Publications by authors named "Marlene Oeffinger"

34 Publications

Choosing the right exit: How functional plasticity of the nuclear pore drives selective and efficient mRNA export.

Wiley Interdiscip Rev RNA 2021 Nov 2;12(6):e1660. Epub 2021 May 2.

Systems Biology, Institut de Recherches Cliniques de Montréal, Montréal, Canada.

The nuclear pore complex (NPC) serves as a central gate for mRNAs to transit from the nucleus to the cytoplasm. The ability for mRNAs to get exported is linked to various upstream nuclear processes including co-transcriptional RNP assembly and processing, and only export competent mRNPs are thought to get access to the NPC. While the nuclear pore is generally viewed as a monolithic structure that serves as a mediator of transport driven by transport receptors, more recent evidence suggests that the NPC might be more heterogenous than previously believed, both in its composition or in the selective treatment of cargo that seek access to the pore, providing functional plasticity to mRNA export. In this review, we consider the interconnected processes of nuclear mRNA metabolism that contribute and mediate export competence. Furthermore, we examine different aspects of NPC heterogeneity, including the role of the nuclear basket and its associated complexes in regulating selective and/or efficient binding to and transport through the pore. This article is categorized under: RNA Export and Localization > Nuclear Export/Import RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.
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http://dx.doi.org/10.1002/wrna.1660DOI Listing
November 2021

Insights into synthesis and function of KsgA/Dim1-dependent rRNA modifications in archaea.

Nucleic Acids Res 2021 02;49(3):1662-1687

Regensburg Center for Biochemistry, Biochemistry III - Institute for Biochemistry, Genetics and Microbiology, University of Regensburg, Universitätsstraße 31, 93053 Regensburg, Germany.

Ribosomes are intricate molecular machines ensuring proper protein synthesis in every cell. Ribosome biogenesis is a complex process which has been intensively analyzed in bacteria and eukaryotes. In contrast, our understanding of the in vivo archaeal ribosome biogenesis pathway remains less characterized. Here, we have analyzed the in vivo role of the almost universally conserved ribosomal RNA dimethyltransferase KsgA/Dim1 homolog in archaea. Our study reveals that KsgA/Dim1-dependent 16S rRNA dimethylation is dispensable for the cellular growth of phylogenetically distant archaea. However, proteomics and functional analyses suggest that archaeal KsgA/Dim1 and its rRNA modification activity (i) influence the expression of a subset of proteins and (ii) contribute to archaeal cellular fitness and adaptation. In addition, our study reveals an unexpected KsgA/Dim1-dependent variability of rRNA modifications within the archaeal phylum. Combining structure-based functional studies across evolutionary divergent organisms, we provide evidence on how rRNA structure sequence variability (re-)shapes the KsgA/Dim1-dependent rRNA modification status. Finally, our results suggest an uncoupling between the KsgA/Dim1-dependent rRNA modification completion and its release from the nascent small ribosomal subunit. Collectively, our study provides additional understandings into principles of molecular functional adaptation, and further evolutionary and mechanistic insights into an almost universally conserved step of ribosome synthesis.
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http://dx.doi.org/10.1093/nar/gkaa1268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7897474PMC
February 2021

Altered rRNA processing disrupts nuclear RNA homeostasis via competition for the poly(A)-binding protein Nab2.

Nucleic Acids Res 2020 11;48(20):11675-11694

Department of Cell Biology, University of Alberta, Edmonton, Canada.

RNA-binding proteins (RBPs) are key mediators of RNA metabolism. Whereas some RBPs exhibit narrow transcript specificity, others function broadly across both coding and non-coding RNAs. Here, in Saccharomyces cerevisiae, we demonstrate that changes in RBP availability caused by disruptions to distinct cellular processes promote a common global breakdown in RNA metabolism and nuclear RNA homeostasis. Our data shows that stabilization of aberrant ribosomal RNA (rRNA) precursors in an enp1-1 mutant causes phenotypes similar to RNA exosome mutants due to nucleolar sequestration of the poly(A)-binding protein (PABP) Nab2. Decreased nuclear PABP availability is accompanied by genome-wide changes in RNA metabolism, including increased pervasive transcripts levels and snoRNA processing defects. These phenotypes are mitigated by overexpression of PABPs, inhibition of rDNA transcription, or alterations in TRAMP activity. Our results highlight the need for cells to maintain poly(A)-RNA levels in balance with PABPs and other RBPs with mutable substrate specificity across nucleoplasmic and nucleolar RNA processes.
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http://dx.doi.org/10.1093/nar/gkaa964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672433PMC
November 2020

It's Not the Destination, It's the Journey: Heterogeneity in mRNA Export Mechanisms.

Adv Exp Med Biol 2019 ;1203:33-81

Institut de recherches cliniques de Montréal, Montréal, QC, Canada.

