Biology subjects

Lapointe, C. P.

Publications and source records attributed to Lapointe, C. P..

18 recordsLinked to original sources

bioRxiv2026

Remodeling of mRNA by eIF4F in human translation initiation

For the ribosome to load onto an mRNA during the early steps of translation initiation, the mRNA must be activated by the eIF4F complex. The mechanism of this activation step has remained elusive. Here we employ multi-perspective real-time single-molecule assays to observe directly mRNA-eIF4F binding near the 5' end, mRNA conformational remodeling, and 40S ribosomal subunit loading. eIFs 4E, 4G, and 4B play distinct roles in promoting eIF4F association and stabilizing eIF4A binding. Binding of eIF4F is the rate-limiting step in mRNA activation: once bound, mRNA conformation is rapidly extended in an ATP-dependent manner. The mRNA extended state is the necessary substrate for 43S PIC loading and perturbations to extension delay loading. Features of the mRNA, such as the 7-methylguanosine cap at the 5' end and secondary structures, modulate these steps and regulate ribosome loading. Our results establish a kinetic and mechanistic framework for the early steps in translation initiation.

biophysics↗

bioRxiv2026

A ribosomal kinetic checkpoint governs selective mRNA recruitment

Translation initiation begins with recruitment of mRNA to the ribosome, yet how mRNA engagement is converted into productive initiation remains unclear. Using real-time fluorescence assays with purified components, we show that mRNA recruitment proceeds through a branched kinetic pathway on the 40S subunit. Following rapid sampling, mRNAs partition into either a productive accommodated state or an arrested state that stabilizes ribosome binding before accommodation. mRNA structure, eIF3, and eIF3j bias recruitment toward arrest, whereas eIF4F promotes accommodation in an ATP-dependent manner coupled to displacement of eIF3j from the mRNA entry channel. Unstructured mRNAs accommodate independently of eIF4E, whereas structured mRNAs require an upstream eIF4E-dependent step, enabling selective recruitment under limiting eIF4E. Arrested complexes can convert directly into the accommodated state without dissociation, revealing a reversible standby intermediate poised for activation. Together, our findings establish mRNA accommodation as a ribosome-intrinsic checkpoint governing initiation and provide a framework for selective translation.

biochemistry↗

bioRxiv2026

A codon-sensitive conformational switch gates commitment to translation start sites

Human translation initiation requires single-nucleotide precision to establish the reading frame, yet initiation at non-AUG codons plays key roles in gene expression. How the initiation machinery balances precision with this regulated flexibility remains unclear. Here, we define a conformational branchpoint governed by the human initiation factor eIF5 that gates commitment to start codons. Using single-molecule and structural approaches, we demonstrate that eIF5 reversibly occupies two conformations, which depends on a strictly conserved loop in the protein that monitors start codon identity. AUG codons favor the conformation that is stabilized by an eIF5-stimulated GTP hydrolysis step, which commits the complex to the start site. Non-AUG codons favor a standby conformation that destabilizes eIF5 and likely overlaps the binding site of an ancient structural homolog. This branchpoint complements enforcement of start codon fidelity by upstream steps and intrinsically controls the efficiency of non-AUG initiation.

biophysics↗

bioRxiv2025

RNA regulates repeat-associated non-AUG (RAN) translation initiation in C9orf72 FTD/ALS

Repeat-associated non-AUG (RAN) translation synthesizes protein in the absence of a cognate AUG start codon 1,2In frontotemporal dementia and amyotrophic lateral sclerosis, a GGGGCC (G4C2) repeat expansion in an intron of C9orf72 leads to synthesis of neurotoxic dipeptide-repeat proteins, underscoring the need to understand the mechanism of C9orf72 RAN translation1-5. RNA sequence and structure have been implicated, but how they direct C9orf72 RAN translation, particularly the rate-limiting, multi-step initiation phase, remains unclear6-10. We applied single-molecule biophysics to a reconstituted human translation initiation system and tracked fluorescently labeled ribosomes and initiation factors in real time. We show that RNA G4C2 repeats and sequence context alter initiation factor dynamics after ribosomal scanning, generating a kinetic bottleneck in the commitment to initiate at a near-cognate CUG start codon. Our model of C9orf72 RAN translation provides a mechanistic framework for how repeat expansions change underlying translation dynamics and may be broadly relevant to other disorders that involve RAN translation.

