All Publications

  • eIF5B and eIF1A reorient initiator tRNA to allow ribosomal subunit joining. Nature Lapointe, C. P., Grosely, R., Sokabe, M., Alvarado, C., Wang, J., Montabana, E., Villa, N., Shin, B., Dever, T. E., Fraser, C. S., Fernandez, I. S., Puglisi, J. D. 2022


    Translation initiation defines the identity and quantity of a synthesized protein. The process is dysregulated in many human diseases1,2. A key commitment step is when the ribosomal subunits join at a translation start site on a messenger RNA to form a functional ribosome. 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 theuniversally conserved eukaryotic initiation factors eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we visualized initiation complexes that contained both eIF1A and eIF5B using single-particle cryo-electron microscopy. The resulting structure revealed how eukaryote-specific contacts between the two proteins remodel the initiation complex to orient the initiator aminoacyl-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 translation initiation in humans.

    View details for DOI 10.1038/s41586-022-04858-z

    View details for PubMedID 35732735

  • Dynamic competition between SARS-CoV-2 NSP1 and mRNA on the human ribosome inhibits translation initiation. Proceedings of the National Academy of Sciences of the United States of America Lapointe, C. P., Grosely, R. n., Johnson, A. G., Wang, J. n., Fernández, I. S., Puglisi, J. D. 2021; 118 (6)


    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a beta-CoV that 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. We demonstrate biochemically that NSP1 inhibits translation of model human and SARS-CoV-2 messenger RNAs (mRNAs). NSP1 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 determine that eukaryotic translation initiation factors (eIFs) allosterically modulate the interaction of NSP1 with ribosomal preinitiation complexes in the absence of mRNA. We further 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 proteins from viruses in at least two subgenera of beta-CoVs associate 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.

    View details for DOI 10.1073/pnas.2017715118

    View details for PubMedID 33479166

  • Records of RNA locations in living yeast revealed through covalent marks PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Medina-Munoz, H. C., Lapointe, C. P., Porter, D. F., Wickens, M. 2020; 117 (38): 23539–47


    RNA movements and localization pervade biology, from embryonic development to disease. To identify RNAs at specific locations, we developed a strategy in which a uridine-adding enzyme is anchored to subcellular sites, where it directly marks RNAs with 3' terminal uridines. This localized RNA recording approach yields a record of RNA locations, and is validated through identification of RNAs localized selectively to the endoplasmic reticulum (ER) or mitochondria. We identify a broad dual localization pattern conserved from yeast to human cells, in which the same battery of mRNAs encounter both ER and mitochondria in both species, and include an mRNA encoding a key stress sensor. Subunits of many multiprotein complexes localize to both the ER and mitochondria, suggesting coordinated assembly. Noncoding RNAs in the course of RNA surveillance and processing encounter both organelles. By providing a record of RNA locations over time, the approach complements those that capture snapshots of instantaneous positions.

    View details for DOI 10.1073/pnas.1921408117

    View details for Web of Science ID 000575888700022

    View details for PubMedID 32907940

    View details for PubMedCentralID PMC7519331

  • A memory of eS25 loss drives resistance phenotypes. Nucleic acids research Johnson, A. G., Flynn, R. A., Lapointe, C. P., Ooi, Y. S., Zhao, M. L., Richards, C. M., Qiao, W. n., Yamada, S. B., Couthouis, J. n., Gitler, A. D., Carette, J. E., Puglisi, J. D. 2020


    In order to maintain cellular protein homeostasis, ribosomes are safeguarded against dysregulation by myriad processes. Remarkably, many cell types can withstand genetic lesions of certain ribosomal protein genes, some of which are linked to diverse cellular phenotypes and human disease. Yet the direct and indirect consequences from these lesions 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 and suggests caution in the interpretation of ribosomal protein gene mutation data.

    View details for DOI 10.1093/nar/gkaa444

    View details for PubMedID 32463448

  • RACK1 on and off the ribosome RNA Johnson, A. G., Lapointe, C. P., Wang, J., Corsepius, N. C., Choi, J., Fuchs, G., Puglisi, J. D. 2019; 25 (7): 881–95
  • Unbiased screen of RNA tailing activities reveals a poly(UG) polymerase NATURE METHODS Preston, M. A., Porter, D. F., Chen, F., Buter, N., Lapointe, C. P., Keles, S., Kimble, J., Wickens, M. 2019; 16 (5): 437-+
  • eIF5B gates the transition from translation initiation to elongation. Nature Wang, J. n., Johnson, A. G., Lapointe, C. P., Choi, J. n., Prabhakar, A. n., Chen, D. H., Petrov, A. N., Puglisi, J. D. 2019