The process of creating a translation-competent mRNA is highly complex and involves numerous steps including transcription, splicing, addition of modifications, and, finally, export to the cytoplasm. Historically, much of the research on regulation of gene expression at the level of the mRNA has been focused on either the regulation of mRNA synthesis (transcription and splicing) or metabolism (translation and degradation). However, in recent years, the advent of new experimental techniques has revealed the export of mRNA to be a major node in the regulation of gene expression, and numerous large-scale and specific mRNA export pathways have been defined. In this chapter, we will begin by outlining the mechanism by which most mRNAs are homeostatically exported ("bulk mRNA export"), involving the recruitment of the NXF1/TAP export receptor by the Aly/REF and THOC5 components of the TREX complex. We will then examine various mechanisms by which this pathway may be controlled, modified, or bypassed in order to promote the export of subset(s) of cellular mRNAs, which include the use of metazoan-specific orthologs of bulk mRNA export factors, specific cis RNA motifs which recruit mRNA export machinery via specific trans-acting-binding factors, posttranscriptional mRNA modifications that act as "inducible" export cis elements, the use of the atypical mRNA export receptor, CRM1, and the manipulation or bypass of the nuclear pore itself. Finally, we will discuss major outstanding questions in the field of mRNA export heterogeneity and outline how cutting-edge experimental techniques are providing new insights into and tools for investigating the intriguing field of mRNA export heterogeneity.
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http://dx.doi.org/10.1007/978-3-030-31434-7_2DOI Listing
December 2019

Structural studies of the eIF4E-VPg complex reveal a direct competition for capped RNA: Implications for translation.

Proc Natl Acad Sci U S A 2019 11 11;116(48):24056-24065. Epub 2019 Nov 11.

Institute of Research in Immunology and Cancer, Department of Pathology and Cell Biology, Université de Montréal, Pavilion Marcelle-Coutu, Chemin Polytechnique, Montréal, QC H3T 1J4, Canada;

Viruses have transformed our understanding of mammalian RNA processing, including facilitating the discovery of the methyl-7-guanosine (mG) cap on the 5' end of RNAs. The mG cap is required for RNAs to bind the eukaryotic translation initiation factor eIF4E and associate with the translation machinery across plant and animal kingdoms. The potyvirus-derived viral genome-linked protein (VPg) is covalently bound to the 5' end of viral genomic RNA (gRNA) and associates with host eIF4E for successful infection. Divergent models to explain these observations proposed either an unknown mode of eIF4E engagement or a competition of VPg for the mG cap-binding site. To dissect these possibilities, we resolved the structure of VPg, revealing a previously unknown 3-dimensional (3D) fold, and characterized the VPg-eIF4E complex using NMR and biophysical techniques. VPg directly bound the cap-binding site of eIF4E and competed for mG cap analog binding. In human cells, VPg inhibited eIF4E-dependent RNA export, translation, and oncogenic transformation. Moreover, VPg formed trimeric complexes with eIF4E-eIF4G, eIF4E bound VPg- RNA conjugates, and these VPg-RNA conjugates were templates for translation. Informatic analyses revealed structural similarities between VPg and the human kinesin EG5. Consistently, EG5 directly bound eIF4E in a similar manner to VPg, demonstrating that this form of engagement is relevant beyond potyviruses. In all, we revealed an unprecedented modality for control and engagement of eIF4E and show that VPg-RNA conjugates functionally engage eIF4E. As such, potyvirus VPg provides a unique model system to interrogate eIF4E.
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http://dx.doi.org/10.1073/pnas.1904752116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883836PMC
November 2019

Live-Cell Imaging of mRNP-NPC Interactions in Budding Yeast.

Methods Mol Biol 2019 ;2038:131-150

Department of Cell Biology, University of Alberta, Edmonton, Canada.

Single-molecule resolution imaging has become an important tool in the study of cell biology. Aptamer-based approaches (e.g., MS2 and PP7) allow for detection of single RNA molecules in living cells and have been used to study various aspects of mRNA metabolism, including mRNP nuclear export. Here we outline an imaging protocol for the study of interactions between mRNPs and nuclear pore complexes (NPCs) in the yeast S. cerevisiae, including mRNP export. We describe in detail the steps that allow for high-resolution live-cell mRNP imaging and measurement of mRNP interactions with NPCs using simultaneous two-color imaging. Our protocol discusses yeast strain construction, choice of marker proteins to label the nuclear pore complex, as well as imaging conditions that allow high signal-to-noise data acquisition. Moreover, we describe various aspects of postacquisition image analysis for single molecule tracking and image registration allowing for the characterization of mRNP-NPC interactions.
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http://dx.doi.org/10.1007/978-1-4939-9674-2_9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392168PMC
April 2020

Senescence-associated ribosome biogenesis defects contributes to cell cycle arrest through the Rb pathway.

Nat Cell Biol 2018 07 25;20(7):789-799. Epub 2018 Jun 25.

Department of Biochemistry and Molecular Medicine, Université de Montréal, Montreal, Quebec, Canada.

