Biology subjects

Russell, R.

Publications and source records attributed to Russell, R..

18 recordsLinked to original sources

bioRxiv2026

A Pooled CRISPR Screen Protocol for Comparative Pathway Analysis Across Multiple Stressors

High content CRISPR screens have become a leading method for the identification of new cellular pathways, proteins functions, and drug targets. However, as the complexity of these screens increase, so does the possibility of artifacts. Here, we describe a refined CRISPR screening protocol that can compare acute cellular responses across multiple cellular treatments and timepoints, while reducing false positives by up to 40% compared to conventional screening approaches. This is achieved through the use of a fluorescence-based reporter line responsive to multiple stressors, as well as a fixation step to preserve acute response differences and avoid sorting-related false positives. We recently performed a version of this workflow to compare kinases regulating different types of selective autophagy, including starvation-induced autophagy, ERphagy, and pexophagy. Beyond autophagy research, this screen approach can be implemented for any fluorescence-based screen where preservation of acute cellular responses is important. Here we present a complete workflow of this optimized screening protocol using our selective autophagy screen as a specific example throughout. Implementation of these strategies should allow researchers to increase the number of stressors tested within a single screen without sacrificing cell health, reliability, or statistical power.

genomics↗

bioRxiv2026

Rho ({rho}) Analysis to Dissect RNA Folding and Assembly Pathways

The modular structure and energetics of RNA simplifies its folding. Leveraging this modularity, we introduce Rho ({rho}) analysis to systematically dissect RNA conformational pathways. {rho} analysis uses isolated RNA secondary or tertiary contacts as external standards to provide insights not possible via the "internal" comparisons of traditional {phi} analysis. Equivalent effects of a mutation on the folding rate constant of the RNA of interest and the thermodynamic stability of the isolated contact indicate that the mutated interaction is fully formed prior to the rate-limiting transition state; the absence of a kinetic effect indicates that the interaction is formed after this transition state. Comparisons with properties of the isolated contact provide additional insights about conformational pathways. We demonstrate {rho} analysis by dissecting Tetrahymena group I intron folding pathways, using a split intron in which the P5abc subdomain assembles with the intron core through three tertiary contacts. We uncover multiple folding pathways and modulation in pathway flux that are readily understood from the energetic properties of the constituent RNA motifs. Extending these concepts to RNA-guided DNA recognition by CRISPR-Cas12a, crRNA-DNA mismatches give substantial {phi} values across much of the target, indicating a late transition state in binding. Thermodynamic penalties from mismatches support modular base-pairing energetics and define an upper bound on DNA target specificity. Our results establish {rho} analysis as a general framework to probe RNA conformational pathways and function. It is straightforward to implement and can be readily applied in vitro and in cells.

biophysics↗

bioRxiv2026

Region- and layer-specific glutamatergic synapse development in the nascent cortical hierarchy

Neocortical synapses are highly dynamic during brain development, undergoing formation, elimination, and maturation before acquiring properties that support adult cognition. Individual neocortical regions develop at different ages and individual layers within these regions contain distinct neuronal subtypes that process unique patterns of local and long-range synaptic input. To better understand the development of the cortical hierarchy we explored the laminar maturation of glutamatergic synapses across cortical regions. Synapse maturation was associated with the upregulation of the postsynaptic density protein PSD95. This maturation occurred in a region- and layer-specific manner -- layers associated with feedforward pathways develop earlier, while layers associated with higher-order circuits develop later. Our findings highlight adolescence as an important period for the cortex-wide maturation of synapses in cortical layer 1, synapses known to receive top-down feedback from higher-order cortices. We propose that this delayed adolescent maturation of top-down input represents a global signature of cortical development and seemingly acts as the final stage of outside-in brain maturation.

