Ribonucleoprotein Interactions Research Articles

Cryo-EM structure of single-layered nucleoprotein-RNA complex from Marburg virus

Marburg virus (MARV) causes lethal hemorrhagic fever in humans, posing a threat to global health. We determined by cryogenic electron microscopy (cryo-EM) the MARV helical ribonucleoprotein (RNP) complex structure in single-layered conformation, which differs from the previously reported structure of a double-layered helix. Our findings illuminate novel RNP interactions and expand knowledge on MARV genome packaging and nucleocapsid assembly, both processes representing attractive targets for the development of antiviral therapeutics against MARV disease.

SARS-CoV-2 infection engenders heterogeneous ribonucleoprotein interactions to impede translation elongation in the lungs.

Translational regulation in tissue environments during in vivo viral pathogenesis has rarely been studied due to the lack of translatomes from virus-infected tissues, although a series of translatome studies using in vitro cultured cells with viral infection have been reported. In this study, we exploited tissue-optimized ribosome profiling (Ribo-seq) and severe-COVID-19 model mice to establish the first temporal translation profiles of virus and host genes in the lungs during SARS-CoV-2 pathogenesis. Our datasets revealed not only previously unknown targets of translation regulation in infected tissues but also hitherto unreported molecular signatures that contribute to tissue pathology after SARS-CoV-2 infection. Specifically, we observed gradual increases in pseudoribosomal ribonucleoprotein (RNP) interactions that partially overlapped the trails of ribosomes, being likely involved in impeding translation elongation. Contemporaneously developed ribosome heterogeneity with predominantly dysregulated 5 S rRNP association supported the malfunction of elongating ribosomes. Analyses of canonical Ribo-seq reads (ribosome footprints) highlighted two obstructive characteristics to host gene expression: ribosome stalling on codons within transmembrane domain-coding regions and compromised translation of immunity- and metabolism-related genes with upregulated transcription. Our findings collectively demonstrate that the abrogation of translation integrity may be one of the most critical factors contributing to pathogenesis after SARS-CoV-2 infection of tissues.

Arabidopsis mTERF9 protein promotes chloroplast ribosomal assembly and translation by establishing ribonucleoprotein interactions in vivo.

The mitochondrial transcription termination factor proteins are nuclear-encoded nucleic acid binders defined by degenerate tandem helical-repeats of ∼30 amino acids. They are found in metazoans and plants where they localize in organelles. In higher plants, the mTERF family comprises ∼30 members and several of these have been linked to plant development and response to abiotic stress. However, knowledge of the molecular basis underlying these physiological effects is scarce. We show that the Arabidopsis mTERF9 protein promotes the accumulation of the 16S and 23S rRNAs in chloroplasts, and interacts predominantly with the 16S rRNA in vivo and in vitro. Furthermore, mTERF9 is found in large complexes containing ribosomes and polysomes in chloroplasts. The comprehensive analysis of mTERF9 in vivo protein interactome identified many subunits of the 70S ribosome whose assembly is compromised in the null mterf9 mutant, putative ribosome biogenesis factors and CPN60 chaperonins. Protein interaction assays in yeast revealed that mTERF9 directly interact with these proteins. Our data demonstrate that mTERF9 integrates protein-protein and protein-RNA interactions to promote chloroplast ribosomal assembly and translation. Besides extending our knowledge of mTERF functional repertoire in plants, these findings provide an important insight into the chloroplast ribosome biogenesis.

Identification of RNase-sensitive LINE-1 Ribonucleoprotein Interactions by Differential Affinity Immobilization.

Long Interspersed Nuclear Element-1 (LINE-1, L1) constitutes a family of autonomous, self-replicating genetic elements known as retrotransposons. Although most are inactive, copious L1 sequences populate the human genome. L1s proliferate in a ‘copy-and-paste’ fashion through an RNA intermediate; a full-length L1 transcript is ~6,000 nucleotides long and functions as a bicistronic mRNA that encodes and assembles in cis with two main polypeptides, ORF1p and ORF2p, forming a ribonucleoprotein (RNP); L1 RNPs also interact with a wide range of host factors in positive and negative regulatory capacities. The following protocol describes an approach to affinity enrich ectopically expressed L1 RNPs and, using RNases, release the fraction of protein that depends upon the presence of intact RNA for retention in the immobilized macromolecules.