biophysics↗

bioRxiv2025

Deaminase-based RNA recording enables high throughput mutational profiling of protein-RNA interactions

Protein-RNA interactions govern nearly every aspect of RNA metabolism and are frequently dysregulated in disease. While individual protein residues and RNA nucleotides critical for these interactions have been characterized, scalable methods that jointly map protein- and RNA-level determinants remain limited. RNA deaminase fusions have emerged as a powerful strategy to identify transcriptome-wide targets of RNA-binding proteins by converting binding events into site-specific nucleotide edits. Here, we demonstrate that this RNA recording approach enables high-throughput mutational scanning of protein-RNA interfaces. Using the {lambda}N-boxB system as a model, we show that editing by a fused TadA adenosine deaminase directly correlates with binding affinity between protein and RNA variants in vitro. Systematic variation of RNA sequence context reveals a strong bias for editing at UA dinucleotides by the engineered TadA8.20, mirroring wild-type TadA preferences. We further demonstrate that stepwise recruitment of the deaminase using nanobody and protein A/G fusions maintains both sequence and binding specificity. Stable expression of the TadA fusion in human cells reproduces in vitro editing patterns across a library of RNA variants. Finally, comprehensive single amino acid mutagenesis of {lambda}N in human cells reveals critical residues mediating RNA binding. Together, our results establish RNA recording as a versatile and scalable tool for dissecting protein-RNA interactions at nucleotide and residue resolution, both in vitro and in cells.

molecular biology↗

bioRxiv2025

Capsule-Based Single-Cell Genome Sequencing

Single-cell genomics methods have unveiled the heterogeneity present in seemingly homogenous populations of cells, however, these techniques require meticulous optimization. How exactly does one handle and manipulate the biological contents from a single cell? Here, we introduce and characterize a novel semi-permeable capsule (SPC), capable of isolating single cells and their contents while facilitating biomolecular exchange based on size-selectivity. These capsules maintain stability under diverse physical and chemical conditions and allow selective diffusion of biomolecules, effectively retaining larger biomolecules including genomic DNA, and cellular complexes, while permitting the exchange of smaller molecules, including primers and enzymes. We demonstrate the utility of SPCs for single cell assays by performing the simultaneous culture of over 500,000 cellular colonies, demonstrating efficient and unbiased nucleic acid amplification, and performing combinatorial indexing-based single-cell whole genome sequencing (sc-WGS). Notably, SPC-based sc-WGS facilitates uniform genome coverage and minimal cross-contamination allowing for the detection of genomic variants with high sensitivity and specificity. Leveraging these properties, we conducted a proof-of-concept lineage tracing experiment using cells harboring the hypermutator polymerase {varepsilon} allele (POLE P286R). Sequencing of 1000 single cell genomes at low depth facilitated the capture of lineage marks deposited throughout the genome during each cell division and the subsequent reconstruction of cellular genealogies. Capsule-based sc-WGS expands the single-cell genomics toolkit and will facilitate the investigation of somatic variants, resolved to single cells at scale.

genomics↗

bioRxiv2025

SARS-CoV-2 nsp1 mediates broad inhibition of translation in mammals

SARS-CoV-2 nonstructural protein 1 (nsp1) promotes innate immune evasion by inhibiting host translation in human cells. However, the role of nsp1 in other host species remains elusive, especially in bats which are natural reservoirs of sarbecoviruses and possess a markedly different innate immune system than humans. Here, we reveal that SARS-CoV-2 nsp1 potently inhibits translation in bat cells from Rhinolophus lepidus, belonging to the same genus as known sarbecovirus reservoirs hosts. We determined a cryo-electron microscopy structure of SARS-CoV-2 nsp1 bound to the Rhinolophus lepidus 40S ribosome and show that it blocks the mRNA entry channel via targeting a highly conserved site among mammals. Accordingly, we found that nsp1 blocked protein translation in mammalian cell lines from several species, underscoring its broadly inhibitory activity and conserved role in numerous SARS-CoV-2 hosts. Our findings illuminate the arms race between coronaviruses and mammalian host immunity (including bats), providing a foundation for understanding the determinants of viral maintenance in bat hosts and spillovers.