neuroscience↗

bioRxiv2025

Structural Basis for Target Discrimination and Activation by Cas13d

CRISPR-Cas13d is increasingly used for RNA knockdowns due to its programmability, but off-target RNA binding and cleavage of near-cognate RNAs hinder its broader adoption. Here, we explore the mechanisms of nuclease activation by solving seven ternary cryo-electron mi-croscopy structures of wild-type Cas13d in complex with matched and mismatched targets. These structures reveal a series of active, intermediate, and inactive states that illustrate a detailed activation mechanism. The crRNA undergoes dramatic conformational changes upon target RNA binding, with the helical-1 domain transitioning from an initially docked state with the N-terminal domain to an allosterically switched conformation that stabilizes the RNA duplex. Quantitative kinetics reveal that a single proximal mismatch preserves nanomolar binding affinity but completely abolishes nuclease activity by trapping Cas13d in an inactive state. We identify an active site loop in the HEPN domains that regulates substrate accessibility, with alanine scanning mutagenesis revealing both hypo- and hyperactivated variants. These findings establish the structural basis for Cas13ds exquisite mismatch surveillance and provide a mechanistic framework for engineering RNA-targeting specificity and activity across HEPN nuclease family members.

biochemistry↗

bioRxiv2024

A high-throughput search for intracellular factors that affect RNA folding identifies E. coli proteins PepA and YagL as RNA chaperones that promote RNA remodeling

General RNA chaperones are RNA-binding proteins (RBPs) that interact transiently and non-specifically with RNA substrates and assist in their folding into native state. In bacteria, these chaperones impact both coding and non-coding RNAs and are particularly important for large, structured RNAs which are prone to becoming kinetically trapped in misfolded states. Currently, due to the limited number of well-characterized examples and the lack of a consensus structural or sequence motif, it is difficult to identify general RNA chaperones in bacteria. Here, we adapted a previously published in vivo RNA regional accessibility probing assay to screen genome wide for intracellular factors in E. coli affecting RNA folding, among which we aimed to uncover novel RNA chaperones. Through this method, we identified and validated eight proteins whose deletion gives changes in regional accessibility within the exogenously expressed Tetrahymena group I intron ribozyme. Further, we purified and measured in vitro properties of two of these proteins, YagL and PepA, which were especially attractive as general chaperone candidates. We showed that both proteins bind RNA and that YagL accelerates native refolding of the ribozyme from a long-lived misfolded state. Further dissection of YagL showed that a putative helix-turn-helix (HTH) domain is responsible for most of its RNA binding activity but only the full protein shows chaperone activity. Altogether, this work expands the current repertoire of known general RNA chaperones in bacteria.

molecular biology↗

bioRxiv2024

Optimal Control Prevents Itself from Eradicating Stochastic Disease Epidemics

The resources available for managing disease epidemics - whether in animals, plants or humans - are limited by a range of practical and financial constraints. This motivates study of where these resources should be allocated to maximise their impact. Optimal control has been widely explored for optimising management of epidemics. The most common approach assumes a deterministic, continuous model based on differential equations to approximate the epidemic dynamics. However, real systems are stochastic and so a range of outcomes are possible for any given epidemic situation and choice of control. The deterministic models are also known to be poor approximations in cases where the number of infected hosts is low - either globally or within a subset of the population - and these cases are highly relevant in the context of control. Hence, this work explores the effectiveness of disease management strategies derived using optimal control theory when applied to a more realistic, stochastic form of disease model. We demonstrate that solutions to the deterministic optimal control are not optimal in cases where the disease is eradicated or close to eradication. The range of potential outcomes in the stochastic models means that optimising the deterministic case will not reliably eradicate disease, even when it is possible. For effective eradication, the rate of control must be higher than optimal control as applied to the deterministic model would predict. Using Model Predictive Control, in which the optimisation is performed repeatedly as the system evolves to correct for deviations from the optimal control predictions, improves performance but does not fix the underlying issue and the level of control calculated at each repeated optimisation is still insufficient. To demonstrate this, we present several very simple heuristics to identify which sites to control which can outperform the strategies calculated by MPC when the control budget is sufficient for eradication to be possible. Our illustration uses examples based on stochastic simulation of the spatial spread of plant disease but similar issues would be expected in any deterministic model where infection is driven close to zero or targeted to remain below some critical value.

