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Proceedings of the National Academy of... Jun 2001Following transcription and splicing, each mRNA of a mammalian cell passes into the cytoplasm where its fate is in the hands of a complex network of ribonucleoproteins... (Review)
Review
Following transcription and splicing, each mRNA of a mammalian cell passes into the cytoplasm where its fate is in the hands of a complex network of ribonucleoproteins (mRNPs). The success or failure of a gene to be expressed depends on the performance of this mRNP infrastructure. The entry, gating, processing, and transit of each mRNA through an mRNP network helps determine the composition of a cell's proteome. The machinery that regulates storage, turnover, and translational activation of mRNAs is not well understood, in part, because of the heterogeneous nature of mRNPs. Recently, subsets of cellular mRNAs clustered as members of mRNP complexes have been identified by using antibodies reactive with RNA-binding proteins, including ELAV/Hu, eIF-4E, and poly(A)-binding proteins. Cytoplasmic ELAV/Hu proteins are involved in the stability and translation of early response gene (ERG) transcripts and are expressed predominately in neurons. mRNAs recovered from ELAV/Hu mRNP complexes were found to have similar sequence elements, suggesting a common structural linkage among them. This approach opens the possibility of identifying transcripts physically clustered in vivo that may have similar fates or functions. Moreover, the proteins encoded by physically organized mRNAs may participate in the same biological process or structural outcome, not unlike operons and their polycistronic mRNAs do in prokaryotic organisms. Our goal is to understand the organization and flow of genetic information on an integrative systems level by analyzing the collective properties of proteins and mRNAs associated with mRNPs in vivo.
Topics: Animals; Gene Expression Regulation; Genome; Protein Biosynthesis; Proteome; RNA Processing, Post-Transcriptional; RNA Splicing; RNA, Messenger; Ribonucleoproteins; Transcription, Genetic; Transcriptional Activation
PubMed: 11416181
DOI: 10.1073/pnas.111145598 -
PLoS Computational Biology Jun 2019A coarse-grain computational method integrates biophysical and structural data to generate models of HIV-1 genomic RNA, nucleocapsid and integrase condensed into a...
A coarse-grain computational method integrates biophysical and structural data to generate models of HIV-1 genomic RNA, nucleocapsid and integrase condensed into a mature ribonucleoprotein complex. Several hypotheses for the initial structure of the genomic RNA and oligomeric state of integrase are tested. In these models, integrase interaction captures features of the relative distribution of gRNA in the immature virion and increases the size of the RNP globule, and exclusion of nucleocapsid from regions with RNA secondary structure drives an asymmetric placement of the dimerized 5'UTR at the surface of the RNP globule.
Topics: Computational Biology; HIV-1; Molecular Dynamics Simulation; RNA, Viral; Ribonucleoproteins; Viral Proteins; Virion; Virus Assembly
PubMed: 31194731
DOI: 10.1371/journal.pcbi.1007150 -
RNA (New York, N.Y.) Sep 2010Nuclear ribonuclease (RNase) P is a ubiquitous essential ribonucleoprotein complex, one of only two known RNA-based enzymes found in all three domains of life. The RNA... (Review)
Review
Nuclear ribonuclease (RNase) P is a ubiquitous essential ribonucleoprotein complex, one of only two known RNA-based enzymes found in all three domains of life. The RNA component is the catalytic moiety of RNases P across all phylogenetic domains; it contains a well-conserved core, whereas peripheral structural elements are diverse. RNA components of eukaryotic RNases P tend to be less complex than their bacterial counterparts, a simplification that is accompanied by a dramatic reduction of their catalytic ability in the absence of protein. The size and complexity of the protein moieties increase dramatically from bacterial to archaeal to eukaryotic enzymes, apparently reflecting the delegation of some structural functions from RNA to proteins and, perhaps, in response to the increased complexity of the cellular environment in the more evolutionarily advanced organisms; the reasons for the increased dependence on proteins are not clear. We review current information on RNase P and the closely related universal eukaryotic enzyme RNase MRP, focusing on their functions and structural organization.
