Stress-Induced Translation Inhibition through Rapid Displacement of Scanning Initiation Factors.

Cellular responses to environmental stress are frequently mediated by RNA-binding proteins (RBPs). Here, we examined global RBP dynamics in Saccharomyces cerevisiae in response to glucose starvation and heat shock. Each stress induced rapid remodeling of the RNA-protein interactome without corresponding changes in RBP abundance. Consistent with general translation shutdown, ribosomal proteins contacting the mRNA showed decreased RNA association. Among translation components, RNA association was most reduced for initiation factors involved in 40S scanning (eukaryotic initiation factor 4A [eIF4A], eIF4B, and Ded1), indicating a common mechanism of translational repression. In unstressed cells, eIF4A, eIF4B, and Ded1 primarily targeted the 5' ends of mRNAs. Following glucose withdrawal, 5' binding was abolished within 30 s, explaining the rapid translation shutdown, but mRNAs remained stable. Heat shock induced progressive loss of 5' RNA binding by initiation factors over ∼16 min and provoked mRNA degradation, particularly for translation-related factors, mediated by Xrn1. Taken together, these results reveal mechanisms underlying translational control of gene expression during stress.

System-wide Profiling of RNA-Binding Proteins Uncovers Key Regulators of Virus Infection.

The compendium of RNA-binding proteins (RBPs) has been greatly expanded by the development of RNA-interactome capture (RIC). However, it remained unknown if the complement of RBPs changes in response to environmental perturbations and whether these rearrangements are important. To answer these questions, we developed "comparative RIC" and applied it to cells challenged with an RNA virus called sindbis (SINV). Over 200 RBPs display differential interaction with RNA upon SINV infection. These alterations are mainly driven by the loss of cellular mRNAs and the emergence of viral RNA. RBPs stimulated by the infection redistribute to viral replication factories and regulate the capacity of the virus to infect. For example, ablation of XRN1 causes cells to be refractory to SINV, while GEMIN5 moonlights as a regulator of SINV gene expression. In summary, RNA availability controls RBP localization and function in SINV-infected cells.

Multitier regulation of the E. coli extreme acid stress response by CsrA.

CsrA is an RNA-binding protein that regulates processes critical for growth and survival, including central carbon metabolism, motility, biofilm formation, stress responses, and expression of virulence factors in pathogens. Transcriptomics studies in Escherichia coli suggested that CsrA repressed genes involved in surviving extremely acidic conditions. Here, we examine the effects of disrupting CsrA-dependent regulation on the expression of genes and circuitry for acid stress survival and demonstrate CsrA-mediated repression at multiple levels. We show that this repression is critical for managing the trade-off between growth and survival; overexpression of acid stress genes caused by csrA disruption enhances survival under extreme acidity but is detrimental for growth under mildly acidic conditions. In vitro studies confirmed that CsrA binds specifically to mRNAs of structural and regulatory genes for acid stress survival, causing translational repression. We also found that translation of the top-tier acid stress regulator, evgA, is coupled to that of a small leader peptide, evgL, which is repressed by CsrA. Unlike dedicated acid stress response genes, csrA and its sRNA antagonists, csrB and csrC, did not exhibit a substantial response to acid shock. Furthermore, disruption of CsrA regulation of acid stress genes impacted host-microbe interactions in Caenorhabditis elegans, alleviating GABA deficiencies. This study expands the known regulon of CsrA to genes of the extreme acid stress response of E. coli and highlights a new facet of the global role played by CsrA in balancing the opposing physiological demands of stress resistance with the capacity for growth and modulating host interactions.IMPORTANCETo colonize/infect the mammalian intestinal tract, bacteria must survive exposure to the extreme acidity of the stomach. E. coli does this by expressing proteins that neutralize cytoplasmic acidity and cope with molecular damage caused by low pH. Because of the metabolic cost of these processes, genes for surviving acid stress are tightly regulated. Here, we show that CsrA negatively regulates the cascade of expression responsible for the acid stress response. Increased expression of acid response genes due to csrA disruption improved survival at extremely low pH but inhibited growth under mildly acidic conditions. Our findings define a new layer of regulation in the acid stress response of E. coli and a novel physiological function for CsrA.

