Improved constructs for bait RNA display in a bacterial three-hybrid assay.

We have previously developed a transcription-based bacterial three-hybrid (B3H) assay as a genetic approach to probe RNA-protein interactions inside of E. coli cells. This system offers a straightforward path to identify and assess the consequences of mutations in RBPs with molecular phenotypes of interest. One limiting factor in detecting RNA-protein interactions in the B3H assay is RNA misfolding arising from incorrect base-pair interactions with neighboring RNA sequences in a hybrid RNA. To support correct folding of hybrid bait RNAs, we have explored the use of a highly stable stem ("GC clamp") to isolate regions of a hybrid RNA as discrete folding units. In this work, we introduce new bait RNA constructs to 1) insulate the folding of individual components of the hybrid RNA with GC clamps and 2) express bait RNAs that do not encode their own intrinsic terminator. We find that short GC clamps (5 or 7 bp long) are more effective than a longer 13bp GC clamp in the B3H assay. These new constructs increase the number of Hfq-sRNA and -5'UTR interactions that are detectable in the B3H system and improve the signal-to-noise ratio of many of these interactions. We therefore recommend the use of constructs containing short GC clamps for the expression of future B3H bait RNAs. With these new constructs, a broader range of RNA-protein interactions are detectable in the B3H assay, expanding the utility and impact of this genetic tool as a platform to search for and interrogate mechanisms of additional RNA-protein interactions.

The guide-RNA sequence dictates the slicing kinetics and conformational dynamics of the Argonaute silencing complex.

The RNA-induced silencing complex (RISC), which powers RNA interference (RNAi), consists of a guide RNA and an Argonaute protein that slices target RNAs complementary to the guide. We find that, for different guide-RNA sequences, slicing rates of perfectly complementary bound targets can be surprisingly different (>250-fold range), and that faster slicing confers better knockdown in cells. Nucleotide sequence identities at guide-RNA positions 7, 10, and 17 underlie much of this variation in slicing rates. Analysis of one of these determinants implicates a structural distortion at guide nucleotides 6-7 in promoting slicing. Moreover, slicing directed by different guide sequences has an unanticipated, 600-fold range in 3'-mismatch tolerance, attributable to guides with weak (AU-rich) central pairing requiring extensive 3' complementarity (pairing beyond position 16) to more fully populate the slicing-competent conformation. Together, our analyses identify sequence determinants of RISC activity and provide biochemical and conformational rationale for their action.

Structural Analysis of the Hepatitis B Virus RNA Encapsidation Signal ε by Selective 2'-Hydroxyl Acylation Analyzed by Primer Extension (SHAPE).

RNA structure is crucial for RNA function, including in viral cis-elements such as the hepatitis B virus (HBV) RNA encapsidation signal ε. Interacting with the viral polymerase ε mediates packaging of the pregenomic (pg) RNA into capsids, initiation of reverse transcription, and it affects the mRNA functions of pgRNA. As free RNA, the 61-nucleotide (nt) ε sequence adopts a bipartite stem-loop structure with a central bulge and an apical loop. Due to stable Watson-Crick base pairing, this was already predicted by early RNA folding programs and confirmed by classical enzymatic and chemical structure probing. A newer, high-resolution probing technique exploits the selective acylation of solvent-accessible 2'-hydroxyls in the RNA backbone by electrophilic compounds such as 2-methylnicotinic acid imidazolide (NAI), followed by mapping of the modified sites by primer extension. This SHAPE principle has meanwhile been extended to numerous applications. Here we provide a basic protocol for NAI-based SHAPE of isolated HBV ε RNA which already provided insights into the impact of mutations, and preliminarily, of polymerase binding on the RNA structural dynamics. While the focus is on NAI modification, we also briefly cover target RNA preparation by in vitro transcription, primer extension using a radiolabeled primer, and analysis of the resulting cDNAs by denaturing polyacrylamide gelelectrophoresis (PAGE). Given the high tolerance of SHAPE chemistry to different conditions, including applicability in live cells, we expect this technique to greatly facilitate deciphering the conformational dynamics underlying the various functions of the ε element, especially in concert with the recently solved three-dimensional structure of the free RNA.

Structure-based mechanism of riboregulation of the metabolic enzyme SHMT1.