plant biology↗

bioRxiv2024

Loss of VHL-mediated pRb regulation promotes clear cell renal cell carcinoma

The von Hippel-Lindau (VHL) tumor suppressor is a component of E3 ubiquitin ligase complexes that target cellular substrates for proteasome-mediated degradation. VHL inactivation by genetic aberrations is observed in most sporadic cases of clear cell renal cell carcinoma (ccRCC). VHL loss leads to constitutive stabilization of E3 ligase targets, including hypoxia inducible factor (HIF), in VHL-associated tumors. HIF stabilization upon VHL loss promotes transactivation of hypoxia responsive genes, which contributes to ccRCC development. However, several HIF-independent VHL targets have also been implicated in the promotion of tumorigenesis. Using proximity labeling to identify proteasomal VHL interactors, we identified retinoblastoma protein (pRb) as a novel substrate of VHL. Mechanistically, VHL interacts with pRb in an oxygen-sensitive manner, promoting its ubiquitin-mediated degradation. Concordantly, VHL-inactivation results in pRb hyperstabilization. Functionally, the hyperstabilization of pRb in ccRCC promoted tumorigenesis in vitro and in mouse models. We also show that downstream transcriptional changes induced by pRb hyperstabilization may contribute to ccRCC tumor development. Together, our findings reveal a novel VHL-related pathway which can be therapeutically targeted to inhibit ccRCC tumor development.

cancer biology↗

bioRxiv2024

The ER-phagy receptor FAM134B is targeted by Salmonella Typhimurium to promote infection

Macroautophagy/autophagy is a key catabolic-recycling pathway that can selectively target damaged organelles or invading pathogens for degradation. The selective autophagic degradation of the endoplasmic reticulum (hereafter referred to as ER-phagy) is a homeostatic mechanism, controlling ER size, the removal of misfolded protein aggregates, and organelle damage. ER-phagy is also stimulated by pathogen infection. However, the link between ER-phagy and bacterial infection remains poorly understood, as are the mechanisms evolved by pathogens to escape the effects of ER-phagy. Here, we show that Salmonella enterica serovar Typhimurium inhibits ER-phagy by targeting the ER-phagy receptor FAM134B, leading to a pronounced increase in Salmonella viability after invasion. Salmonella prevents FAM134B oligomerization, which is required for efficient ER-phagy. FAM134B knock-out raises intracellular Salmonella number, while FAM134B activation reduces Salmonella burden. Additionally, we found that Salmonella targets FAM134B through the bacterial effector SopF to enhance intracellular survival through ER-phagy inhibition. Furthermore, FAM134B knock-out mice infected with Salmonella presented severe intestinal damage and increased bacterial burden. These results provide new mechanistic insight into the interplay between ER-phagy and bacterial infection, highlighting a key role for FAM134B in innate immunity.

molecular biology↗

bioRxiv2023

Kinetic dissection of pre-crRNA binding and processing by CRISPR-Cas12a

CRISPR-Cas12a binds and processes a single pre-crRNA during maturation, providing a simple tool for genome editing applications. Here, we constructed a kinetic and thermodynamic framework for pre-crRNA processing by Cas12a in vitro, and we measured the contributions of distinct regions of the pre-crRNA to this reaction. We find that the pre-crRNA binds rapidly and extraordinarily tightly to Cas12a (Kd = 0.6 pM), such that pre-crRNA binding is fully rate limiting for processing and therefore determines the specificity of Cas12a for different pre-crRNAs. The guide sequence contributes 10-fold to the affinities of both the precursor and mature forms of the crRNA, while deletion of an upstream sequence had no significant effect on affinity of the pre-crRNA. After processing, the mature crRNA remains very tightly bound to Cas12a, with a half-life of [~]1 day and a Kd value of 60 pM. Addition of a 5-phosphoryl group, which is normally lost during the processing reaction as the scissile phosphate, tightens binding of the mature crRNA by [~]10-fold by accelerating binding and slowing dissociation. Using a direct competition assay, we found that pre-crRNA binding specificity is robust to other changes in RNA sequence, including tested changes in the guide sequence, addition of a 3 extension, and secondary structure within the guide region. Together our results provide a quantitative framework for pre-crRNA binding and processing by Cas12a and suggest strategies for optimizing crRNA design in some genome editing applications.