Topics: Animals; Bacteria; Endoribonucleases; Evolution, Molecular; Humans; Ribonuclease P; Ribonucleoproteins
PubMed: 20627997
DOI: 10.1261/rna.2214510 -
Methods in Molecular Biology (Clifton,... 2016Long INterspersed Element-1 (LINE-1 or L1) retrotransposons encode two proteins (ORF1p and ORF2p) that are required for retrotransposition. The L1 element amplification...
Long INterspersed Element-1 (LINE-1 or L1) retrotransposons encode two proteins (ORF1p and ORF2p) that are required for retrotransposition. The L1 element amplification protocol (LEAP) assays the ability of L1 ORF2p to reverse transcribe L1 RNA in vitro. Ultracentrifugation or immunoprecipitation is used to isolate L1 ribonucleoprotein particle (RNP) complexes from cultured human cells transfected with an engineered L1 expression construct. The isolated RNPs are incubated with an oligonucleotide that contains a unique sequence at its 5' end and a thymidine-rich sequence at its 3' end. The addition of dNTPs to the reaction allows L1 ORF2p bound to L1 RNA to generate L1 cDNA. The resultant L1 cDNAs then are amplified using polymerase chain reaction (PCR) and the products are visualized by gel electrophoresis. Sequencing the resultant PCR products then allows product verification. The LEAP assay has been instrumental in determining how mutations in L1 ORF1p and ORF2p affect L1 reverse transcriptase (RT) activity. Furthermore, the LEAP assay has revealed that the L1 ORF2p RT can extend a DNA primer with mismatched 3' terminal bases when it is annealed to an L1 RNA template. As the LINE-1 biology field gravitates toward studying cellular proteins that regulate LINE-1, molecular genetic and biochemical approaches such as LEAP, in conjunction with the LINE-1-cultured cell retrotransposition assay, are essential to dissect the molecular mechanism of L1 retrotransposition.
Topics: HeLa Cells; Humans; Immunoprecipitation; Long Interspersed Nucleotide Elements; Open Reading Frames; Polymerase Chain Reaction; Ribonucleoproteins; Ultracentrifugation
PubMed: 26895063
DOI: 10.1007/978-1-4939-3372-3_21 -
STAR Protocols Mar 2021Here, we describe a generic protocol for monitoring protein-RNA interaction using a cleavable GFP fusion of a recombinant RNA-binding protein. We detail each expression...
Here, we describe a generic protocol for monitoring protein-RNA interaction using a cleavable GFP fusion of a recombinant RNA-binding protein. We detail each expression and purification step, including high salt and heparin column for contaminant RNA removal. After the assembly of RNA into the ribonucleoprotein complex, the MicroScale Thermophoresis assay enables the binding affinity to be obtained quickly with a small amount of sample. Further Gaussian accelerated molecular dynamics simulations allow us to analyze protein:RNA interactions in detail. For complete details on the use and execution of this protocol, please refer to Gao et al. (2020).
Topics: Biological Assay; Molecular Dynamics Simulation; Protein Binding; RNA; Ribonucleoproteins; Thermodynamics
PubMed: 33659898
DOI: 10.1016/j.xpro.2021.100315 -
Methods (San Diego, Calif.) Oct 2021Cataract is the leading cause of blindness worldwide. Congenital or paediatric cataract can result in permanent visual impairment or blindness even with best attempts at...