Understudied proteins and understudied functions in the model bacterium Bacillus subtilis-A major challenge in current research.

Model organisms such as the Gram-positive bacterium Bacillus subtilis have been studied intensively for decades. However, even for model organisms, no function has been identified for about one fourth of all proteins. It has recently been realized that such understudied proteins as well as poorly studied functions set a limitation to our understanding of the requirements for cellular life, and the Understudied Proteins Initiative has been launched. Of poorly studied proteins, those that are strongly expressed are likely to be important to the cell and should therefore be considered high priority in further studies. Since the functional analysis of unknown proteins can be extremely laborious, a minimal knowledge is required prior to targeted functional studies. In this review, we discuss strategies to obtain such a minimal annotation, for example, from global interaction, expression, or localization studies. We present a set of 41 highly expressed and poorly studied proteins of B. subtilis. Several of these proteins are thought or known to bind RNA and/or the ribosome, some may control the metabolism of B. subtilis, and another subset of particularly small proteins may act as regulatory elements to control the expression of downstream genes. Moreover, we discuss the challenges of poorly studied functions with a focus on RNA-binding proteins, amino acid transport, and the control of metabolic homeostasis. The identification of the functions of the selected proteins not only will strongly advance our knowledge on B. subtilis, but also on other organisms since many of the proteins are conserved in many groups of bacteria.

Protein-RNA interaction prediction with deep learning: structure matters.

Protein-RNA interactions are of vital importance to a variety of cellular activities. Both experimental and computational techniques have been developed to study the interactions. Because of the limitation of the previous database, especially the lack of protein structure data, most of the existing computational methods rely heavily on the sequence data, with only a small portion of the methods utilizing the structural information. Recently, AlphaFold has revolutionized the entire protein and biology field. Foreseeably, the protein-RNA interaction prediction will also be promoted significantly in the upcoming years. In this work, we give a thorough review of this field, surveying both the binding site and binding preference prediction problems and covering the commonly used datasets, features and models. We also point out the potential challenges and opportunities in this field. This survey summarizes the development of the RNA-binding protein-RNA interaction field in the past and foresees its future development in the post-AlphaFold era.

The importance of virion-incorporated cellular RNA-Binding Proteins in viral particle assembly and infectivity.

RNA is a central molecule in RNA virus biology due to its dual function as messenger and genome. However, the small number of proteins encoded by viral genomes is insufficient to enable virus infection. Hence, viruses hijack cellular RNA-binding proteins (RBPs) to aid replication and spread. In this review we discuss the 'knowns' and 'unknowns' regarding the contribution of host RBPs to the formation of viral particles and the initial steps of infection in the newly infected cell. Through comparison of the virion proteomes of ten different human RNA viruses, we confirm that a pool of cellular RBPs are typically incorporated into viral particles. We describe here illustrative examples supporting the important functions of these RBPs in viral particle formation and infectivity and we propose that the role of host RBPs in these steps can be broader than previously anticipated. Understanding how cellular RBPs regulate virus infection can lead to the discovery of novel therapeutic targets against viruses.

An updated dataset and a structure-based prediction model for protein-RNA binding affinity.

Understanding the process of protein-RNA interaction is essential for structural biology. The thermodynamic process is an important part to uncover the protein-RNA interaction mechanism. The regulatory networks between protein and RNA in organisms are dominated by the binding or dissociation in the cells. Therefore, determining the binding affinity for protein-RNA complexes can help us to understand the regulation mechanism of protein-RNA interaction. Since it is time-consuming and labor-intensive to determine the binding affinity for protein-RNA complexes by experimental methods, it is necessary and urgent to develop computational methods to predict that. To develop a binding affinity prediction model, first we update the dataset of protein-RNA binding affinity benchmark (PRBAB), which includes 145 complexes now. Second, we extract the structural features based on complex structure, and then we analyze and select the representative structural features to train the regression model. Third, we random select the subset from the PRBAB2.0 to fit the protein-RNA binding affinity determined by experiment. In the end, we tested our model on the nonredundant PDBbind dataset, and the results showed that Pearson correlation coefficient r = .57 and RMSE = 2.51 kcal/mol. The Pearson correlation coefficient achieves 0.7 while removing 5 complex structures with modified residues/nucleotides and metal ions. While testing on ProNAB, the results showed that 71.60% of the prediction achieves Pearson correlation coefficient r = .61 and RMSE = 1.56 kcal/mol with experiment values.