biochemistry↗

bioRxiv2023

Structural basis of Cas12a R-loop propagation on pathway to DNA cleavage

Cas12a is a CRISPR RNA-guided nuclease that engages target DNA through protein-DNA and RNA-DNA contacts. Initial PAM binding by Cas12a leads to formation of a 20bp R-loop between the complementary crRNA guide and target strand. Following specificity-determining R-loop formation, both DNA strands undergo RuvC-mediated cleavage. Current structures of Cas12a bound to its target only show the R-loop after formation, leaving an important gap in knowledge as to how Cas12a accommodates the extending R-loop and coordinates R-loop formation with nuclease activation. Here, we use cryo-EM to capture a series of kinetically trapped Cas12a R-loop intermediates and observe how Cas12a delivers each DNA strand into the RuvC active site. We show that Cas12a first interrogates target DNA via a 5bp seed, followed by dramatic Rec domain conformational flexibility to accommodate R-loop extension. Only during formation of the final R-loop base pairs do the Rec and BH domains engage in the majority of contacts with the R-loop. R-loop completion leads the nontarget strand to displace the RuvC lid and kink into the active site via a base stacking interaction. Following nontarget strand cleavage, we observe substantial Rec2 and Nuc domain dynamics as the TS is brought to the RuvC active site. Our kinetics-guided structural snapshots provide a comprehensive model describing Cas12a DNA targeting and highlight mechanistic differences between Cas12a and Cas9.

biochemistry↗

bioRxiv2022

Species-specific roles for the MAFA and MAFB transcription factors in regulatingislet β cell identity

Type 2 diabetes (T2D) is associated with compromised identity of insulin-producing pancreatic islet beta ({beta}) cells, characterized by inappropriate production of other islet cell-enriched hormones. Here we examined how hormone misexpression was influenced by the MAFA and MAFB transcription factors, closely related proteins that maintain islet cell function. Mice specifically lacking MafA in {beta} cells demonstrated broad, population-wide changes in hormone gene expression with an overall gene signature closely resembling islet gastrin (Gast)-positive cells generated under conditions of chronic hyperglycemia and obesity. A human {beta} cell line deficient in MAFB, but not one lacking MAFA, also produced a gastrin (GAST)-positive gene expression pattern. In addition, GAST was detected in human T2D {beta} cells with low levels of MAFB. Moreover, evidence is provided that human MAFB can directly repress GAST gene transcription. These results support a novel, species-specific role for MafA and MAFB in maintaining adult mouse and human {beta} cell identity, respectively, by repressing expression of Gast/GAST and other non-{beta} cell hormones.

cell biology↗

bioRxiv2022

Group II Intron-Like Reverse TranscriptasesFunction in Double-Strand Break Repair by Microhomology-Mediated End Joining

Bacteria encode free-standing reverse transcriptases (RTs) of unknown function that are closely related to group II intron-encoded RTs. Here, we found that a Pseudomonas aeruginosa group II intron-like RT (G2L4 RT) with YIDD instead of YADD at its active site functions in DNA repair in its native host and when transferred into Escherichia coli. G2L4 RT has biochemical activities strikingly similar to those of human DNA repair polymerase{theta} and uses them for translesion DNA synthesis and double-strand break repair (DSBR) via microhomology-mediated end-joining (MMEJ) in vitro and in vivo. We also found that a group II intron RT can function similarly to G2L4 RT in DNA repair, with reciprocal substitutions at the active site showing an I residue favors MMEJ and an A residue favors primer extension in both enzymes. The DNA repair functions of these enzymes utilize conserved structural features of non-LTR-retroelement RTs, including human LINE-1 and other eukaryotic non-LTR-retrotransposon RTs, suggesting such enzymes may have an inherent ability to function in DSBR in a wide range of organisms.

molecular biology↗

bioRxiv2021

Pro-IL-18 secreted by keratinocytes detects the group A streptococcal protease SpeB