    Translation initiation determines both the quantity and identity of the protein that is encoded in an mRNA by establishing the reading frame for protein synthesis. In eukaryotic cells, numerous translation initiation factors prepare ribosomes for polypeptide synthesis; however, the underlying dynamics of this process remain unclear1,2. A central question is how eukaryotic ribosomes transition from translation initiation to elongation. Here we use in vitro single-molecule fluorescence microscopy approaches in a purified yeast Saccharomyces cerevisiae translation system to monitor directly, in real time, the pathways of late translation initiation and the transition to elongation. This transition was slower in our eukaryotic system than that reported for Escherichia coli3-5. The slow entry to elongation was defined by a long residence time of eukaryotic initiation factor 5B (eIF5B) on the 80S ribosome after the joining of individual ribosomal subunits-a process that is catalysed by this universally conserved initiation factor. Inhibition of the GTPase activity of eIF5B after the joining of ribosomal subunits prevented the dissociation of eIF5B from the 80S complex, thereby preventing elongation. Our findings illustrate how the dissociation of eIF5B serves as a kinetic checkpoint for the transition from initiation to elongation, and how its release may be governed by a change in the conformation of the ribosome complex that triggers GTP hydrolysis.

    View details for DOI 10.1038/s41586-019-1561-0

    View details for PubMedID 31534220

  • How Messenger RNA and Nascent Chain Sequences Regulate Translation Elongation. Annual review of biochemistry Choi, J., Grosely, R., Prabhakar, A., Lapointe, C. P., Wang, J., Puglisi, J. D. 2018; 87: 421–49


    Translation elongation is a highly coordinated, multistep, multifactor process that ensures accurate and efficient addition of amino acids to a growing nascent-peptide chain encoded in the sequence of translated messenger RNA (mRNA). Although translation elongation is heavily regulated by external factors, there is clear evidence that mRNA and nascent-peptide sequences control elongation dynamics, determining both the sequence and structure of synthesized proteins. Advances in methods have driven experiments that revealed the basic mechanisms of elongation as well as the mechanisms of regulation by mRNA and nascent-peptide sequences. In this review, we highlight how mRNA and nascent-peptide elements manipulate the translation machinery to alter the dynamics and pathway of elongation.

    View details for PubMedID 29925264

  • Multi-omics Reveal Specific Targets of the RNA-Binding Protein Puf3p and Its Orchestration of Mitochondrial Biogenesis CELL SYSTEMS Lapointe, C. P., Stefely, J. A., Jochem, A., Hutchins, P. D., Wilson, G. M., Kwiecien, N. W., Coon, J. J., Wickens, M., Pagliarini, D. J. 2018; 6 (1): 125-+


    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 regulates CoQ biosynthesis. To establish the mechanism for this regulation, we employed a multi-omic 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 Puf3p target: Puf3p regulates the abundance of Coq5p and prevents its detrimental hyperaccumulation, thereby enabling efficient CoQ production. More broadly, 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 they demonstrate the power of multi-omics for defining genuine targets of RBPs.

    View details for DOI 10.1016/j.cels.2017.11.012

    View details for Web of Science ID 000423444900015

    View details for PubMedID 29248374

    View details for PubMedCentralID PMC5799006

  • How Messenger RNA and Nascent Chain Sequences Regulate Translation Elongation ANNUAL REVIEW OF BIOCHEMISTRY, VOL 87 Choi, J., Grosely, R., Prabhakar, A., Lapointe, C. P., Wang, J., Puglisi, J. D., Kornberg, R. D. 2018; 87: 421–49
  • RNA Tagging: Preparation of High-Throughput Sequencing Libraries. Methods in molecular biology (Clifton, N.J.) Lapointe, C. P., Wickens, M. 2018; 1649: 455–71


    Protein-RNA networks, in which a single protein binds and controls multiple mRNAs, are central in biological control. As a result, methods to identify protein-RNA interactions that occur in vivo are valuable. The "RNA Tagging" approach enables the investigator to unambiguously identify global protein-RNA interactions in vivo and is independent of protein purification, cross-linking, and radioactive labeling steps. Here, we provide a protocol to prepare high-throughput sequencing libraries for RNA Tagging experiments.

    View details for DOI 10.1007/978-1-4939-7213-5_30

    View details for PubMedID 29130216

  • Architecture and dynamics of overlapped RNA regulatory networks RNA Lapointe, C. P., Preston, M. A., Wilinski, D., Saunders, H. J., Campbell, Z. T., Wickens, M. 2017; 23 (11): 1636–47


    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 subnetworks: Puf3p, Puf4p, and Puf5p subnetworks, and one controlled by both Puf4p and Puf5p. The architecture of individual subnetworks, and thus the super-network, is 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.

    View details for DOI 10.1261/rna.062687.117

    View details for Web of Science ID 000412996100004

    View details for PubMedID 28768715

    View details for PubMedCentralID PMC5648032

  • Recurrent rewiring and emergence of RNA regulatory networks PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wilinski, D., Buter, N., Klocko, A. D., Lapointe, C. P., Selker, E. U., Gasch, A. P., Wickens, M. 2017; 114 (14): E2816–E2825


    Alterations in regulatory networks contribute to evolutionary change. Transcriptional networks are reconfigured by changes in the binding specificity of transcription factors and their cognate sites. The evolution of RNA-protein regulatory networks is far less understood. The PUF (Pumilio and FBF) family of RNA regulatory proteins controls the translation, stability, and movements of hundreds of mRNAs in a single species. We probe the evolution of PUF-RNA networks by direct identification of the mRNAs bound to PUF proteins in budding and filamentous fungi and by computational analyses of orthologous RNAs from 62 fungal species. Our findings reveal that PUF proteins gain and lose mRNAs with related and emergent biological functions during evolution. We demonstrate at least two independent rewiring events for PUF3 orthologs, independent but convergent evolution of PUF4/5 binding specificity and the rewiring of the PUF4/5 regulons in different fungal lineages. These findings demonstrate plasticity in RNA regulatory networks and suggest ways in which their rewiring occurs.