Cellular senescence is a tumour suppressor programme characterized by a stable cell cycle arrest. Here we report that cellular senescence triggered by a variety of stimuli leads to diminished ribosome biogenesis and the accumulation of both rRNA precursors and ribosomal proteins. These defects were associated with reduced expression of several ribosome biogenesis factors, the knockdown of which was also sufficient to induce senescence. Genetic analysis revealed that Rb but not p53 was required for the senescence response to altered ribosome biogenesis. Mechanistically, the ribosomal protein S14 (RPS14 or uS11) accumulates in the soluble non-ribosomal fraction of senescent cells, where it binds and inhibits CDK4 (cyclin-dependent kinase 4). Overexpression of RPS14 is sufficient to inhibit Rb phosphorylation, inducing cell cycle arrest and senescence. Here we describe a mechanism for maintaining the senescent cell cycle arrest that may be relevant for cancer therapy, as well as biomarkers to identify senescent cells.
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http://dx.doi.org/10.1038/s41556-018-0127-yDOI Listing
July 2018

Nol12 is a multifunctional RNA binding protein at the nexus of RNA and DNA metabolism.

Nucleic Acids Res 2017 Dec;45(21):12509-12528

Institut de Recherches Cliniques de Montréal, 110 Avenue des Pins Ouest, Montréal, Québec H2W 1R7, Canada.

To counteract the breakdown of genome integrity, eukaryotic cells have developed a network of surveillance pathways to prevent and resolve DNA damage. Recent data has recognized the importance of RNA binding proteins (RBPs) in DNA damage repair (DDR) pathways. Here, we describe Nol12 as a multifunctional RBP with roles in RNA metabolism and genome maintenance. Nol12 is found in different subcellular compartments-nucleoli, where it associates with ribosomal RNA and is required for efficient separation of large and small subunit precursors at site 2; the nucleoplasm, where it co-localizes with the RNA/DNA helicase Dhx9 and paraspeckles; as well as GW/P-bodies in the cytoplasm. Loss of Nol12 results in the inability of cells to recover from DNA stress and a rapid p53-independent ATR-Chk1-mediated apoptotic response. Nol12 co-localizes with DNA repair proteins in vivo including Dhx9, as well as with TOPBP1 at sites of replication stalls, suggesting a role for Nol12 in the resolution of DNA stress and maintenance of genome integrity. Identification of a complex Nol12 interactome, which includes NONO, Dhx9, DNA-PK and Stau1, further supports the protein's diverse functions in RNA metabolism and DNA maintenance, establishing Nol12 as a multifunctional RBP essential for genome integrity.
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http://dx.doi.org/10.1093/nar/gkx963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716212PMC
December 2017

The RNA chaperone La promotes pre-tRNA maturation via indiscriminate binding of both native and misfolded targets.

Nucleic Acids Res 2017 Nov;45(19):11341-11355

Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada.

Non-coding RNAs have critical roles in biological processes, and RNA chaperones can promote their folding into the native shape required for their function. La proteins are a class of highly abundant RNA chaperones that contact pre-tRNAs and other RNA polymerase III transcripts via their common UUU-3'OH ends, as well as through less specific contacts associated with RNA chaperone activity. However, whether La proteins preferentially bind misfolded pre-tRNAs or instead engage all pre-tRNA substrates irrespective of their folding status is not known. La deletion in yeast is synthetically lethal when combined with the loss of tRNA modifications predicted to contribute to the native pre-tRNA fold, such as the N2, N2-dimethylation of G26 by the methyltransferase Trm1p. In this work, we identify G26 containing pre-tRNAs that misfold in the absence of Trm1p and/or La (Sla1p) in Schizosaccharomyces pombe cells, then test whether La preferentially associates with such tRNAs in vitro and in vivo. Our data suggest that La does not discriminate a native from misfolded RNA target, and highlights the potential challenges faced by RNA chaperones in preferentially binding defective substrates.
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http://dx.doi.org/10.1093/nar/gkx764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737608PMC
November 2017

High-throughput RNA structure probing reveals critical folding events during early 60S ribosome assembly in yeast.

Nat Commun 2017 09 28;8(1):714. Epub 2017 Sep 28.

Centre for Synthetic and Systems Biology (SynthSys), University of Edinburgh, Edinburgh, EH9 3BF, UK.

While the protein composition of various yeast 60S ribosomal subunit assembly intermediates has been studied in detail, little is known about ribosomal RNA (rRNA) structural rearrangements that take place during early 60S assembly steps. Using a high-throughput RNA structure probing method, we provide nucleotide resolution insights into rRNA structural rearrangements during nucleolar 60S assembly. Our results suggest that many rRNA-folding steps, such as folding of 5.8S rRNA, occur at a very specific stage of assembly, and propose that downstream nuclear assembly events can only continue once 5.8S folding has been completed. Our maps of nucleotide flexibility enable making predictions about the establishment of protein-rRNA interactions, providing intriguing insights into the temporal order of protein-rRNA as well as long-range inter-domain rRNA interactions. These data argue that many distant domains in the rRNA can assemble simultaneously during early 60S assembly and underscore the enormous complexity of 60S synthesis.Ribosome biogenesis is a dynamic process that involves the ordered assembly of ribosomal proteins and numerous RNA structural rearrangements. Here the authors apply ChemModSeq, a high-throughput RNA structure probing method, to quantitatively measure changes in RNA flexibility during the nucleolar stages of 60S assembly in yeast.
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http://dx.doi.org/10.1038/s41467-017-00761-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5620067PMC
September 2017

DDX54 regulates transcriptome dynamics during DNA damage response.