Group A Streptococcus (GAS, Streptococcus pyogenes) is a professional human pathogen that commonly infects the skin. Keratinocytes are one of the first cells to contact GAS, and by inducing inflammation, they can initiate the earliest immune responses to pathogen invasion. Here, we characterized the proinflammatory cytokine repertoire produced by primary human keratinocytes and surrogate cell lines commonly used in vitro. Infection induces several cytokines and chemokines, but keratinocytes constitutively secrete IL-18 in a form that is inert (pro-IL-18) and lacks proinflammatory activity. Canonically, IL-18 activation and secretion are coupled through a single proteolytic event that is regulated intracellularly by the inflammasome protease caspase-1 in myeloid cells. The pool of extracellular pro-IL-18 generated by keratinocytes is poised to sense extracellular proteases and is directly processed into a mature active form by SpeB, a secreted GAS protease that is a critical virulent factor during skin infection. This mechanism contributes to the proinflammatory response against GAS, resulting in T cell activation and the secretion of IFN-{gamma} that restrict GAS growth. Other major bacterial pathogens and microbiota of this skin did not have significant IL-18-maturing ability. Taken together, these results suggest keratinocyte-secreted IL-18 is a sentinel that sounds an early alarm that is highly sensitive to GAS, yet tolerant to non-invasive members of the microbiota.

microbiology↗

bioRxiv2021

Structural basis for template switching by a group II intron-encoded non-LTR-retroelement reverse transcriptase

Reverse transcriptases (RTs) can template switch during cDNA synthesis, enabling them to join discontinuous nucleic acid sequences. Template switching plays crucial roles in retroviral replication and recombination, is used for adapter addition in RNA-seq, and may contribute to retroelement fitness by enabling continuous cDNA synthesis on damaged templates. Here, we determined an X-ray crystal structure of a template-switching complex of a group II intron RT bound simultaneously to an acceptor RNA and donor RNA template/DNA heteroduplex with a 1-nt 3-DNA overhang. The latter mimics a completed cDNA after non-templated addition (NTA) of a nucleotide complementary to the 3 nucleotide of the acceptor as required for efficient template switching. The structure showed that the 3 end of the acceptor RNA binds in a pocket formed by an N-terminal extension (NTE) present in non-long-terminal-repeat (LTR)-retroelement RTs and the RT fingertips loop, with the 3 nucleotide of the acceptor base paired to the 1-nt 3-DNA overhang and its penultimate nucleotide base paired to the incoming dNTP at the RT active site. Analysis of structure-guided mutations identified amino acids that contribute to acceptor RNA binding and a phenylalanine near the RT active site that mediates NTA. Mutation of the latter residue decreased multiple sequential template switches in RNA-seq. Our results provide new insights into the mechanisms of template switching and NTA by RTs, suggest how these reactions could be improved for RNA-seq, and reveal common structural features for template switching by non-LTR-retroelement RTs and viral RNA-dependent RNA polymerases.

biochemistry↗

bioRxiv2020

Inhibition of CRISPR-Cas12a DNA Targeting by Nucleosomes and Chromatin

Genome engineering nucleases, including CRISPR-Cas12a, must access chromatinized DNA. Here, we investigate how Acidaminococcus sp. Cas12a cleaves DNA within human nucleosomes and phase-condensed nucleosome arrays. Using quantitative kinetics approaches, we show that dynamic nucleosome unwrapping regulates DNA target accessibility to Cas12a. Nucleosome unwrapping determines the extent to which both steps of Cas12a binding-PAM recognition and R-loop formation-are inhibited by the nucleosome. Nucleosomes inhibit Cas12a binding even beyond the canonical core particle. Relaxing DNA wrapping within the nucleosome by reducing DNA bendability, adding histone modifications, or introducing a target-proximal nuclease-inactive Cas9 enhances DNA cleavage rates over 10-fold. Surprisingly, Cas12a readily cleaves DNA linking nucleosomes within chromatin-like phase separated nucleosome arrays--with DNA targeting reduced only ~4-fold. This work provides a mechanism for the observation that on-target cleavage within nucleosomes occurs less often than off-target cleavage within nucleosome-depleted regions of cells. We conclude that nucleosome wrapping restricts accessibility to CRISPR-Cas nucleases and anticipate that increasing nucleosome breathing dynamics will improve DNA binding and cleavage in eukaryotic cells.

biochemistry↗

bioRxiv2020

Group A Streptococcus Infection of the Nasopharynx Requires Proinflammatory Signaling Through the Interleukin-1 Receptor