An In Vitro RNA Synthesis Assay for Rabies Virus Defines Ribonucleoprotein Interactions Critical for Polymerase Activity.

The current understanding of the structural and functional significance of the components of the rabies virus replication machinery is incomplete. Although structures are available for the nucleocapsid protein in complex with RNA, and also for portions of P, information on both the structure and function of the L protein is lacking. This study reports the expression and purification of the full-length L protein of RABV and the characterization of its RdRP activity in vitro The study provides a new assay that has utility for screening inhibitors and understanding their mechanisms of action, as well as defining new interactions that are required for RdRP activity.

Constitutive patterns of gene expression regulated by RNA-binding proteins

BackgroundRNA-binding proteins regulate a number of cellular processes, including synthesis, folding, translocation, assembly and clearance of RNAs. Recent studies have reported that an unexpectedly large number of proteins are able to interact with RNA, but the partners of many RNA-binding proteins are still uncharacterized.ResultsWe combined prediction of ribonucleoprotein interactions, based on catRAPID calculations, with analysis of protein and RNA expression profiles from human tissues. We found strong interaction propensities for both positively and negatively correlated expression patterns. Our integration of in silico and ex vivo data unraveled two major types of protein–RNA interactions, with positively correlated patterns related to cell cycle control and negatively correlated patterns related to survival, growth and differentiation. To facilitate the investigation of protein–RNA interactions and expression networks, we developed the catRAPID express web server.ConclusionsOur analysis sheds light on the role of RNA-binding proteins in regulating proliferation and differentiation processes, and we provide a data exploration tool to aid future experimental studies.

Neurodegenerative diseases: Quantitative predictions of protein–RNA interactions

Increasing evidence indicates that RNA plays an active role in a number of neurodegenerative diseases. We recently introduced a theoretical framework, catRAPID, to predict the binding ability of protein and RNA molecules. Here, we use catRAPID to investigate ribonucleoprotein interactions linked to inherited intellectual disability, amyotrophic lateral sclerosis, Creutzfeuld-Jakob, Alzheimer's, and Parkinson's diseases. We specifically focus on (1) RNA interactions with fragile X mental retardation protein FMRP; (2) protein sequestration caused by CGG repeats; (3) noncoding transcripts regulated by TAR DNA-binding protein 43 TDP-43; (4) autogenous regulation of TDP-43 and FMRP; (5) iron-mediated expression of amyloid precursor protein APP and α-synuclein; (6) interactions between prions and RNA aptamers. Our results are in striking agreement with experimental evidence and provide new insights in processes associated with neuronal function and misfunction.

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

Predictions of protein–RNA interactions

AbstractRibonucleoprotein interactions play important roles in a wide variety of cellular processes, ranging from transcriptional and posttranscriptional regulation of gene expression to host defense against pathogens. High throughput experiments to identify RNA–protein interactions provide information about the complexity of interaction networks, but require time and considerable efforts. Thus, there is need for reliable computational methods for predicting ribonucleoprotein interactions. In this review, we discuss a number of approaches that have been developed to predict the ability of proteins and RNA molecules to associate. © 2012 John Wiley & Sons, Ltd.This article is categorized under: Molecular and Statistical Mechanics > Molecular Dynamics and Monte-Carlo Methods

Genus-specific recruitment of filovirus ribonucleoprotein complexes into budding particles

The filoviral matrix protein VP40 orchestrates virus morphogenesis and budding. To do this it interacts with both the glycoprotein (GP1,2) and the ribonucleoprotein (RNP) complex components; however, these interactions are still not well understood. Here we show that for efficient VP40-driven formation of transcription and replication-competent virus-like particles (trVLPs), which contain both an RNP complex and GP1,2, the RNP components and VP40, but not GP1,2 and VP40, must be from the same genus. trVLP preparations contained both spherical and filamentous particles, but only the latter were able to infect target cells and to lead to genome replication and transcription. Interestingly, the genus specificity of the VP40-RNP interactions was specific to the formation of filamentous trVLPs, but not to spherical particles. These results not only further our understanding of VP40 interactions, but also suggest that special care is required when using trVLP or VLP systems to model virus morphogenesis.