Genome Res 2017 08 8;27(8):1344-1359. Epub 2017 Jun 8.

Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin Institute for Medical Systems Biology, 13125 Berlin, Germany.

The cellular response to genotoxic stress is mediated by a well-characterized network of DNA surveillance pathways. The contribution of post-transcriptional gene regulatory networks to the DNA damage response (DDR) has not been extensively studied. Here, we systematically identified RNA-binding proteins differentially interacting with polyadenylated transcripts upon exposure of human breast carcinoma cells to ionizing radiation (IR). Interestingly, more than 260 proteins, including many nucleolar proteins, showed increased binding to poly(A) RNA in IR-exposed cells. The functional analysis of DDX54, a candidate genotoxic stress responsive RNA helicase, revealed that this protein is an immediate-to-early DDR regulator required for the splicing efficacy of its target IR-induced pre-mRNAs. Upon IR exposure, DDX54 acts by increased interaction with a well-defined class of pre-mRNAs that harbor introns with weak acceptor splice sites, as well as by protein-protein contacts within components of U2 snRNP and spliceosomal B complex, resulting in lower intron retention and higher processing rates of its target transcripts. Because DDX54 promotes survival after exposure to IR, its expression and/or mutation rate may impact DDR-related pathologies. Our work indicates the relevance of many uncharacterized RBPs potentially involved in the DDR.
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http://dx.doi.org/10.1101/gr.218438.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538551PMC
August 2017

The sole LSm complex in associates with pre-mRNA splicing and mRNA degradation factors.

RNA 2017 06 21;23(6):952-967. Epub 2017 Mar 21.

Department of Chemistry, University of Northern British Columbia, Prince George, BC V2N 4Z9, Canada.

Proteins of the Sm and Sm-like (LSm) families, referred to collectively as (L)Sm proteins, are found in all three domains of life and are known to promote a variety of RNA processes such as base-pair formation, unwinding, RNA degradation, and RNA stabilization. In eukaryotes, (L)Sm proteins have been studied, inter alia, for their role in pre-mRNA splicing. In many organisms, the LSm proteins form two distinct complexes, one consisting of LSm1-7 that is involved in mRNA degradation in the cytoplasm, and the other consisting of LSm2-8 that binds spliceosomal U6 snRNA in the nucleus. We recently characterized the splicing proteins from the red alga and found that it has only seven LSm proteins. The identities of CmLSm2-CmLSm7 were unambiguous, but the seventh protein was similar to LSm1 and LSm8. Here, we use in vitro binding measurements, microscopy, and affinity purification-mass spectrometry to demonstrate a canonical splicing function for the LSm complex and experimentally validate our bioinformatic predictions of a reduced spliceosome in this organism. Copurification of Pat1 and its associated mRNA degradation proteins with the LSm proteins, along with evidence of a cytoplasmic fraction of CmLSm complexes, argues that this complex is involved in both splicing and cytoplasmic mRNA degradation. Intriguingly, the Pat1 complex also copurifies with all four snRNAs, suggesting the possibility of a spliceosome-associated pre-mRNA degradation complex in the nucleus.
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http://dx.doi.org/10.1261/rna.058487.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5435867PMC
June 2017

Nucleolin and nucleophosmin: nucleolar proteins with multiple functions in DNA repair.

Biochem Cell Biol 2016 Oct 29;94(5):419-432. Epub 2016 Jun 29.

a Laboratory of RNP Biochemistry, Institut de recherches cliniques de Montréal (IRCM), Montréal, QC H2W 1R7, Canada.

The nucleolus represents a highly multifunctional intranuclear organelle in which, in addition to the canonical ribosome assembly, numerous processes such as transcription, DNA repair and replication, the cell cycle, and apoptosis are coordinated. The nucleolus is further a key hub in the sensing of cellular stress and undergoes major structural and compositional changes in response to cellular perturbations. Numerous nucleolar proteins have been identified that, upon sensing nucleolar stress, deploy additional, non-ribosomal roles in the regulation of varied cell processes including cell cycle arrest, arrest of DNA replication, induction of DNA repair, and apoptosis, among others. The highly abundant proteins nucleophosmin (NPM1) and nucleolin (NCL) are two such factors that transit to the nucleoplasm in response to stress, and participate directly in the repair of numerous different DNA damages. This review discusses the contributions made by NCL and (or) NPM1 to the different DNA repair pathways employed by mammalian cells to repair DNA insults, and examines the implications of such activities for the regulation, pathogenesis, and therapeutic targeting of NPM1 and NCL.
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http://dx.doi.org/10.1139/bcb-2016-0068DOI Listing
October 2016

Structural biology: Moulding the ribosome.

