development in an acoel. Acoels represent an enigmatic phylum-level lineage of animals, the Xenacoelomorpha, which is placed either as a sister-group to all other animals with bilateral symmetry or as an early-diverging ambulacrarian, two alternative phylogenetic placements that both position acoels equally well to inform the evolution of developmental mechanisms. We focused on the acoel Hofstenia miamia, a.k.a. the three-banded panther worm, which has emerged as a new laboratory research organism for whole-body regeneration that also enables the study of development from zygote to hatching. We profiled chromatin landscapes over a time course encompassing many major morphological events, including gastrulation, axial patterning, and differentiation of tissues such as epidermis and muscle. Broad patterns of chromatin accessibility and predicted binding of various transcription factor (TF) motifs identified major biological processes and their putative regulators, and we noted that differential accessibility tended to precede major developmental transitions in embryogenesis. Focused analysis of TF binding combined with single-cell RNA-seq data provided regulatory linkages for genes in a previously hypothesized differentiation trajectory for epidermis and generated new hypotheses for gene regulatory networks associated with the formation of muscle. This work provides a platform for the identification of developmental mechanisms in Hofstenia and enables comparisons of embryogenesis in acoels to other animals as well as comparisons of embryogenesis to regeneration.

developmental biology↗

bioRxiv2023

PCP components control anterior and posterior regeneration, with a Prickle homolog impacting muscle organization, in the acoel Hofstenia miamia

Whole-body regeneration requires wound response signals to control patterning programs to enable replacement of structures in their correct locations. While a number of molecular mechanisms underlying anterior-posterior regeneration have been identified, how small fragments of animals first re-establish polarity is less well understood, with non-canonical Wnt signaling recently emerging as a potential regulator. Here, we used the acoel worm Hofstenia miamia, a new research organism capable of robust whole-body regeneration, to assess functions of the components of the Planar Cell Polarity (PCP) pathway in establishing regeneration polarity. We identified homologs of Prickle (pk-1) and Diego (dgo-1) to be required for head and tail regeneration, respectively. RNA-sequencing analysis and experimental corroboration revealed that pk-1 RNAi resulted in diminished expression of early wound response genes as well as of wound-induced expression of the anterior-specific marker fz-7, specifically in tail fragments. In contrast, dgo-1 RNAi impacted wound-induced expression of the posterior-specific marker tf7l2, specifically in head fragments. Furthermore, pk-1 and dgo-1 are enriched in longitudinal muscle, with muscle fibers showing disorganized morphology at anterior-facing wound sites of tail fragments under pk-1 RNAi. These findings suggest that pk-1 and dgo-1 are needed for wound-induced expression of anterior- and posterior-specific genes, and raise the possibility that this action is mediated via the control of muscle fiber orientation. Our work expands the study of PCP genes by revealing their functions in the process of whole-body regeneration in acoels, the sister-group to all other animals with bilateral symmetry, and will enable future studies of PCP components in controlling cellular and tissue-wide regeneration polarity.

developmental biology↗

bioRxiv2023

SARS-CoV-2 Nsp13 is a viral RHIM protein promoting cell death linked to Z-RNA sensing and ZBP1-RIPK3 signaling