Signatures of tRNAGlx -specificity in proteobacterial glutamyl-tRNA synthetases.

The canonical function of glutamyl-tRNA synthetase (GluRS) is to glutamylate tRNAGlu . Yet not all bacterial GluRSs glutamylate tRNAGlu ; many glutamylate both tRNAGlu and tRNAGln , while some glutamylate only tRNAGln and not the cognate substrate tRNAGlu . Understanding the basis of the unique specificity of tRNAGlx is important. Mutational studies have hinted at hotspot residues, both on tRNAGlx and GluRS, which play crucial roles in tRNAGlx -specificity. However, its underlying structural basis remains unexplored. The majority of biochemical studies related to tRNAGlx -specificity have been performed on GluRS from Escherichia coli and other proteobacterial species. However, since the early crystal structures of GluRS and tRNAGlu -bound GluRS were from non-proteobacterial species (Thermus thermophilus), proteobacterial biochemical data have often been interpreted in the context of non-proteobacterial GluRS structures. Marked differences between proteobacterial and non-proteobacterial GluRSs have been demonstrated; therefore, it is important to understand tRNAGlx -specificity vis-a-vis proteobacterial GluRS structures. To this end, we solved the crystal structure of a double mutant GluRS from E. coli. Using the solved structure and several other currently available proteo- and non-proteobacterial GluRS crystal structures, we probed the structural basis of the tRNAGlx -specificity of bacterial GluRSs. Specifically, our analyses suggest a unique role played by the tRNAGlx D-helix contacting loop of GluRS in the modulation of tRNAGln -specificity. While earlier studies have identified functional hotspots on tRNAGlx that control the tRNAGlx -specificity of GluRS, this is the first report of complementary signatures of tRNAGlx -specificity in GluRS.

Spirocyclic Chromenopyrazole Inhibitors Disrupting the Interaction between the RNA-Binding Protein LIN28 and Let-7.

Small-molecule inhibitors of the RNA-binding and regulating protein LIN28 have the potential to be developed as chemical probes for biological perturbation and as therapeutic candidates. Reported small molecules disrupting the interaction between LIN28 and let-7 miRNA suffer from moderate to weak inhibitory activity and flat structure-activity relationship, which hindered the development of next-generation LIN28 inhibitors that warrant further evaluations. We report herein the identification of new LIN28 inhibitors utilizing a spirocyclization strategy based on a chromenopyrazole scaffold. Representative compounds 2-5 showed potent in vitro inhibitory activity against LIN28-let-7 interaction and single-digit micromolar potency in inhibiting the proliferation of LIN28-expressing JAR cancer cells. The spirocyclic compound 5 incorporated a position that is amenable for functional group appendage and further structural modifications. The binding mode of compound 5 with the LIN28 cold shock domain was rationalized via a molecular docking analysis.

The human long non-coding RNA LINC00941 and its modes of action in health and disease.

Long non-coding RNAs have gained attention in recent years as they were shown to play crucial roles in the regulation of cellular processes, but the understanding of the exact mechanisms is still incomplete in most cases. This is also true for long non-coding RNA LINC00941, which was recently found to be highly upregulated in various types of cancer influencing cell proliferation and metastasis. Initial studies could not elucidate the mode of action to understand the role and real impact of LINC00941 in tissue homeostasis and cancer development. However, recent analyses have demonstrated multiple potential modes of action of LINC00941 influencing the functionality of various cancer cell types. Correspondingly, LINC00941 was proposed to be involved in regulation of mRNA transcription and modulation of protein stability, respectively. In addition, several experimental approaches suggest a function of LINC00941 as competitive endogenous RNA, thus acting in a post-transcriptional regulatory fashion. This review summarizes our recent knowledge about the mechanisms of action of LINC00941 elucidated so far and discusses its putative role in miRNA sequestering processes. In addition, the functional role of LINC00941 in regulating human keratinocytes is discussed to also highlight its role in normal tissue homeostasis tissue aside from its involvement in cancer.