Nature 2016 09;537(7618):38-40

Department for Systems Biology, Institut de recherches cliniques de Montréal, Montreal, Quebec H2W 1R7, Canada.

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http://dx.doi.org/10.1038/537038aDOI Listing
September 2016

Imaging single mRNAs to study dynamics of mRNA export in the yeast Saccharomyces cerevisiae.

Methods 2016 Apr 16;98:104-114. Epub 2016 Jan 16.

Département de Biochimieet médecine moléculaire, Faculté de médecine, Université de Montréal, Montréal, Québec H3C 3J7, ​Canada. Electronic address:

Regulation of mRNA and protein expression occurs at many levels, initiated at transcription and followed by mRNA processing, export, localization, translation and mRNA degradation. The ability to study mRNAs in living cells has become a critical tool to study and analyze how the various steps of the gene expression pathway are carried out. Here we describe a detailed protocol for real time fluorescent RNA imaging using the PP7 bacteriophage coat protein, which allows mRNA detection with high spatial and temporal resolution in the yeast Saccharomyces cerevisiae, and can be applied to study various stages of mRNA metabolism. We describe the different parameters required for quantitative single molecule imaging in yeast, including strategies for genomic integration, expression of a PP7 coat protein GFP fusion protein, microscope setup and analysis strategies. We illustrate the method's use by analyzing the behavior of nuclear mRNA in yeast and the role of the nuclear basket in mRNA export.
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http://dx.doi.org/10.1016/j.ymeth.2016.01.006DOI Listing
April 2016

The nuclear basket mediates perinuclear mRNA scanning in budding yeast.

J Cell Biol 2015 Dec;211(6):1131-40

Departement de Biochimie et Medecine Moleculaire, Faculte de Medecine, Universite de Montreal, H3T 1J4 Montreal, Quebec, Canada

After synthesis and transit through the nucleus, messenger RNAs (mRNAs) are exported to the cytoplasm through the nuclear pore complex (NPC). At the NPC, messenger ribonucleoproteins (mRNPs) first encounter the nuclear basket where mRNP rearrangements are thought to allow access to the transport channel. Here, we use single mRNA resolution live cell microscopy and subdiffraction particle tracking to follow individual mRNAs on their path toward the cytoplasm. We show that when reaching the nuclear periphery, RNAs are not immediately exported but scan along the nuclear periphery, likely to find a nuclear pore allowing export. Deletion or mutation of the nuclear basket proteins MLP1/2 or the mRNA binding protein Nab2 changes the scanning behavior of mRNPs at the nuclear periphery, shortens residency time at nuclear pores, and results in frequent release of mRNAs back into the nucleoplasm. These observations suggest a role for the nuclear basket in providing an interaction platform that keeps RNAs at the periphery, possibly to allow mRNP rearrangements before export.
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http://dx.doi.org/10.1083/jcb.201503070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687876PMC
December 2015

Targeted cross-linking-mass spectrometry determines vicinal interactomes within heterogeneous RNP complexes.

Nucleic Acids Res 2016 Feb 10;44(3):1354-69. Epub 2015 Dec 10.

Department for Systems Biology, Institut de recherches cliniques de Montréal, Montréal, Québec H2W 1R7, Canada Département de biochimie, Faculté de médecine, Université de Montréal, Montréal, Québec H3T 1J4, Canada Division of Experimental Medicine, Faculty of Medicine, McGill University, Montréal, Québec H3A 1A3, Canada

Proteomic and RNomic approaches have identified many components of different ribonucleoprotein particles (RNPs), yet still little is known about the organization and protein proximities within these heterogeneous and highly dynamic complexes. Here we describe a targeted cross-linking approach, which combines cross-linking from a known anchor site with affinity purification and mass spectrometry (MS) to identify the changing vicinity interactomes along RNP maturation pathways. Our method confines the reaction radius of a heterobifunctional cross-linker to a specific interaction surface, increasing the probability to capture low abundance conformations and transient vicinal interactors too infrequent for identification by traditional cross-linking-MS approaches, and determine protein proximities within RNPs. Applying the method to two conserved RNA-associated complexes in Saccharomyces cerevisae, the mRNA export receptor Mex67:Mtr2 and the pre-ribosomal Nop7 subcomplex, we identified dynamic vicinal interactomes within those complexes and along their changing pathway milieu. Our results therefore show that this method provides a new tool to study the changing spatial organization of heterogeneous dynamic RNP complexes.
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http://dx.doi.org/10.1093/nar/gkv1366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4756821PMC
February 2016

Single-Step Affinity Purification (ssAP) and Mass Spectrometry of Macromolecular Complexes in the Yeast S. cerevisiae.

Methods Mol Biol 2016 ;1361:265-87

Institut de recherches cliniques de Montréal, 110 Avenue des Pins Ouest, Montréal, QC, Canada, H2W 1R7.