Group A Streptococcus (GAS) is the etiologic agent of numerous high morbidity and high mortality diseases which commonly have a highly proinflammatory pathology. One factor contributing to this inflammation is the GAS protease SpeB, which directly activates the proinflammatory cytokine interleukin-1{beta} (IL-1{beta}), independent of the canonical inflammasome pathway. IL-1{beta} drives neutrophil activation and recruitment that limits bacterial growth and invasion during invasive skin and soft tissue infections like necrotizing fasciitis. GAS also causes pharyngitis (strep throat), and the upper respiratory tract is its primary nidus for growth and transmission. Since the fitness selection for the species is likely primarily for this site, we examined the process of IL-1{beta} activation in the murine nasopharynx. SpeB still activated IL-1{beta}, which was required for neutrophil migration, but this inflammation instead increased GAS replication. Inhibiting IL-1{beta} or depleting neutrophils, which both promote invasive infection, prevented GAS infection of the nasopharynx. Prior antibiotic exposure increased GAS growth in the murine nasopharynx, and antibiotics were sufficient to reverse the attenuation previously observed when IL-1{beta}, neutrophils, or SpeB were not present to drive inflammation. Therefore, the same fundamental mechanism has opposing effects on virulence at different body sites. Invasive disease may be limited in part due to specific adaptations for inducing host inflammation that are beneficial for pharyngitis. IMPORTANCEOur previous reports showed that Group A Streptococcus (GAS) protease SpeB directly activates the host proinflammatory cytokine IL-1{beta} and this restricts invasive skin infection. The upper respiratory tract is the primary site of GAS colonization and infection, but the host-pathogen interactions at this site are still largely unknown. We provide the first evidence that IL-1{beta}-mediated inflammation promotes upper respiratory tract infection. This provides experimental evidence that the notable inflammation of strep throat, which presents with significant swelling, pain, and neutrophil influx, is not an ineffectual immune response, but rather is a GAS-directed remodeling of this niche for its pathogenic benefit.

microbiology↗

bioRxiv2019

Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq

The reverse transcriptases (RTs) encoded by mobile group II intron and other non-LTR-retro-elements differ from retroviral RTs in being able to template switch from the 5 end of one template to the 3 end of another without pre-existing complementarity between the donor and acceptor nucleic acids. Here, we used the ability of a thermostable group II intron RT (TGIRT; GsI-IIC RT) to template switch directly from synthetic RNA template/DNA primer duplexes having either a blunt end or a 3-DNA overhang end to establish a complete kinetic framework for the reaction and identify conditions that more efficiently capture acceptor RNAs or DNAs. The rate and amplitude of template switching are optimal from starter duplexes with a single nucleotide 3-DNA overhang complementary to the 3 nucleotide of the acceptor RNA, suggesting a role for non-templated nucleotide addition of a complementary nucleotide to the 3 end of cDNAs synthesized from natural templates. Longer 3-DNA overhangs progressively decrease the rate of template switching, even when complementary to the 3 end of the acceptor template. The reliance on a single base pair with the 3 nucleotide of the acceptor together with discrimination against mismatches and the high processivity of the enzyme enable synthesis of full-length DNA copies of nucleic acids beginning directly at their 3 end. We discuss possible biological functions of the template-switching activity of group II intron and other non-LTR-retroelements RTs, as well as the optimization of this activity for adapter addition in RNA-and DNA-seq.

molecular biology↗

bioRxiv2018

Kinetic basis for DNA target specificity of CRISPR-Cas12a

Class II CRISPR-Cas nucleases are programmable via a single guide RNA, enabling genome editing applications in nearly all organisms. However, DNA cleavage at off-target sites that resemble the target sequence is a pervasive problem that remains poorly understood mechanistically. Here, we use quantitative kinetics to dissect the reaction steps of DNA targeting by Acidaminococcus sp Cas12a (also known as Cpf1). We show that Cas12a binds DNA tightly in two kinetically-separable steps. Protospacer-adjacent motif (PAM) recognition is followed by rate-limiting R-loop propagation, leading to inevitable DNA cleavage of both strands. Despite the functionally irreversible binding, Cas12a discriminates strongly against mismatches along most of the DNA target sequence, implying substantial reversibility during R-loop formation -a late transition state- and the absence of a seed region. Our results provide a quantitative underpinning for the DNA cleavage patterns measured in vivo and observations of greater reported target specificity of Cas12a than the Cas9 nuclease.

molecular biology↗