A structure-based model for the prediction of protein-RNA binding affinity.

Protein-RNA recognition is highly affinity-driven and regulates a wide array of cellular functions. In this study, we have curated a binding affinity data set of 40 protein-RNA complexes, for which at least one unbound partner is available in the docking benchmark. The data set covers a wide affinity range of eight orders of magnitude as well as four different structural classes. On average, we find the complexes with single-stranded RNA have the highest affinity, whereas the complexes with the duplex RNA have the lowest. Nevertheless, free energy gain upon binding is the highest for the complexes with ribosomal proteins and the lowest for the complexes with tRNA with an average of -5.7 cal/mol/Å2 in the entire data set. We train regression models to predict the binding affinity from the structural and physicochemical parameters of protein-RNA interfaces. The best fit model with the lowest maximum error is provided with three interface parameters: relative hydrophobicity, conformational change upon binding and relative hydration pattern. This model has been used for predicting the binding affinity on a test data set, generated using mutated structures of yeast aspartyl-tRNA synthetase, for which experimentally determined ΔG values of 40 mutations are available. The predicted ΔGempirical values highly correlate with the experimental observations. The data set provided in this study should be useful for further development of the binding affinity prediction methods. Moreover, the model developed in this study enhances our understanding on the structural basis of protein-RNA binding affinity and provides a platform to engineer protein-RNA interfaces with desired affinity.

Improved library preparation with the new iCLIP2 protocol.

Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) is a state-of-the-art technology to map the RNA interaction sites of an RNA-binding protein (RBP) across the transcriptome. Here, we present the new iCLIP2 protocol that allows to obtain high-quality iCLIP libraries in a fast and efficient manner. The new protocol comprises separate adapter ligations, two cDNA amplification steps and bead-based size selection. The full procedure can be completed within four days. Our advances significantly increase the complexity of the iCLIP2 libraries, resulting in a more comprehensive representation of RBP binding sites. Overall, the methodological advances in iCLIP2 allow efficient library generation and thereby promote the versatile and flexible application of this important technology.

An Analytical Review of the Structural Features of Pentatricopeptide Repeats: Strategic Amino Acids, Repeat Arrangements and Superhelical Architecture.

Tricopeptide repeats are common in natural proteins, and are exemplified by 34- and 35-residue repeats, known respectively as tetratricopeptide repeats (TPRs) and pentatricopeptide repeats (PPRs). In both classes, each repeat unit forms an antiparallel bihelical structure, so that multiple such units in a polypeptide are arranged in a parallel fashion. The primary structures of the motifs are nonidentical, but amino acids of similar properties occur in strategic positions. The focus of the present work was on PPR, but TPR, its better-studied cousin, is often included for comparison. The analyses revealed that critical amino acids, namely Gly, Pro, Ala and Trp, were placed at distinct locations in the higher order structure of PPR domains. While most TPRs occur in repeats of three, the PPRs exhibited a much greater diversity in repeat numbers, from 1 to 30 or more, separated by spacers of various sequences and lengths. Studies of PPR strings in proteins showed that the majority of PPR units are single, and that the longer tandems (i.e., without space in between) occurred in decreasing order. The multi-PPR domains also formed superhelical vortices, likely governed by interhelical angles rather than the spacers. These findings should be useful in designing and understanding the PPR domains.

Development of bis-ANS-based modified fluorescence titration assay for IFIT/RNA studies.

The interferon-induced proteins with tetratricopeptide repeats (IFITs) are a family of RNA-binding proteins that are very highly expressed during antiviral response of immune system. IFIT proteins recognize and tightly bind foreign RNA particles. These are primarily viral RNAs ended with triphosphate at the 5' or lacking methylation of the first cap-proximal nucleotide but also in vitro transcribed RNA synthesized in the laboratory. Recognition of RNA by IFIT proteins leads to the formation of stable RNA/IFIT complexes and translational shut off of non-self transcripts. Here, we present a fluorescent-based assay to study the interaction between RNA molecules and IFIT family proteins. We have particularly focused on two representatives of this family: IFIT1 and IFIT5. We found a probe that competitively with RNA binds the positively charged tunnel in these IFIT proteins. The use of this probe for IFIT titration allowed us to evaluate the differences in binding affinities of mRNAs with different variants of 5' ends.