Cellular functions are mostly defined by the dynamic interactions of proteins within macromolecular networks. Deciphering the composition of macromolecular complexes and their dynamic rearrangements is the key to getting a comprehensive picture of cellular behavior and to understanding biological systems. In the last decade, affinity purification coupled to mass spectrometry has emerged as a powerful tool to comprehensively study interaction networks and their assemblies. However, the study of these interactomes has been hampered by severe methodological limitations. In particular, the affinity purification of intact complexes from cell lysates suffers from protein and RNA degradation, loss of transient interactors, and poor overall yields. In this chapter, we describe a rapid single-step affinity purification method for the efficient isolation of dynamic macromolecular complexes. The technique employs cell lysis by cryo-milling, which ensures nondegraded starting material in the submicron range, and magnetic beads, which allow for dense antibody-conjugation and thus rapid complex isolation, while avoiding loss of transient interactions. The method is epitope tag-independent, and overcomes many of the previous limitations to produce large interactomes with almost no contamination. The protocol described here has been optimized for the yeast S. cerevisiae.
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http://dx.doi.org/10.1007/978-1-4939-3079-1_15DOI Listing
August 2016

Emerging properties of nuclear RNP biogenesis and export.

Curr Opin Cell Biol 2015 Jun 15;34:46-53. Epub 2015 May 15.

Department of Cell Biology, University of Alberta, 5-14 Medical Sciences Building, Edmonton, Alberta, Canada T6G 2H7. Electronic address:

RNA biology has recently seen an explosion of data due to advances in RNA sequencing, proteomic, and RNA imaging technologies. In this review, we highlight progress that has been made using these approaches in the area of nuclear RNP biogenesis and export. Excitingly, the ability to collect quantitative data at the 'omics' scale combined with measurements of transcription, decay, and transport kinetics is providing the information needed to address RNP biogenesis at a systems level. We believe this to be a necessary and critical next step that will lead to a better understanding of how RNP quality, diversity, and fate emerge from a defined set of nuclear RNP assembly and maturation steps.
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http://dx.doi.org/10.1016/j.ceb.2015.04.007DOI Listing
June 2015

A robust pipeline for rapid production of versatile nanobody repertoires.

Nat Methods 2014 Dec 2;11(12):1253-60. Epub 2014 Nov 2.

Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York, USA.

Nanobodies are single-domain antibodies derived from the variable regions of Camelidae atypical immunoglobulins. They show promise as high-affinity reagents for research, diagnostics and therapeutics owing to their high specificity, small size (∼15 kDa) and straightforward bacterial expression. However, identification of repertoires with sufficiently high affinity has proven time consuming and difficult, hampering nanobody implementation. Our approach generates large repertoires of readily expressible recombinant nanobodies with high affinities and specificities against a given antigen. We demonstrate the efficacy of this approach through the production of large repertoires of nanobodies against two antigens, GFP and mCherry, with Kd values into the subnanomolar range. After mapping diverse epitopes on GFP, we were also able to design ultrahigh-affinity dimeric nanobodies with Kd values as low as ∼30 pM. The approach presented here is well suited for the routine production of high-affinity capture reagents for various biomedical applications.
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http://dx.doi.org/10.1038/nmeth.3170DOI Listing
December 2014

Sumoylation of the THO complex regulates the biogenesis of a subset of mRNPs.

Nucleic Acids Res 2014 Apr 5;42(8):5043-58. Epub 2014 Feb 5.

Institut Jacques Monod, CNRS, UMR 7592, Univ Paris Diderot, Sorbonne Paris Cité, F-75205 Paris, France, Ecole Doctorale Gènes Génomes Cellules, Université Paris Sud-11, Orsay, France, Proteomics facility, Institut Jacques Monod, CNRS, UMR 7592, Univ Paris Diderot, Sorbonne Paris Cité, F-75205 Paris, France, Department for Systems Biology, Institut de recherches cliniques de Montréal (IRCM), Montreal, Québec, Canada H2W 1R7, Département de Biochimie et Médecine Moléculaire, Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada H3T 1J4, Division of Experimental Medicine, Faculty of Medicine, McGill University, Montréal, Québec, Canada H3A 1A3, Université Pierre et Marie Curie, UMR7238, 15, rue de l'Ecole de Médecine, 75006 Paris, France and CNRS, UMR7238, Laboratoire de Génomique des Microorganismes, 75006 Paris, France.

Assembly of messenger ribonucleoparticles (mRNPs) is a pivotal step in gene expression, but only a few molecular mechanisms contributing to its regulation have been described. Here, through a comprehensive proteomic survey of mRNP assembly, we demonstrate that the SUMO pathway specifically controls the association of the THO complex with mRNPs. We further show that the THO complex, a key player in the interplay between gene expression, mRNA export and genetic stability, is sumoylated on its Hpr1 subunit and that this modification regulates its association with mRNPs. Altered recruitment of the THO complex onto mRNPs in sumoylation-defective mutants does not affect bulk mRNA export or genetic stability, but impairs the expression of acidic stress-induced genes and, consistently, compromises viability in acidic stress conditions. Importantly, inactivation of the nuclear exosome suppresses the phenotypes of the hpr1 non-sumoylatable mutant, showing that SUMO-dependent mRNP assembly is critical to allow a specific subset of mRNPs to escape degradation. This article thus provides the first example of a SUMO-dependent mRNP-assembly event allowing a refined tuning of gene expression, in particular under specific stress conditions.
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http://dx.doi.org/10.1093/nar/gku124DOI Listing
April 2014

Two steps forward--one step back: advances in affinity purification mass spectrometry of macromolecular complexes.