RHIM is a protein motif in cell death proteins that assembles higher-order signaling complexes and triggers regulated cell death, which in itself limits virus spread and additionally triggers inflammation for mounting immune responses. A few DNA viruses employ viral RHIMs mimicking host RHIMs. However, these viral RHIMs counteract host cell death by interacting with host RHIM proteins and blocking complex formation to alleviate antiviral defenses. Whether RNA viruses operate such viral RHIMs remains unknown. RHIM-protein signaling promotes lung damage and cytokine storm in respiratory RNA virus infections, arguing the presence of viral RHIMs. Here, we report the novel viral RHIMs in Nsp13 and Nsp14 of SARS-CoV-2 and other bat RNA viruses, providing the basis for bats as the hosts for their evolution. Nsp13 promoted cell death in bat and human cells, however, viral RHIM of Nsp13 is more critical for human cell death than bat cells, suggesting species-specific regulation. The conformation of RNA-binding channel in Nsp13 is critical for cell death in bat and human cells. Nsp13 showed RHIM-dependent interactions with ZBP1 and RIPK3 and promoted the formation of large insoluble complexes of ZBP1 and RIPK3. Also, Nsp13 promoted ZBP1-RIPK3 signaling-mediated cell death dependent on intracellular RNA ligands. Intriguingly, the SARS-CoV-2 genome consists of bona fide Z-RNA-forming segments. These SARS-CoV-2 Z-RNA segments promoted Nsp13-dependent cell death, further revealing Nsp13s association with Z-RNA sensing and ZBP1-RIPK3 signaling. Our findings reveal the functional viral RHIMs of bat-originated RNA viruses regulating host cell death associated with Z-RNA sensing and ZBP1-RIPK3 signaling activation. These observations allow the understanding of mechanisms of cellular damage and cytokine storm in SARS-CoV-2 and other bat-originated RNA virus infections. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/554434v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@f71c5aorg.highwire.dtl.DTLVardef@1666aa5org.highwire.dtl.DTLVardef@e37d82org.highwire.dtl.DTLVardef@145020f_HPS_FORMAT_FIGEXP M_FIG C_FIG One-sentence summaryBat-associated RNA viruses employ viral RHIMs and regulate host cell death.

immunology↗

bioRxiv2023

Cross-site reproducibility of human cortical organoids reveals consistent cell type composition and architecture

BackgroundReproducibility of human cortical organoid (hCO) phenotypes remains a concern for modeling neurodevelopmental disorders. While guided hCO protocols reproducibly generate cortical cell types in multiple cell lines at one site, variability across sites using a harmonized protocol has not yet been evaluated. We present an hCO cross-site reproducibility study examining multiple phenotypes. MethodsThree independent research groups generated hCOs from one induced pluripotent stem cell (iPSC) line using a harmonized miniaturized spinning bioreactor protocol. scRNA-seq, 3D fluorescent imaging, phase contrast imaging, qPCR, and flow cytometry were used to characterize the 3 month differentiations across sites. ResultsIn all sites, hCOs were mostly cortical progenitor and neuronal cell types in reproducible proportions with moderate to high fidelity to the in vivo brain that were consistently organized in cortical wall-like buds. Cross-site differences were detected in hCO size and morphology. Differential gene expression showed differences in metabolism and cellular stress across sites. Although iPSC culture conditions were consistent and iPSCs remained undifferentiated, primed stem cell marker expression prior to differentiation correlated with cell type proportions in hCOs. ConclusionsWe identified hCO phenotypes that are reproducible across sites using a harmonized differentiation protocol. Previously described limitations of hCO models were also reproduced including off-target differentiations, necrotic cores, and cellular stress. Improving our understanding of how stem cell states influence early hCO cell types may increase reliability of hCO differentiations. Cross-site reproducibility of hCO cell type proportions and organization lays the foundation for future collaborative prospective meta-analytic studies modeling neurodevelopmental disorders in hCOs.

neuroscience↗

bioRxiv2023

A wound-induced differentiation trajectory for neurons

Animals capable of whole-body regeneration can replace any missing cell type and regenerate fully-functional new organs, de novo. The regeneration of a new brain requires the formation of diverse neuronal cell types and their assembly into an organized structure and correctly-wired circuits. Recent work in various regenerative animals has revealed transcriptional programs required for the differentiation of distinct neuronal subpopulations, however how these transcriptional programs are initiated upon amputation remains unknown. Here, we focused on the highly regenerative acoel worm, Hofstenia miamia, to study wound-induced transcriptional regulatory events that lead to the production of neurons. Footprinting analysis using chromatin accessibility data on an improved genome assembly revealed that binding sites for the NFY transcription factor complex were significantly bound during regeneration, showing a dynamic increase in binding within one hour upon amputation specifically in tail fragments, which will regenerate a new brain. Strikingly, NFY targets were highly enriched for genes with neuronal functional. Single-cell transcriptome analysis combined with functional studies identified sox4+stem cells as the likely progenitor population for multiple neuronal subtypes. Further, we found that wound-induced sox4 expression is likely under direct transcriptional control by NFY, uncovering a mechanism for how early wound-induced binding of a transcriptional regulator results in the initiation of a neuronal differentiation pathway. HighlightsO_LIA new chromosome-scale assembly for Hofstenia enables comprehensive analysis of transcription factor binding during regeneration C_LIO_LINFY motifs become dynamically bound by 1hpa in regenerating tail fragments, particularly in the loci of neural genes C_LIO_LIA sox4+ neural-specialized stem cell is identified using scRNA-seq C_LIO_LIsox4 is wound-induced and required for differentiation of multiple neural cell types C_LIO_LINFY regulates wound-induced expression of sox4 during regeneration C_LI