biochemistry↗

bioRxiv2024

eIF1 and eIF5 dynamically control translation start site fidelity

Translation initiation defines the identity of a synthesized protein through selection of a translation start site on a messenger RNA. This process is essential to well-controlled protein synthesis, modulated by stress responses, and dysregulated in many human diseases. The eukaryotic initiation factors eIF1 and eIF5 interact with the initiator methionyl-tRNAiMet on the 40S ribosomal subunit to coordinate start site selection. Here, using single-molecule analysis of in vitro reconstituted human initiation combined with translation assays in cells, we examine eIF1 and eIF5 function. During translation initiation on a panel of RNAs, we monitored both proteins directly and in real time using single-molecule fluorescence. As expected, eIF1 loaded onto mRNAs as a component of the 43S initiation complex. Rapid ([~] 2 s) eIF1 departure required a translation start site and was delayed by alternative start sites and a longer 5 untranslated region (5UTR). After its initial departure, eIF1 rapidly and transiently sampled initiation complexes, with more prolonged sampling events on alternative start sites. By contrast, eIF5 only transiently bound initiation complexes late in initiation immediately prior to association of eIF5B, which allowed joining of the 60S ribosomal subunit. eIF5 association required the presence of a translation start site and was inhibited and destabilized by alternative start sites. Using both knockdown and overexpression experiments in human cells, we validated that eIF1 and eIF5 have opposing roles during initiation. Collectively, our findings demonstrate how multiple eIF1 and eIF5 binding events control start-site selection fidelity throughout initiation, which is tuned in response to changes in the levels of both proteins.

biophysics↗

bioRxiv2021

eIF5B and eIF1A remodel human translation initiation complexes to mediate ribosomal subunit joining

Joining of the ribosomal subunits at a translation start site on a messenger RNA during initiation commits the ribosome to synthesize a protein. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when universally-conserved eukaryotic initiation factors (eIFs) eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we examined initiation complexes that contained both eIF1A and eIF5B using single-particle electron cryo-microscopy. The resulting structure illuminated how eukaryote-specific contacts between eIF1A and eIF5B remodel the initiation complex to orient initiator tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during human translation initiation.

biophysics↗

bioRxiv2021

Multiplexed single-molecule analysis of human telomerase synthesizing DNA

Genomic stability in proliferating cells critically depends on telomere maintenance by telomerase reverse transcriptase. Here we developed a real-time single-molecule RNA sequencing approach that visualizes telomerase catalysis and structural dynamics at single-nucleotide resolution using FRET and zero-mode waveguides. The method permits direct detection of dynamic steps and structural states throughout the telomerase catalytic cycle and can be generalized to other nucleic acid polymerase systems.

biophysics↗

bioRxiv2020

Dynamic competition between SARS-CoV-2 NSP1 and mRNA on the human ribosome inhibits translation initiation

SARS-CoV-2 recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic. A molecular framework of how the virus manipulates host cellular machinery to facilitate infection remains unclear. Here, we focus on SARS-CoV-2 NSP1, which is proposed to be a virulence factor that inhibits protein synthesis by directly binding the human ribosome. Using extract-based and reconstitution experiments, we demonstrate that NSP1 inhibits translation initiation on model human and SARS-CoV-2 mRNAs. NSP1 also specifically binds to the small (40S) ribosomal subunit, which is required for translation inhibition. Using single-molecule fluorescence assays to monitor NSP1-40S subunit binding in real time, we demonstrate that eukaryotic translation initiation factors (eIFs) modulate the interaction: NSP1 rapidly and stably associates with most ribosomal pre-initiation complexes in the absence of mRNA, with particular enhancement and inhibition by eIF1 and eIF3j, respectively. Using model mRNAs and an inter-ribosomal-subunit FRET signal, we elucidate that NSP1 competes with RNA segments downstream of the start codon to bind the 40S subunit and that the protein is unable to associate rapidly with 80S ribosomes assembled on an mRNA. Collectively, our findings support a model where NSP1 associates with the open head conformation of the 40S subunit to inhibit an early step of translation, by preventing accommodation of mRNA within the entry channel. SIGNIFICANCE STATEMENTSARS-CoV-2 is the causative agent of the COVID-19 pandemic. A molecular framework for how SARS-CoV-2 manipulates host cellular machinery to facilitate infection is needed. Here, we integrate biochemical and single-molecule strategies to reveal molecular insight into how NSP1 from SARS-CoV-2 inhibits translation initiation. NSP1 directly binds to the small (40S) subunit of the human ribosome, which is modulated by human initiation factors. Further, NSP1 and mRNA compete with each other to bind the ribosome. Our findings suggest that the presence of NSP1 on the small ribosomal subunit prevents proper accommodation of the mRNA. How this competition disrupts the many steps of translation initiation is an important target for future studies.