Proteomics 2012 May;12(10):1591-608

Institut de Recherches Cliniques de Montréal, Montréal, Québec, Canada.

Cellular functions are defined by the dynamic interactions of proteins within macromolecular networks. Deciphering these complex interplays is the key to getting a comprehensive picture of cellular behavior and to understanding biological systems, from a simple bacterial cell to highly regulated neuronal cells or cancerous tissue. In the last decade, affinity purification (AP) coupled to mass spectrometry has emerged as a powerful tool to comprehensively study interaction networks and their macromolecular assemblies. This review discusses recent advances in AP approaches, from cell lysis to the importance of sample preparation and the choice of AP matrix as well as the development of different epitope tags and strategies to study dynamic interactions, with an emphasis on RNA-protein interaction networks.
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http://dx.doi.org/10.1002/pmic.201100509DOI Listing
May 2012

To the pore and through the pore: a story of mRNA export kinetics.

Biochim Biophys Acta 2012 Jun 22;1819(6):494-506. Epub 2012 Feb 22.

Institut de recherches cliniques de Montréal, 110 Avenue des Pins Ouest, Montréal, Québec, Canada.

The evolutionary 'decision' to store genetic information away from the place of protein synthesis, in a separate compartment, has forced eukaryotic cells to establish a system to transport mRNAs from the nucleus to the cytoplasm for translation. To ensure export to be fast and efficient, cells have evolved a complex molecular interplay that is tightly regulated. Over the last few decades, many of the individual players in this process have been described, starting with the composition of the nuclear pore complex to proteins that modulate co-transcriptional events required to prepare an mRNP for export to the cytoplasm. How the interplay between all the factors and processes results in the efficient and selective export of mRNAs from the nucleus and how the export process itself is executed within cells, however, is still not fully understood. Recent advances in using proteomic and single molecule microscopy approaches have provided important insights into the process and its kinetics. This review summarizes these recent advances and how they led to the current view on how cells orchestrate the export of mRNAs. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.
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http://dx.doi.org/10.1016/j.bbagrm.2012.02.011DOI Listing
June 2012

Joining the interface: a site for Nmd3 association on 60S ribosome subunits.

J Cell Biol 2010 Jun;189(7):1071-3

Laboratoire de Biochimie des ribonucléoprotéines, Institut de recherches cliniques de Montréal, Montréal, Québec H2W 1R7, Canada.

The adaptor protein Nmd3 is required for Crm1-dependent export of large ribosomal subunits from the nucleus. In this issue, Sengupta et al. (2010. J. Cell Biol. doi:10.1083/jcb.201001124) identify a binding site for yeast Nmd3 on 60S ribosomal subunits using cryoelectron microscopy and suggest a conformational model for its release in the cytoplasm. The study provides the first detailed structural description of a ribosome biogenesis factor in complex with the large subunit.
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http://dx.doi.org/10.1083/jcb.201006033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2894455PMC
June 2010

Rrp17p is a eukaryotic exonuclease required for 5' end processing of Pre-60S ribosomal RNA.

Mol Cell 2009 Dec;36(5):768-81

Rockefeller University, New York, NY 10065, USA.

Ribosomal processing requires a series of endo- and exonucleolytic steps for the production of mature ribosomes, of which most have been described. To ensure ribosome synthesis, 3' end formation of rRNA uses multiple nucleases acting in parallel; however, a similar parallel mechanism had not been described for 5' end maturation. Here, we identify Rrp17p as a previously unidentified 5'-3' exonuclease essential for ribosome biogenesis, functioning with Rat1p in a parallel processing pathway analogous to that of 3' end formation. Rrp17p is required for efficient exonuclease digestion of the mature 5' ends of 5.8S(S) and 25S rRNAs, contains a catalytic domain close to its N terminus, and is highly conserved among higher eukaryotes, being a member of a family of exonucleases. We show that Rrp17p binds late pre-60S ribosomes, accompanying them from the nucleolus to the nuclear periphery, and provide evidence for physical and functional links between late 60S subunit processing and export.
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http://dx.doi.org/10.1016/j.molcel.2009.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2806520PMC
December 2009

Assembly factors Rpf2 and Rrs1 recruit 5S rRNA and ribosomal proteins rpL5 and rpL11 into nascent ribosomes.

Genes Dev 2007 Oct;21(20):2580-92

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.