RNA can directly control protein activity in a process called riboregulation; only a few mechanisms of riboregulation have been described in detail, none of which have been characterized on structural grounds. Here, we present a comprehensive structural, functional, and phylogenetic analysis of riboregulation of cytosolic serine hydroxymethyltransferase (SHMT1), the enzyme interconverting serine and glycine in one-carbon metabolism. We have determined the cryoelectron microscopy (cryo-EM) structure of human SHMT1 in its free- and RNA-bound states, and we show that the RNA modulator competes with polyglutamylated folates and acts as an allosteric switch, selectively altering the enzyme's reactivity vs. serine. In addition, we identify the tetrameric assembly and a flap structural motif as key structural elements necessary for binding of RNA to eukaryotic SHMT1. The results presented here suggest that riboregulation may have played a role in evolution of eukaryotic SHMT1 and in compartmentalization of one-carbon metabolism. Our findings provide insights for RNA-based therapeutic strategies targeting this cancer-linked metabolic pathway.

A primer for junior trainees: Recognition of RNA modifications by RNA-binding proteins.

The complexity of RNA cannot be fully expressed with the canonical A, C, G, and U alphabet. To date, over 170 distinct chemical modifications to RNA have been discovered in living systems. RNA modifications can profoundly impact the cellular outcomes of messenger RNAs (mRNAs), transfer and ribosomal RNAs, and noncoding RNAs. Additionally, aberrant RNA modifications are associated with human disease. RNA modifications are a rising topic within the fields of biochemistry and molecular biology. The role of RNA modifications in gene regulation, disease pathogenesis, and therapeutic applications increasingly captures the attention of the scientific community. This review aims to provide undergraduates, junior trainees, and educators with an appreciation for the significance of RNA modifications in eukaryotic organisms, alongside the skills required to identify and analyze fundamental RNA-protein interactions. The pumilio RNA-binding protein and YT521-B homology (YTH) family of modified RNA-binding proteins serve as examples to highlight the fundamental biochemical interactions that underlie the specific recognition of both unmodified and modified ribonucleotides, respectively. By instilling these foundational, textbook concepts through practical examples, this review contributes an analytical toolkit that facilitates engagement with RNA modifications research at large.

Nucleotide Resolution Mapping of Rift Valley Fever Virus Nucleoprotein-Genome RNA Interactions.

Rift Valley fever virus (RVFV; genus Phlebovirus, family Phenuiviridae, order Bunyavirales) is a mosquito-borne zoonotic pathogen endemic in Africa. Its negative-stranded genomic RNA (vRNA) is divided into three segments termed L, M, and S. Both vRNAs and antigenomic cRNAs are encapsidated by viral nucleoprotein (N) to form nucleocapsids, which constitute the template for genome transcription and replication. Based on a number of electron microscopy and structural studies, the viral RNAs of negative-strand RNA viruses, including phleboviruses, are commonly considered to be entirely and uniformly covered by N protein. However, high resolution data supporting this notion was missing to date.Here, we describe a method how to globally map all N-RNA interactions of RVFV by using iCLIP (individual-nucleotide resolution UV cross-linking and immunoprecipitation). The protocol is based on covalent cross-linking of direct protein-RNA interactions by UV irradiation. Following sample lysis, a selective isolation of N in complex with its RNA targets is achieved by immunoprecipitation. Then, N-RNA complexes are separated by SDS-PAGE, and after membrane transfer, RNA is isolated and subjected to library preparation and high-throughput sequencing. We explain how the standard iCLIP protocol can be adapted to RVFV N-RNA interaction studies. The protocol describes mapping of all N interactions with the vRNAs and cRNAs derived either from RVFV particles or from infected cells.

Identification of Host Proteins Involved in Hepatitis B Virus Genome Packaging.

A critical part of the hepatitis B virus (HBV) life cycle is the packaging of the pregenomic RNA (pgRNA) into nucleocapsids. While this process is known to involve several viral elements, much less is known about the identities and roles of host proteins in this process. To better understand the role of host proteins, we isolated pgRNA and characterized its protein interactome in cells expressing either packaging-competent or packaging-incompetent HBV genomes. We identified over 250 host proteins preferentially associated with pgRNA from the packaging-competent version of the virus. These included proteins already known to support capsid formation, enhance viral gene expression, catalyze nucleocapsid dephosphorylation, and bind to the viral genome, demonstrating the ability of the approach to effectively reveal functionally significant host-virus interactors. Three of these host proteins, AURKA, YTHDF2, and ATR, were selected for follow-up analysis. RNA immunoprecipitation qPCR (RIP-qPCR) confirmed pgRNA-protein association in cells, and siRNA knockdown of the proteins showed decreased encapsidation efficiency. This study provides a template for the use of comparative RNA-protein interactome analysis in conjunction with virus engineering to reveal functionally significant host-virus interactions.

