RESUMO
mRNA export factor 1 (Rae1) and nucleoporin 98 (Nup98) are host cell targets for the matrix (M) protein of vesicular stomatitis virus (VSV). How Rae1 functions in mRNA export and how M protein targets both Rae1 and Nup98 are not understood at the molecular level. To obtain structural insights, we assembled a 1:1:1 complex of Mâ¢Rae1â¢Nup98 and established a crystal structure at 3.15-Å resolution. We found that the M protein contacts the Rae1â¢Nup98 heterodimer principally by two protrusions projecting from the globular domain of M like a finger and thumb. Both projections clamp to the side of the ß-propeller of Rae1, with the finger also contacting Nup98. The most prominent feature of the finger is highly conserved Methionine 51 (Met51) with upstream and downstream acidic residues. The complementary surface on Rae1 displays a deep hydrophobic pocket, into which Met51 fastens like a bolt, and a groove of basic residues on either side, which bond to the acidic residues of the finger. Notably, the M protein competed for in vitro binding of various oligonucleotides to Rae1â¢Nup98. We localized this competing activity of M to its finger using a synthetic peptide. Collectively, our data suggest that Rae1 serves as a binding protein for the phosphate backbone of any nucleic acid and that the finger of M mimics this ligand. In the context of mRNA export, we propose that a given mRNA segment, after having been deproteinated by helicase, is transiently reproteinated by Nup98-tethered Rae1. We suggest that such repetitive cycles provide cytoplasmic stopover sites required for ratcheting mRNA across the nuclear pore.
Assuntos
Complexos Multiproteicos/química , Proteínas Associadas à Matriz Nuclear/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Proteínas de Transporte Nucleocitoplasmático/química , Vesiculovirus/química , Proteínas da Matriz Viral/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Estrutura Quaternária de Proteína , Vesiculovirus/genética , Vesiculovirus/metabolismo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismoRESUMO
Release of properly processed and assembled mRNPs from the actively transcribing genes, movement of the mRNPs through the interchromatin and interaction with the Nuclear Pore Complexes, leading to cytoplasmic export, are essential steps of eukaryotic gene expression. Here, we review these intranuclear gene expression steps.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Regulação da Expressão Gênica , Humanos , Modelos Genéticos , Poro Nuclear/metabolismo , Transporte de RNA , RNA Mensageiro/genética , Ribonucleoproteínas/genéticaRESUMO
The flow of genetic information from sites of transcription within the nucleus to the cytoplasmic translational machinery of eukaryotic cells is obstructed by a physical blockade, the nuclear double membrane, which must be overcome in order to adhere to the central dogma of molecular biology, DNA makes RNA makes protein. Advancement in the field of cellular and molecular biology has painted a detailed picture of the molecular mechanisms from transcription of genes to mRNAs and their processing that is closely coupled to export from the nucleus. The rules that govern delivering messenger transcripts from the nucleus must be obeyed by influenza A virus, a member of the Orthomyxoviridae that has adopted a nuclear replication cycle. The negative-sense genome of influenza A virus is segmented into eight individual viral ribonucleoprotein (vRNP) complexes containing the viral RNA-dependent RNA polymerase and single-stranded RNA encapsidated in viral nucleoprotein. Influenza A virus mRNAs fall into three major categories, intronless, intron-containing unspliced and spliced. During evolutionary history, influenza A virus has conceived a way of negotiating the passage of viral transcripts from the nucleus to cytoplasmic sites of protein synthesis. The major mRNA nuclear export NXF1 pathway is increasingly implicated in viral mRNA export and this review considers and discusses the current understanding of how influenza A virus exploits the host mRNA export pathway for replication.
Assuntos
Núcleo Celular/metabolismo , Vírus da Influenza A/genética , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Transporte Ativo do Núcleo Celular , Vírus da Influenza A/metabolismo , Transporte de RNA , RNA Mensageiro/genética , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/genéticaRESUMO
BACKGROUND: Understanding gene transcription and mRNA-protein (mRNP) dynamics in single cells in a multicellular organism has been challenging. The catalytically dead CRISPR-Cas13 (dCas13) system has been used to visualize RNAs in live cells without genetic manipulation. We optimize this system to track developmentally expressed mRNAs in zebrafish embryos and to understand features of endogenous transcription kinetics and mRNP export. RESULTS: We report that zygotic microinjection of purified CRISPR-dCas13-fluorescent proteins and modified guide RNAs allows single- and dual-color tracking of developmentally expressed mRNAs in zebrafish embryos from zygotic genome activation (ZGA) until early segmentation period without genetic manipulation. Using this approach, we uncover non-synchronized de novo transcription between inter-alleles, synchronized post-mitotic re-activation in pairs of alleles, and transcriptional memory as an extrinsic noise that potentially contributes to synchronized post-mitotic re-activation. We also reveal rapid dCas13-engaged mRNP movement in the nucleus with a corralled and diffusive motion, but a wide varying range of rate-limiting mRNP export, which can be shortened by Alyref and Nxf1 overexpression. CONCLUSIONS: This optimized dCas13-based toolkit enables robust spatial-temporal tracking of endogenous mRNAs and uncovers features of transcription and mRNP motion, providing a powerful toolkit for endogenous RNA visualization in a multicellular developmental organism.
Assuntos
RNA , Peixe-Zebra , Animais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Transporte Ativo do Núcleo Celular , RNA/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
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.