More than 170 proteins are necessary for assembly of ribosomes in eukaryotes. However, cofactors that function with each of these proteins, substrates on which they act, and the precise functions of assembly factors--e.g., recruiting other molecules into preribosomes or triggering structural rearrangements of pre-rRNPs--remain mostly unknown. Here we investigated the recruitment of two ribosomal proteins and 5S ribosomal RNA (rRNA) into nascent ribosomes. We identified a ribonucleoprotein neighborhood in preribosomes that contains two yeast ribosome assembly factors, Rpf2 and Rrs1, two ribosomal proteins, rpL5 and rpL11, and 5S rRNA. Interactions between each of these four proteins have been confirmed by binding assays in vitro. These molecules assemble into 90S preribosomal particles containing 35S rRNA precursor (pre-rRNA). Rpf2 and Rrs1 are required for recruiting rpL5, rpL11, and 5S rRNA into preribosomes. In the absence of association of these molecules with pre-rRNPs, processing of 27SB pre-rRNA is blocked. Consequently, the abortive 66S pre-rRNPs are prematurely released from the nucleolus to the nucleoplasm, and cannot be exported to the cytoplasm.
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http://dx.doi.org/10.1101/gad.1569307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2000323PMC
October 2007

Comprehensive analysis of diverse ribonucleoprotein complexes.

Nat Methods 2007 Nov 7;4(11):951-6. Epub 2007 Oct 7.

Rockefeller University, 1230 York Avenue, New York, New York 10021, USA.

The study of the dynamic interactome of cellular ribonucleoprotein (RNP) particles has been hampered by severe methodological limitations. In particular, the affinity purification of intact RNP complexes from cell lysates suffers from RNA degradation, loss of interacting macromolecules and poor overall yields. Here we describe a rapid affinity-purification method for efficient isolation of the subcomplexes that dynamically organize different RNP biogenesis pathways in Saccharomyces cerevisiae. Our method overcomes many of the previous limitations to produce large RNP interactomes with almost no contamination.
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http://dx.doi.org/10.1038/nmeth1101DOI Listing
November 2007

Yeast Rrp14p is required for ribosomal subunit synthesis and for correct positioning of the mitotic spindle during mitosis.

Nucleic Acids Res 2007 1;35(4):1354-66. Epub 2007 Feb 1.

Wellcome Trust Centre for Cell Biology, University of Edinburgh Edinburgh EH9 3JR, Scotland, UK.

Here we report that Rrp14p/Ykl082p is associated with pre-60S particles and to a lesser extent with earlier 90S pre-ribosomes. Depletion of Rrp14p inhibited pre-rRNA synthesis on both the 40S and 60S synthesis pathways. Synthesis of the 20S precursor to the 18S rRNA was largely blocked, as was maturation of the 27SB pre-rRNA to the 5.8S and 25S rRNAs. Unexpectedly, Rrp14p-depleted cells also showed apparently specific cell-cycle defects. Following release from synchronization in S phase, Rrp14p-depleted cells uniformly arrested in metaphase with short mitotic spindles that were frequently incorrectly aligned with the site of bud formation. In the absence of Bub2p, which is required for the spindle orientation checkpoint, this metaphase arrest was not seen in Rrp14p-depleted cells, which then arrested with multiple buds, several SPBs and binucleate mother cells. These data suggest that Rrp14p may play some role in cell polarity and/or spindle positioning, in addition to its function in ribosome synthesis.
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http://dx.doi.org/10.1093/nar/gkl824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1849896PMC
April 2007

I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions.

J Proteome Res 2005 Sep-Oct;4(5):1752-6

The Rockefeller University, New York, NY 10021, USA.

Isolation of protein complexes via affinity-tagged proteins provides a powerful tool for studying biological systems, but the technique is often compromised by co-enrichment of nonspecifically interacting proteins. We describe a new technique (I-DIRT) that distinguishes contaminants from bona fide interactors in immunopurifications, overcoming this most challenging problem in defining protein complexes. I-DIRT will be of broad value for studying protein complexes in biological systems that can be metabolically labeled.
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http://dx.doi.org/10.1021/pr050225eDOI Listing
January 2006

A pre-ribosome-associated HEAT-repeat protein is required for export of both ribosomal subunits.

Genes Dev 2004 Jan 16;18(2):196-209. Epub 2004 Jan 16.

Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland, UK.

Rrp12p (Ypl012w) is unusual among characterized ribosome synthesis factors in being associated with late precursors to both the 40S and 60S subunits. Rrp12p is predominantly nuclear with nucleolar enrichment at steady state, but shuttled between the nucleus and cytoplasm in a heterokaryon assay. Strains depleted of Rrp12p are impaired in the nuclear export of both ribosomal subunits. Sequence analysis combined with fold recognition and modeling showed that Rrp12p is a member of a family of pre-ribosome-associated HEAT-repeat proteins. Like other HEAT-repeat transport factors, Rrp12p binds in vitro to nucleoporin FG-repeats of both the GLFG and FXFG families and to the GTPase Gsp1p (yeast RAN). Rrp12p also showed robust in vitro binding to a pre-rRNA transcript, in addition to poly(A) and poly(U). We propose that Rrp12p binds to the RNA components of the pre-ribosomes and promotes export of both subunits via its interactions with the nucleoporins and Gsp1p.
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http://dx.doi.org/10.1101/gad.285604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC324425PMC
January 2004
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