Ribonucleoprotein dynamics connects mRNA networks with drug mechanisms

Mol Syst Biol. 5: 289 Beyond transcription, gene expression is potently controlled through processes that affect pre‐mRNA splicing and maturation, as well as mRNA transport, turnover, storage, and translation. These events are tightly regulated by two main classes of trans ‐binding factors, RNA‐binding proteins (RBPs) and non‐coding RNAs (ncRNAs) (Moore, 2005; Mattick and Makunin, 2006). RBPs bind to target transcripts and influence virtually all facets of post‐transcriptional gene expression; ncRNAs, particularly microRNAs (miRNAs), mainly lower the translation and stability of target mRNAs (Keene, 2007; Bartel, 2009). As mRNAs transit through the cell, their association with RBPs and miRNAs determines their recruitment to cellular sites such as processing bodies (PBs), stress granules (SGs), polysomes, the exosome, and the RNA‐induced silencing complex (RISC), each specialized in distinct aspects of mRNA metabolism (Filipowicz et al , 2008). In this manner, ribonucleoprotein (RNP) interactions directly affect the types, location, and abundance of expressed proteins. In a report published in Molecular Systems Biology , Mukherjee et al (2009) use quantitative RNA dynamics to present a fundamentally new approach to study global RNP networks. The study bridges two key and reciprocal concepts in post‐transcriptional gene regulation. The first is that each individual mRNA is capable of associating with numerous RBPs. Several studies have shown that RBPs can interact combinatorially on a single mRNA and thereby alter its post‐transcriptional fate and ultimately the levels of expressed protein; for example, an RBP can compete or cooperate with another RBP for binding to a shared target mRNA, while miRNAs can promote or prevent the action of RBPs on a given mRNA (e.g., Lal et al , 2004; George and …

Analysis of Turnover and Translation Regulatory RNA-Binding Protein Expression through Binding to Cognate mRNAs

RNA-binding proteins (RBPs) that associate with specific mRNA sequences and function as mRNA turnover and translation regulatory (TTR) RBPs are emerging as pivotal posttranscriptional regulators of gene expression. However, little is known about the mechanisms that govern the expression of TTR-RBPs. Here, we employed human cervical carcinoma HeLa cells to test the hypothesis that TTR-RBP expression is influenced posttranscriptionally by TTR-RBPs themselves. Systematic testing of the TTR-RBPs AUF1, HuR, KSRP, NF90, TIA-1, and TIAR led to three key discoveries. First, each TTR-RBP was found to associate with its cognate mRNA and with several other TTR-RBP-encoding mRNAs, as determined by testing both endogenous and biotinylated transcripts. Second, silencing of individual TTR-RBPs influenced the expression of other TTR-RBPs at the mRNA and/or protein level. Third, further analysis of two specific ribonucleoprotein (RNP) complexes revealed that TIA-1 expression was controlled via HuR-enhanced mRNA stabilization and TIAR-repressed translation. Together, our findings underscore the notion that TTR-RBP expression is controlled, at least in part, at the posttranscriptional level through a complex circuitry of self- and cross-regulatory RNP interactions.