    View details for DOI 10.1073/pnas.1617777114

    View details for Web of Science ID 000398159000005

    View details for PubMedID 28320951

    View details for PubMedCentralID PMC5389312

  • Protein-RNA networks revealed through covalent RNA marks NATURE METHODS Lapointe, C. P., Wilinski, D., Saunders, H. J., Wickens, M. 2015; 12 (12): 1163-+


    Protein-RNA networks are ubiquitous and central in biological control. We present an approach termed RNA Tagging that enables the user to identify protein-RNA interactions in vivo by analyzing purified cellular RNA, without protein purification or cross-linking. An RNA-binding protein of interest is fused to an enzyme that adds uridines to the end of RNA. RNA targets bound by the chimeric protein in vivo are covalently marked with uridines and subsequently identified from extracted RNA via high-throughput sequencing. We used this approach to identify hundreds of RNAs bound by a Saccharomyces cerevisiae PUF protein, Puf3p. The results showed that although RNA-binding proteins productively bind specific RNAs to control their function, they also 'sample' RNAs without exerting a regulatory effect. We used the method to uncover hundreds of new and likely regulated targets for a protein without canonical RNA-binding domains, Bfr1p. RNA Tagging is well suited to detect and analyze protein-RNA networks in vivo.

    View details for DOI 10.1038/nmeth.3651

    View details for Web of Science ID 000365890700025

    View details for PubMedID 26524240

    View details for PubMedCentralID PMC4707952

  • RNA regulatory networks diversified through curvature of the PUF protein scaffold NATURE COMMUNICATIONS Wilinski, D., Qiu, C., Lapointe, C. P., Nevil, M., Campbell, Z. T., Hall, T., Wickens, M. 2015; 6: 8213


    Proteins bind and control mRNAs, directing their localization, translation and stability. Members of the PUF family of RNA-binding proteins control multiple mRNAs in a single cell, and play key roles in development, stem cell maintenance and memory formation. Here we identified the mRNA targets of a S. cerevisiae PUF protein, Puf5p, by ultraviolet-crosslinking-affinity purification and high-throughput sequencing (HITS-CLIP). The binding sites recognized by Puf5p are diverse, with variable spacer lengths between two specific sequences. Each length of site correlates with a distinct biological function. Crystal structures of Puf5p-RNA complexes reveal that the protein scaffold presents an exceptionally flat and extended interaction surface relative to other PUF proteins. In complexes with RNAs of different lengths, the protein is unchanged. A single PUF protein repeat is sufficient to induce broadening of specificity. Changes in protein architecture, such as alterations in curvature, may lead to evolution of mRNA regulatory networks.

    View details for DOI 10.1038/ncomms9213

    View details for Web of Science ID 000362854500003

    View details for PubMedID 26364903

    View details for PubMedCentralID PMC4570272

  • The Nucleic Acid-binding Domain and Translational Repression Activity of a Xenopus Terminal Uridylyl Transferase JOURNAL OF BIOLOGICAL CHEMISTRY Lapointe, C. P., Wickens, M. 2013; 288 (28): 20723–33


    Terminal uridylyl transferases (TUTs) catalyze the addition of uridines to the 3' ends of RNAs and are implicated in the regulation of both messenger RNAs and microRNAs. To better understand how TUTs add uridines to RNAs, we focused on a putative TUT from Xenopus laevis, XTUT7. We determined that XTUT7 catalyzed the addition of uridines to RNAs. Mutational analysis revealed that a truncated XTUT7 enzyme, which contained solely the nucleotidyl transferase and poly(A) polymerase-associated domains, was sufficient for catalytic activity. XTUT7 activity decreased upon removal of the CCHC zinc finger domains and a short segment of basic amino acids (the basic region). This basic region bound nucleic acids in vitro. We also demonstrated that XTUT7 repressed translation of a polyadenylated RNA, to which it added a distinct number of uridines. We generated a predicted structure of the XTUT7 catalytic core that indicated histidine 1269 was likely important for uridine specificity. Indeed, mutation of histidine 1269 broadened the nucleotide specificity of XTUT7 and abolished XTUT7-dependent translational repression. Our data reveal key aspects of how XTUT7 adds uridines to RNAs, highlight the role of the basic region, illustrate that XTUT7 can repress translation, and identify an amino acid important for uridine specificity.

    View details for DOI 10.1074/jbc.M113.455451

    View details for Web of Science ID 000321715100057

    View details for PubMedID 23709223

    View details for PubMedCentralID PMC3711335