Cataract is the leading cause of blindness worldwide. Congenital or paediatric cataract can result in permanent visual impairment or blindness even with best attempts at treatment. A significant proportion of paediatric cataract has a genetic cause. Therefore, identifying the genes that lead to cataract formation is essential for understanding the pathological process of inherited paediatric cataract as well as to the development of new therapies. Despite clear progress in genomics technologies, verification of the biological effects of newly identified candidate genes and variants is still challenging. Here, we provide a step-by-step pipeline to evaluate cataract candidate genes in F0 zebrafish using CRISPR-Cas9 ribonucleoprotein complexes (RNP). Detailed descriptions of CRISPR-Cas9 RNP design and formulation, microinjection, optimization of CRISPR-Cas9 RNP reagent dose and delivery route, editing efficacy analysis as well as cataract formation evaluation are included. Following this protocol, any cataract candidates can be readily and efficiently evaluated within 2 weeks using basic laboratory supplies.
Topics: Animals; Blindness; CRISPR-Cas Systems; Cataract; Gene Editing; Humans; Ribonucleoproteins; Zebrafish
PubMed: 33418061
DOI: 10.1016/j.ymeth.2020.12.004 -
FEBS Letters Feb 1997The hnRNP K protein is a versatile molecule that interacts with RNA, DNA, the proto-oncoprotein VaV, Src-like tyrosine and inducible serine/threonine kinases, the... (Review)
Review
The hnRNP K protein is a versatile molecule that interacts with RNA, DNA, the proto-oncoprotein VaV, Src-like tyrosine and inducible serine/threonine kinases, the transcription factor TBP and a number of zinc-finger transcriptional repressors. The interaction of K protein with some of its protein partners is modulated by nucleic acids and K protein can alter the in vivo and in vitro rate of transcription. K protein can simultaneously engage several proteins and may facilitate molecular cross-talk. Taken together these diverse interactions suggest that K protein may act as a nucleic acid-regulated docking platform.
Topics: Amino Acid Sequence; DNA; Gene Expression; Heterogeneous-Nuclear Ribonucleoprotein K; Heterogeneous-Nuclear Ribonucleoproteins; Humans; RNA; Ribonucleoproteins; Signal Transduction
PubMed: 9042948
DOI: 10.1016/s0014-5793(97)00041-0 -
The Journal of Cell Biology May 1988
Review
Topics: Heterogeneous-Nuclear Ribonucleoproteins; RNA Precursors; RNA Splicing; RNA, Messenger; Ribonucleoproteins; Ribonucleoproteins, Small Nuclear; Signal Recognition Particle; Transcription, Genetic
PubMed: 2836428
DOI: 10.1083/jcb.106.5.1419 -
Journal of Bacteriology Sep 1993
Review
Topics: Peptidyl Transferases; Proteins; RNA; Ribonucleoproteins
PubMed: 7690022
DOI: 10.1128/jb.175.17.5297-5300.1993 -
Nucleic Acids Research Nov 2021Understanding the mRNA life cycle requires information about the dynamics and macromolecular composition and stoichiometry of mRNPs. Fluorescence correlation and...
Understanding the mRNA life cycle requires information about the dynamics and macromolecular composition and stoichiometry of mRNPs. Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS) are appealing technologies to study these macromolecular structures because they have single molecule sensitivity and readily provide information about their molecular composition and dynamics. Here, we demonstrate how FCS can be exploited to study cytoplasmic mRNPs with high accuracy and reproducibility in cell lysates. Cellular lysates not only recapitulate data from live cells but provide improved readings and allow investigation of single mRNP analysis under particular conditions or following enzymatic treatments. Moreover, FCCS employing minute amounts of cells closely corroborated previously reported RNA dependent interactions and provided estimates of the relative overlap between factors in the mRNPs, thus depicting their heterogeneity. The described lysate-based FCS and FCCS analysis may not only complement current biochemical approaches but also provide novel opportunities for the quantitative analysis of the molecular composition and dynamics of single mRNPs.
Topics: Cytoplasm; HeLa Cells; Humans; Ribonucleoproteins; Single Molecule Imaging; Spectrometry, Fluorescence
PubMed: 34478550
DOI: 10.1093/nar/gkab751