Structure of the Base of the L7/L12 Stalk of the Haloarcula marismortui Large Ribosomal Subunit: Analysis of L11 Movements

Initiation factors, elongation factors, and release factors all interact with the L7/L12 stalk of the large ribosomal subunit during their respective GTP-dependent cycles on the ribosome. Electron density corresponding to the stalk is not present in previous crystal structures of either 50 S subunits or 70 S ribosomes. We have now discovered conditions that result in a more ordered factor-binding center in the Haloarcula marismortui (H.ma) large ribosomal subunit crystals and consequently allows the visualization of the full-length L11, the N-terminal domain (NTD) of L10 and helices 43 and 44 of 23 S rRNA. The resulting model is currently the most complete reported structure of a L7/L12 stalk in the context of a ribosome. This region contains a series of intermolecular interfaces that are smaller than those typically seen in other ribonucleoprotein interactions within the 50 S subunit. Comparisons of the L11 NTD position between the current structure, which is has an NTD splayed out with respect to previous structures, and other structures of ribosomes in different functional states demonstrates a dynamic range of L11 NTD movements. We propose that the L11 NTD moves through three different relative positions during the translational cycle: apo-ribosome, factor-bound pre-GTP hydrolysis and post-GTP hydrolysis. These positions outline a pathway for L11 NTD movements that are dependent on the specific nucleotide state of the bound ligand. These three states are represented by the orientations of the L11 NTD relative to the ribosome and suggest that L11 may play a more specialized role in the factor binding cycle than previously appreciated.

Structural aspects of ribonucleoprotein interactions in ribosomes

Impressive progress has been made towards the determination of the molecular structure of the ribosome and the illumination of its functional features. The highlights include reaching almost atomic resolution in crystallography of intact ribosomal particles and isolated ribosomal components and the assignments of several functional centers.

TRNA identity: A hair of the dogma that bit us

Those drawn to the calling of molecular biology will inevitably be touched when a universal step in the flow of genetic information is clarified. That clarification is underway for the interaction of tRNAs and aminoacyl-tRNA synthetases (AARS) in vivo and in vitro. These new data also apply to the more general topic of RNA-protein association and may illuminate other little-understood, though ubiquitous, ribonucleoprotein interactions. The Data, Old and New Distinction among tRNAs by AARS is sometimes simple. From work begun almost two decades ago, we know that a single additional genetic mutation in the CCA arm of amber (UAG) suppressor tRNATyrcun (the subscript shows a changed anticodon) alters this tRNA so that it inserts glutamine (Ghysen and Celis, JMB 83, 333-351, 1974). A single C-U mutation in the middle position of the anticodon of tRNATrp simultaneously converts it to an amber suppressor and makes it a glutaminyl-tRNA (Yaniv et al., JMB 86,245260,1974). Thus GlnRS depends heavily on one or two nucleotides to determine acceptance or rejection of some tRNAs. The anticodon plainly contains sufficient information to uniquely determine a tRNA’s associated amino acid, and can be the predominant determinant of amino acid specificity. The importance of the anticodon to E. coli MetRS in vitro was shown by work begun shortly after the early genetic experiments. Schulman and Pelka (PNAS 80, 6755-6759, 1983) ligated anticodon oligoribonucleotides into RNAase-gapped tRNAMet to demonstrate that MetRS depends heavily on the three nucleotides of the anticodon (34,35, and 36 in the figure, marked M). These same workers (Schulman and Pelka, Science, in press, 1988) have now transformed tRNAVarI into tRNAVa$,cAu, which accepts methionine at the same rate as bona fide tRNAMet. This represents an increase of 600,000-fold in acylation rate (V,,/K,) as a result of the substitution of two anticodon nucleotides. Similarly, tRNATrPCAu is acylated by MetRS only tenfold more slowly than tRNAMet. Other experiments of Schulman and Pelka show that ValRS displays the same type of dependence on the anticodon (sites marked V in the figure): tRNAMet,,unc accepts valine at a rate only tenfold lower than that of tRNAV$. Much recent work takes its inspiration from Normanly et al. (Nature 327, 213-219, 1986) whose experiments initially suggested that tRNA discrimination could be studied by changing a relatively small part of the tRNA sequence. These workers introduced a now widely used approach for determining the “identity set” of a tRNA and AARS (identity set is defined as the minimal set of nucleotide substitutions that changes the amino acid inserted into cellular Minireview