biophysics↗

bioRxiv2019

A memory of RPS25 loss drives resistance phenotypes

In order to maintain cellular protein homeostasis, ribosomes are safeguarded against dysregulation by myriad processes. Many cell types can nonetheless withstand genetic lesions of certain ribosomal protein genes, some of which are linked to diverse cellular phenotypes and human disease. However, the direct and indirect consequences from sustained alterations in ribosomal protein levels are poorly understood. To address this knowledge gap, we studied in vitro and cellular consequences that follow genetic knockout of the ribosomal proteins RPS25 or RACK1 in a human cell line, as both proteins are implicated in direct translational control. Prompted by the unexpected detection of an off-target ribosome alteration in the RPS25 knockout, we closely interrogated cellular phenotypes. We found that multiple RPS25 knockout clones display viral- and toxin-resistance phenotypes that cannot be rescued by functional cDNA expression, suggesting that RPS25 loss elicits a cell state transition. We characterized this state and found that it underlies pleiotropic phenotypes and has a common rewiring of gene expression. Rescuing RPS25 expression by genomic locus repair failed to correct for the phenotypic and expression hysteresis. Our findings illustrate how the elasticity of cells to a ribosome perturbation can drive specific phenotypic outcomes that are indirectly linked to translation.

molecular biology↗

bioRxiv2019

Records of RNA localization through covalent tagging

RNA movements and localization pervade biology, from embryonic development to disease. To identify RNAs at specific subcellular locations, we anchored a uridine-adding enzyme at those sites, which then marked RNAs in its vicinity with 3 terminal uridines. RNAs were tagged independent of their translation status, and included not only mRNAs, but also ncRNAs and ncRNA processing intermediates. A battery of RNAs, including the stress sensor, IRE1, were tagged at both ER and mitochondria, and reveal RNAs whose dual localization is conserved from yeast to human cells.

genomics↗

bioRxiv2019

eIF5B gates the transition from translation initiation to elongation

Translation initiation determines both the quantity and identity of the protein product by establishing the reading frame for protein synthesis. In eukaryotic cells, numerous translation initiation factors (eIFs) prepare ribosomes for polypeptide elongation, yet the underlying dynamics of this process remain enigmatic1-4. A central question is how eukaryotic ribosomes transition from translation initiation to elongation. Here, we applied in vitro single-molecule fluorescence microscopy approaches to monitor directly in real time the pathways of late translation initiation and the transition to elongation using a purified yeast Saccharomyces cerevisiae translation system. This transition was remarkably slower in our eukaryotic system than that reported for Escherichia coli5-7. The slow entry to elongation was defined by a long residence time of eIF5B on the 80S ribosome after joining of individual ribosomal subunits, which is catalyzed by this universally conserved initiation factor. Inhibition of eIF5B GTPase activity following subunit joining prevented eIF5B dissociation from the 80S complex, thereby preventing elongation. Our findings illustrate how eIF5B dissociation serves as a kinetic checkpoint for the transition from initiation to elongation, and its release may be governed by a conformation of the ribosome complex that triggers GTP hydrolysis.

biochemistry↗

bioRxiv2019

RACK1 on and off the ribosome

Receptor for activated C kinase 1 (RACK1) is a eukaryote-specific ribosomal protein implicated in diverse biological functions. To engineer ribosomes for specific fluorescent labeling, we selected RACK1 as an target given its location on the small ribosomal subunit and other properties. However, prior results suggested that RACK1 has roles both on and off the ribosome, and such an exchange might be related to its various cellular functions and hinder our ability to use RACK1 as a stable fluorescent tag for the ribosome. In addition, the kinetics of spontaneous exchange of RACK1 or any ribosomal protein from a mature ribosome in vitro remain unclear. To address these issues, we engineered fluorescently-labeled human ribosomes via RACK1, and applied bulk and single-molecule biochemical analyses to track RACK1 on and off the human ribosome. Our results demonstrate that, despite its cellular non-essentiality from yeast to humans, RACK1 readily re-associates with the ribosome, displays limited conformational dynamics, and remains stably bound to the ribosome for hours in vitro. This work sheds insight onto the biochemical basis of ribosomal protein exchange on and off a mature ribosome and provides tools for single-molecule analysis of human translation.