developmental biology↗

bioRxiv2023

Derivation of trophoblast stem cells unveils unrestrained potential of mouse ESCs and epiblast

mESCs and epiblast are considered to follow strict lineage adherence and lack the potential to contribute to trophoectoderm. Here, we report the derivation of trophoblast stem cells (ESTS) from the mESCs. The single-cell transcriptome and molecular characterization of ESTS show similarity with TSCs. They efficiently integrate into the TE compartment of the blastocyst and contribute to the placenta during development. We discovered GSK3{beta} as a critical regulator of the TE fate of ESCs. It plays a vital stage-specific role during ESTS derivation. We further show {beta}-CATENIN and an intron-I regulatory element of Cdx2 are essential for the TE fate of ESCs. We further show that the mouse epiblast can readily differentiate into TE lineage. In contrast to the paradigm of the restricted potential of pluripotent ESCs and epiblast, our data shows that murine ESCs and epiblast have the unrestrained developmental potential for extraembryonic lineages.

developmental biology↗

bioRxiv2022

Soluble VE-cadherin disrupts endothelial barrier function via VE-PTP/RhoA signalling

AimIncreased levels of soluble Vascular endothelial (VE)-cadherin fragments (sVE-cadherin) have previously been linked with inflammation-induced loss of endothelial barrier function. We tested whether sVE-cadherin is critically involved in the onset of endothelial barrier dysfunction. Methods and ResultsApplication of recombinant human sVE-cadherin (extracellular domains EC1-5) on human microvascular endothelial cells in vitro and in a rat model in vivo induced loss of endothelial barrier function and reduced microcirculatory flow. sVE-cadherinEC1-5 led to decreased localization of VE-cadherin at cell borders. Additionally, sVE-cadherinEC1-5 perturbed VE-protein tyrosine phosphatase (VE-PTP)/VE-cadherin interaction. VE-PTP inhibitor AKB9778 blunted all sVE-cadherinEC1-5-induced effects in vitro and in vivo. Downstream effects involve VE-PTP-dependent RhoA activation which was attenuated by AKB9778. Rho-kinase inhibitor Y27632 blocked sVE-cadherinEC1-5-induced loss of endothelial barrier function. ConclusionsVE-cadherin disrupts endothelial barrier function by dismantling the VE-cadherin complex at cell borders via VE-PTP-dependent RhoA activation. This uncovers a novel pathophysiological role of sVE-cadherin in the context of endothelial barrier dysfunction in inflammation.

physiology↗

bioRxiv2022

Modeling and Simulation of Rat Non-Barrel Somatosensory Cortex. Part I: Modeling Anatomy

The function of the neocortex is fundamentally determined by its repeating microcircuit motif, but also by its rich, interregional connectivity. We present a data-driven computational model of the anatomy of non-barrel primary somatosensory cortex of juvenile rat, integrating whole-brain scale data while providing cellular and subcellular specificity. The model consists of 4.2 million morphologically detailed neurons, placed in a digital brain atlas. They are connected by 14.2 billion synapses, comprising local, mid-range and extrinsic connectivity. We delineated the limits of determining connectivity from neuron morphology and placement, finding that it reproduces targeting by Sst+ neurons, but requires additional specificity to reproduce targeting by PV+ and VIP+ interneurons. Globally, connectivity was characterized by local clusters tied together through hub neurons in layer 5, demonstrating how local and interegional connectivity are complicit, inseparable networks. The model is suitable for simulation-based studies, and a 211,712 neuron subvolume is made openly available to the community.