Zooming in on protein-RNA interactions: a multi-level workflow to identify interaction partners.

Interactions between proteins and RNA are at the base of numerous cellular regulatory and functional phenomena. The investigation of the biological relevance of non-coding RNAs has led to the identification of numerous novel RNA-binding proteins (RBPs). However, defining the RNA sequences and structures that are selectively recognised by an RBP remains challenging, since these interactions can be transient and highly dynamic, and may be mediated by unstructured regions in the protein, as in the case of many non-canonical RBPs. Numerous experimental and computational methodologies have been developed to predict, identify and verify the binding between a given RBP and potential RNA partners, but navigating across the vast ocean of data can be frustrating and misleading. In this mini-review, we propose a workflow for the identification of the RNA binding partners of putative, newly identified RBPs. The large pool of potential binders selected by in-cell experiments can be enriched by in silico tools such as catRAPID, which is able to predict the RNA sequences more likely to interact with specific RBP regions with high accuracy. The RNA candidates with the highest potential can then be analysed in vitro to determine the binding strength and to precisely identify the binding sites. The results thus obtained can furthermore validate the computational predictions, offering an all-round solution to the issue of finding the most likely RNA binding partners for a newly identified potential RBP.

Defining the RNA interactome by total RNA-associated protein purification.

The RNA binding proteome (RBPome) was previously investigated using UV crosslinking and purification of poly(A)-associated proteins. However, most cellular transcripts are not polyadenylated. We therefore developed total RNA-associated protein purification (TRAPP) based on 254 nm UV crosslinking and purification of all RNA-protein complexes using silica beads. In a variant approach (PAR-TRAPP), RNAs were labelled with 4-thiouracil prior to 350 nm crosslinking. PAR-TRAPP in yeast identified hundreds of RNA binding proteins, strongly enriched for canonical RBPs. In comparison, TRAPP identified many more proteins not expected to bind RNA, and this correlated strongly with protein abundance. Comparing TRAPP in yeast and E. coli showed apparent conservation of RNA binding by metabolic enzymes. Illustrating the value of total RBP purification, we discovered that the glycolytic enzyme enolase interacts with tRNAs. Exploiting PAR-TRAPP to determine the effects of brief exposure to weak acid stress revealed specific changes in late 60S ribosome biogenesis. Furthermore, we identified the precise sites of crosslinking for hundreds of RNA-peptide conjugates, using iTRAPP, providing insights into potential regulation. We conclude that TRAPP is a widely applicable tool for RBPome characterization.

PremPRI: Predicting the Effects of Missense Mutations on Protein-RNA Interactions.

Protein-RNA interactions are crucial for many cellular processes, such as protein synthesis and regulation of gene expression. Missense mutations that alter protein-RNA interaction may contribute to the pathogenesis of many diseases. Here, we introduce a new computational method PremPRI, which predicts the effects of single mutations occurring in RNA binding proteins on the protein-RNA interactions by calculating the binding affinity changes quantitatively. The multiple linear regression scoring function of PremPRI is composed of three sequence- and eight structure-based features, and is parameterized on 248 mutations from 50 protein-RNA complexes. Our model shows a good agreement between calculated and experimental values of binding affinity changes with a Pearson correlation coefficient of 0.72 and the corresponding root-mean-square error of 0.76 kcal·mol-1, outperforming three other available methods. PremPRI can be used for finding functionally important variants, understanding the molecular mechanisms, and designing new protein-RNA interaction inhibitors.

RMalign: an RNA structural alignment tool based on a novel scoring function RMscore.