biochemistry↗

bioRxiv2018

Unbiased screen of RNA tailing enzymes at single-nucleotide resolution reveals a poly(UG) polymerase required for genome integrity and RNA silencing

Ribonucleotidyl transferases (rNTases) add non-templated ribonucleotides to diverse RNAs. We developed a screening strategy in S. cerevisiae to identify sequences added by candidate enzymes from different organisms at single-nucleotide resolution. The rNTase activities of 19 previously unexplored enzymes were determined. In addition to poly(A)- and poly(U)-adding enzymes, we identified a C-adding enzyme that is likely part of a two-enzyme system that adds CCA to tRNAs in a eukaryote; a nucleotidyl transferase that adds nucleotides to RNA without apparent nucleotide preference; and a poly(UG) polymerase, C. elegans MUT-2, which adds alternating U and G nucleotides to form poly(UG) tails. MUT-2 is known to be required for certain forms of RNA silencing, and mutations in the enzyme that are defective in silencing also fail to add poly(UG) tails in our assay. We propose that MUT-2 poly(UG) polymerase activity is required to promote genome integrity and RNA silencing.

molecular biology↗

bioRxiv2017

Architecture and dynamics of overlapped RNA regulatory networks

A single protein can bind and regulate many mRNAs. Multiple proteins with similar specificities often bind and control overlapping sets of mRNAs. Yet, little is known about the architecture or dynamics of overlapped networks. We focused on three proteins with similar structures and related RNA-binding specificities - Puf3p, Puf4p, and Puf5p of S. cerevisiae. Using RNA Tagging, we identified a \"super-network\" comprised of four sub-networks: Puf3p, Puf4p, and Puf5p sub-networks, and one controlled by both Puf4p and Puf5p. The architecture of individual subnetworks, and thus the super-network, are determined by competition among particular PUF proteins to bind mRNAs, their affinities for binding elements, and the abundances of the proteins. The super-network responds dramatically: the remaining network can either expand or contract. These strikingly opposite outcomes are determined by an interplay between the relative abundance of the RNAs and proteins, and their affinities for one another. The diverse interplay between overlapping RNA-protein networks provides versatile opportunities for regulation and evolution.

molecular biology↗

bioRxiv2017

Post-Transcriptional Control of Coenzyme Q Biosynthesis Revealed by Transomic Analysis of the RNA-Binding Protein Puf3p

Coenzyme Q (CoQ) is a redox active lipid required for mitochondrial oxidative phosphorylation (OxPhos). How CoQ biosynthesis is coordinated with the biogenesis of OxPhos protein complexes is unclear. Here, we show that the Saccharomyces cerevisiae RNA-binding protein (RBP) Puf3p directly regulates CoQ biosynthesis. To establish the mechanism for this regulation, we employed a transomic strategy to identify mRNAs that not only bind Puf3p, but also are regulated by Puf3p in vivo. The CoQ biosynthesis enzyme Coq5p is a critical Put3p target: Puf3p regulates the level of Coq5p and prevents its toxicity, thereby enabling efficient CoQ production. In parallel, Puf3p represses a specific set of proteins involved in mitochondrial protein import, translation, and OxPhos complex assembly -- pathways essential to prime mitochondrial biogenesis. Our data reveal a mechanism for post-transcriptionally coordinating CoQ production with OxPhos biogenesis and, more broadly, demonstrate the power of transomics for defining genuine targets of RBPs.\n\nHIGHLIGHTSO_LIThe RNA binding protein (RBP) Puf3p regulates coenzyme Q (CoQ) biosynthesis\nC_LIO_LITransomic analysis of RNAs, proteins, lipids, and metabolites defines RBP targets\nC_LIO_LIPuf3p regulates the potentially toxic CoQ biosynthesis enzyme Coq5p\nC_LIO_LIPuf3p couples regulation of CoQ with a broader program for controlling mitochondria\nC_LI

biochemistry↗