neuroscience↗

bioRxiv2022

Visualization of Retroplacental Clear Space Disruption in a Mouse Model of Placental Accreta

IntroductionPrior preclinical studies established the utility of liposomal nanoparticle blood-pool contrast agents in visualizing the retroplacental clear space (RPCS), a marker of normal placentation, while sparing fetuses from exposure because the agent does not cross the placental barrier. In this work we characterized RPCS disruption in a mouse model of placenta accreta spectrum (PAS) using these agents. MethodsContrast-enhanced MRI (CE-MRI) and computed tomography (CE-CT) using liposomal nanoparticles bearing gadolinium (liposomal-Gd) and iodine were performed in pregnant Gab3-/- and wild type (WT) mice at day 16 of gestation. CE-MRI was performed on a 1T scanner using a 2D T1-weighted sequence (100x100x600 {micro}m3 voxels) and CE-CT was performed at a higher resolution (70x70x70 {micro}m3 voxels). Animals were euthanized post-imaging and feto-placental units (FPUs) were harvested for histological examination. RPCS conspicuity was scored through blinded assessment of images. ResultsPregnant Gab3-/- mice show elevated rates of complicated pregnancy. Contrast-enhanced imaging demonstrated frank infiltration of the RPCS of Gab3-/- FPUs. RPCS in Gab3-/- FPUs was smaller in volume, demonstrated a heterogeneous signal profile, and received lower conspicuity scores than WT FPUs. Histology confirmed in vivo findings and demonstrated staining consistent with a thinner RPCS in Gab3-/- FPUs. DiscussionImaging of the Gab3-/- mouse model at late gestation with liposomal contrast agents enabled in vivo characterization of morphological differences in the RPCS that could cause the observed pregnancy complications. An MRI-based method for visualizing the RPCS would be valuable for early detection of invasive placentation.

developmental biology↗

bioRxiv2022

Acoel single-cell atlas reveals expression dynamics and heterogeneity of a pluripotent stem cell population

Pluripotent adult stem cell populations underlie whole-body regeneration in many distantly related animal lineages. These collectively pluripotent populations of cells share some features across species, such as the expression of piwi and other germline-related genes. Studies of how these cells operate during regeneration are needed in diverse systems to determine how underlying cellular and molecular mechanisms of renewal and differentiation compare. Here, we sought to characterize stem cells and their dynamics in the acoel Hofstenia miamia, a highly regenerative marine worm with a piwi-expressing stem cell population called neoblasts. Transcriptome profiling at single cell resolution revealed cell types shared across postembryonic stages, including stem cells and differentiated cell types such as neural, epidermal, muscle, and digestive cells. Reconstruction of single-cell differentiation trajectories followed by functional studies confirmed that neoblasts are the source of differentiated cells and identified transcription factors needed for the formation of major cell types. Next, analysis of single-cell transcriptomes from regenerating worms showed that both differentiated cells and stem cells dynamically alter gene expression in response to amputation. Further analysis of the stem cells recovered subpopulations of neoblasts, each with specific transcriptional profiles suggesting that the majority of neoblasts are specialized to differentiated lineages, reflecting putatively lineage-primed progenitors. Notably, neoblast subsets in Hofstenia were identifiable consistently across postembryonic stages and also displayed differential expression dynamics in response to wounding. Altogether, these data suggest that whole-body regeneration is accomplished by the coordination of cells with distinct and dynamic transcriptomic profiles through time. Furthermore, the data generated here will enable the study of how this coordination is achieved, enhancing our understanding of pluripotent stem cells and their evolution across metazoans.