BACKGROUND: RNA-protein 3D complex structure prediction is still challenging. Recently, a template-based approach PRIME is proposed in our team to build RNA-protein 3D complex structure models with a higher success rate than computational docking software. However, scoring function of RNA alignment algorithm SARA in PRIME is size-dependent, which limits its ability to detect templates in some cases. RESULTS: Herein, we developed a novel RNA 3D structural alignment approach RMalign, which is based on a size-independent scoring function RMscore. The parameter in RMscore is then optimized in randomly selected RNA pairs and phase transition points (from dissimilar to similar) are determined in another randomly selected RNA pairs. In tRNA benchmarking, the precision of RMscore is higher than that of SARAscore (0.88 and 0.78, respectively) with phase transition points. In balance-FSCOR benchmarking, RMalign performed as good as ESA-RNA with a non-normalized score measuring RNA structural similarity. In balance-x-FSCOR benchmarking, RMalign achieves much better than a state-of-the-art RNA 3D structural alignment approach SARA due to a size-independent scoring function. Take the advantage of RMalign, we update our RNA-protein modeling approach PRIME to version 2.0. The PRIME2.0 significantly improves about 10% success rate than PRIME. CONCLUSION: Based on a size-independent scoring function RMscore, a novel RNA 3D structural alignment approach RMalign is developed and integrated into PRIME2.0, which could be useful for the biological community in modeling protein-RNA interaction.

An in vivo cell-based assay for investigating the specific interaction between the SARS-CoV N-protein and its viral RNA packaging sequence.

The SARS-CoV nucleocapsid (N) protein serves multiple functions in viral replication, transcription, and assembly of the viral genome complex. Coronaviruses specifically package genomic RNA into assembled virions, and in SARS-CoV, it is reported that this process is driven by an interaction between the N-protein and a packaging signal encoded within the viral RNA. While recent studies have uncovered the sequence of this packaging signal, little is known about the specific interaction between the N-protein and the packaging signal sequence, and the mechanisms by which this interaction drives viral genome packaging. In this study, we developed a novel in vivo cell-based assay for examining this interaction between the N-protein and packaging signal RNA for SARS-CoV, as well as other viruses within the coronaviridae family. Our results demonstrate that the N-protein specifically recognizes the SARS-CoV packaging signal with greater affinity compared to signals from other coronaviruses or non-coronavirus species. We also use deletion mapping to identify a 151-nt region within the packaging signal sequence that is critical for N-protein-RNA binding, and conversely, we show that both the N-terminal and C-terminal domains of the N protein are necessary for recognizing the packaging RNA. These results describe, for the first time, in vivo evidence for an interaction between the SARS-CoV N-protein and its packaging signal RNA, and demonstrate the feasibility of using this cell-based assay to further probe viral RNA-protein interactions in future studies.

A conformational switch balances viral RNA accessibility and protection in a nucleocapsid ring model.

Virus from the Mononegavirales order share common features ranging from virion structure arrangement to mechanisms of replication and transcription. One of them is the way the nucleoprotein (N) wraps and protects the RNA genome from degradation by forming a highly ordered helical nucleocapsid. However, crystal structures from numerous Mononegavirales reveal that binding to the nucleoprotein results in occluded nucleotides that hinder base pairing necessary for transcription and replication. This hints at the existence of alternative conformations of the N protein that would impact on the protein-RNA interface, allowing for transient exposure of the nucleotides without complete RNA release. Moreover, the regulation between the alternative conformations should be finely tuned. Recombinant expression of N from the respiratory syncytial virus form regular N/RNA common among all Mononegavirales, and these constitute an ideal minimal unit for investigating the mechanisms through which these structures protect RNA so efficiently while allowing for partial accessibility during transcription and replication. Neither pH nor high ionic strength could dissociate the RNA but led to irreversible aggregation of the nucleoprotein. Low concentrations of guanidine chloride dissociated the RNA moiety but leading to irreversible aggregation of the protein moiety. On the other hand, high concentrations of urea and long incubation periods were required to remove bound RNA. Both denaturants eventually led to unfolding but converged in the formation of an RNA-free ß-enriched intermediate species that remained decameric even at high denaturant concentrations. Although the N-RNA rings interact with the phosphoprotein P, the scaffold of the RNA polymerase complex, this interaction did not lead to RNA dissociation from the rings in vitro. Thus, we have uncovered complex equilibria involving changes in secondary structure of N and RNA loosening, processes that must take place in the context of RNA transcription and replication, whose detailed mechanisms and cellular and viral participants need to be established.