developmental biology↗

bioRxiv2022

The transformability of genotype-phenotype landscapes

The mapping from genotype to phenotype to fitness typically involves multiple nonlinearities that can transform the effects of mutations. For example, mutations may contribute additively to a phenotype, but their effects on fitness may combine non-additively because selection favors a low or intermediate value of that phenotype. This can cause incongruence between the topographical properties of a fitness landscape and its underlying genotype-phenotype landscape. Yet, genotype-phenotype landscapes are often used as a proxy for fitness landscapes to study the dynamics and predictability of evolution. Here, we use theoretical models and empirical data on transcription factor-DNA interactions to systematically study the incongruence of genotype-phenotype and fitness landscapes when selection favors a low or intermediate phenotypic value. Using the theoretical models, we prove a number of fundamental results. For example, selection for low or intermediate phenotypic values does not change simple sign epistasis into reciprocal sign epistasis, yet it changes reciprocal sign epistasis into simple sign epistasis and no sign epistasis with equal probability. More broadly, we show that such selection tends to create fitness landscapes that are more rugged than the underlying genotype-phenotype landscape, but this increased ruggedness typically does not frustrate adaptive evolution because the local adaptive peaks in the fitness landscape tend to be nearly as tall as the global peak. Many of these results carry forward to the empirical genotype-phenotype landscapes, which may help to explain why low- and intermediate-affinity transcription factor-DNA interactions are so prevalent in eukaryotic gene regulation. AUTHOR SUMMARYHow do mutations change phenotypic traits and organismal fitness? This question is often addressed in the context of a classic metaphor of evolutionary theory -- the fitness landscape. A fitness landscape is akin to a physical landscape, in which genotypes define spatial coordinates, and fitness defines the elevation of each coordinate. Evolution then acts like a hill-climbing process, in which populations ascend fitness peaks as a consequence of mutation and selection. It is becoming increasingly common to construct such landscapes using experimental data from high-throughput sequencing technologies and phenotypic assays, in systems such as macromolecules and gene regulatory circuits. Although these landscapes are typically defined by molecular phenotypes, and are therefore more appropriately referred to as genotype-phenotype landscapes, they are often used to study evolutionary dynamics. This requires the assumption that the molecular phenotype is a reasonable proxy for fitness, which need not be the case. For example, selection may favor a low or intermediate phenotypic value, causing incongruence between a fitness landscape and its underlying genotype-phenotype landscape. Here, we study such incongruence using a diversity of theoretical models and experimental data from gene regulatory systems. We regularly find incongruence, in that fitness landscapes tend to comprise more peaks than their underlying genotype-phenotype landscapes. However, using evolutionary simulations, we show that this increased ruggedness need not impede adaptation.

evolutionary biology↗

bioRxiv2022

SUMO/deSUMOylation of the BRI1 brassinosteroid receptor modulates plant growth responses to temperature

Brassinosteroids (BRs) are a class of steroid molecules perceived at the cell surface that act as plant hormones. The BR receptor BRI1 offers a model to understand receptor-mediated signaling in plants and the role of post-translational modifications. Here we identify SUMOylation as a new modification, targeting BRI1 to regulate its activity. BRI1 is SUMOylated in planta on two lysine residues and the levels of BRI1-SUMO conjugates are controlled by the Desi3a SUMO protease. We demonstrate that BRI1 is deSUMOylated at elevated temperature by Desi3a, leading to increased BRI1 interaction with the negative regulator of BR signaling BIK1 and enhancing BRI1 endocytosis. Loss of Desi3a or BIK1 results in increased response to temperature elevation, indicating that BRI1 deSUMOylation acts as a safety mechanism necessary to keep temperature responses in check. Altogether, our work establishes BRI1 deSUMOylation as a molecular crosstalk mechanism between temperature and BR signaling, allowing plants to translate environmental inputs into growth response. SIGNIFICANCE STATEMENTThe brassinosteroid (BR) receptor BRI1 provides a paradigm for understanding receptor-mediated signaling in plants and contribution of post-translational modifications. Here, we show that BRI carries SUMO modifications in planta on two intracellular lysine residues and that temperature elevation triggers BRI1 deSUMOylation mediated by the Desi3a SUMO protease. Importantly, BRI1 deSUMOylation leads to downregulation of BR signaling via increased BRI1 interaction with the BIK1 negative regulator and increased BRI1 endocytosis. Loss of BRI1 deSUMOylation in desi3a mutants boosts plant responses to heat, indicating that BRI1 deSUMOylation acts as a brake to keep temperature responses in check. Our study uncovers a new post-translational modification targeting BRI1 and sheds light on its functional outcome for environmentally-controlled plant growth.

plant biology↗

bioRxiv2021

MK2 Expression Promotes Non-Small Cell Lung Cancer Cell Death and Predicts Survival

Non-small cell lung cancers demonstrate intrinsic resistance to cell death even in response to chemotherapy. Previous work suggested that defective nuclear translocation of active caspase 3 may play a role in resistance to cell death. Separately, our group has identified that mitogen activated protein kinase activated protein kinase 2 (MK2) is required for nuclear translocation of active caspase 3 in the execution of apoptosis. This study demonstrates a relatively low expression of MK2 in non-small cell lung carcinoma cell lines compared to small cell carcinoma cell lines. Further, overexpression of MK2 in non-small cell lung carcinoma cell lines results in increased caspase 3 activity and caspase 3 mediated cell death. Higher MK2 transcript levels were observed in patients with earlier-stage non-small cell lung cancer. Higher expression of MK2 is associated with better survival in patients with early stage non-small cell lung cancer across two independent clinical datasets. Using data sets spanning multiple cancer types, we observed improved survival with higher MK2 expression was unique to lung adenocarcinoma. Mechanistically, MK2 promotes nuclear translocation of caspase 3 leading to PARP1 cleavage and execution of cell death. While MK2 can directly phosphorylate caspase 3, neither phosphorylation status of caspase 3 nor the kinase activity of MK2 impacts caspase 3 activation, nuclear translocation and execution of cell death. Rather, a non-kinase function of MK2, specifically trafficking via its nuclear localization sequence, is required for caspase 3 mediated cell death. In summary this study highlights the importance of a non-enzymatic function of MK2 in the execution of apoptosis, which may be leveraged in the adjunctive treatment of NSCLC or other conditions where regulation of apoptosis is crucial.

cancer biology↗

bioRxiv2021

CASowary: CRISPR-Cas13 guide RNA predictor for transcript depletion

Recent discovery of the gene editing system -CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) associated proteins (Cas), has resulted in its widespread use for improved understanding of a variety of biological systems. Cas13, a lesser studied Cas protein, has been repurposed to allow for efficient and precise editing of RNA molecules. The Cas13 system utilizes base complementarity between a crRNA/sgRNA (crispr RNA or single guide RNA) and a target RNA transcript, to preferentially bind to only the target transcript. Unlike targeting the upstream regulatory regions of protein coding genes on the genome, the transcriptome is significantly more redundant, leading to many transcripts having wide stretches of identical nucleotide sequences. Transcripts also exhibit complex three-dimensional structures and interact with an array of RBPs (RNA Binding Proteins), both of which further limit the scope of effective target sequences. As a result, there currently exists no method to predict whether a specific sgRNA will effectively knockdown a transcript. Here we present a novel machine learning and computational tool, CASowary, to predict the efficacy of a sgRNA. We used publicly available RNA knockdown data from Cas13 characterization experiments for 555 sgRNAs targeting the transcriptome in HEK293 cells, in conjunction with transcriptome-wide protein occupancy information on RNA. Our model utilizes a Decision Tree architecture with a set of 112 sequence and target availability features, to classify sgRNA efficacy into one of four classes, based upon expected level of target transcript knockdown. After accounting for noise in the training data set, the noise-normalized accuracy exceeds 70%. Additionally, highly effective sgRNA predictions have been experimentally validated using an independent RNA targeting Cas system -CIRTS, confirming the robustness and reproducibility of our models sgRNA predictions. Utilizing transcriptome wide protein occupancy map generated using POP-seq in Hela cells against publicly available protein-RNA interaction map in Hek293 cells, we show that CASowary can predict high quality guides for numerous transcripts in a cell line specific manner. Application of CASowary to whole transcriptomes should enable rapid deployment of CRISPR/Cas13 systems, facilitating the development of therapeutic interventions linked with aberrations in RNA regulatory processes.

bioinformatics↗