Spatiotemporal constrained RNA-protein heterogeneous network for protein complex identification.

The identification of protein complexes from protein interaction networks is crucial in the understanding of protein function, cellular processes and disease mechanisms. Existing methods commonly rely on the assumption that protein interaction networks are highly reliable, yet in reality, there is considerable noise in the data. In addition, these methods fail to account for the regulatory roles of biomolecules during the formation of protein complexes, which is crucial for understanding the generation of protein interactions. To this end, we propose a SpatioTemporal constrained RNA-protein heterogeneous network for Protein Complex Identification (STRPCI). STRPCI first constructs a multiplex heterogeneous protein information network to capture deep semantic information by extracting spatiotemporal interaction patterns. Then, it utilizes a dual-view aggregator to aggregate heterogeneous neighbor information from different layers. Finally, through contrastive learning, STRPCI collaboratively optimizes the protein embedding representations under different spatiotemporal interaction patterns. Based on the protein embedding similarity, STRPCI reweights the protein interaction network and identifies protein complexes with core-attachment strategy. By considering the spatiotemporal constraints and biomolecular regulatory factors of protein interactions, STRPCI measures the tightness of interactions, thus mitigating the impact of noisy data on complex identification. Evaluation results on four real PPI networks demonstrate the effectiveness and strong biological significance of STRPCI. The source code implementation of STRPCI is available from https://github.com/LI-jasm/STRPCI.

The guide RNA sequence dictates the slicing kinetics and conformational dynamics of the Argonaute silencing complex.

The RNA-induced silencing complex (RISC), which powers RNA interference (RNAi), consists of a guide RNA and an Argonaute protein that slices target RNAs complementary to the guide. We find that for different guide-RNA sequences, slicing rates of perfectly complementary, bound targets can be surprisingly different (>250-fold range), and that faster slicing confers better knockdown in cells. Nucleotide sequence identities at guide-RNA positions 7, 10, and 17 underlie much of this variation in slicing rates. Analysis of one of these determinants implicates a structural distortion at guide nucleotides 6-7 in promoting slicing. Moreover, slicing directed by different guide sequences has an unanticipated, 600-fold range in 3'-mismatch tolerance, attributable to guides with weak (AU-rich) central pairing requiring extensive 3' complementarity (pairing beyond position 16) to more fully populate the slicing-competent conformation. Together, our analyses identify sequence determinants of RISC activity and provide biochemical and conformational rationale for their action.

The Post-Transcriptional Regulatory Protein CsrA Amplifies Its Targetome through Direct Interactions with Stress-Response Regulatory Hubs: The EvgA and AcnA Cases.

Global rewiring of bacterial gene expressions in response to environmental cues is mediated by regulatory proteins such as the CsrA global regulator from E. coli. Several direct mRNA and sRNA targets of this protein have been identified; however, high-throughput studies suggest an expanded RNA targetome for this protein. In this work, we demonstrate that CsrA can extend its network by directly binding and regulating the evgA and acnA transcripts, encoding for regulatory proteins. CsrA represses EvgA and AcnA expression and disrupting the CsrA binding sites of evgA and acnA, results in broader gene expression changes to stress response networks. Specifically, altering CsrA-evgA binding impacts the genes related to acidic stress adaptation, and disrupting the CsrA-acnA interaction affects the genes involved in metal-induced oxidative stress responses. We show that these interactions are biologically relevant, as evidenced by the improved tolerance of evgA and acnA genomic mutants depleted of CsrA binding sites when challenged with acid and metal ions, respectively. We conclude that EvgA and AcnA are intermediate regulatory hubs through which CsrA can expand its regulatory role. The indirect CsrA regulation of gene networks coordinated by EvgA and AcnA likely contributes to optimizing cellular resources to promote exponential growth in the absence of stress.

Optimizing ChIRP-MS for Comprehensive Profiling of RNA-Protein Interactions in Arabidopsis thaliana: A Telomerase RNA Case Study.

The current repertoire of methods available for studying RNA-protein interactions in plants is somewhat limited. Employing an RNA-centric approach, particularly with less abundant RNAs, presents various challenges. Many of the existing methods were initially designed for different model systems, with their application in plants receiving limited attention thus far. The Comprehensive Identification of RNA-Binding Proteins by Mass Spectrometry (ChIRP-MS) technique, initially developed for mammalian cells, has been adapted in this study for application in Arabidopsis thaliana. The procedures have been meticulously modified and optimized for telomerase RNA, a notable example of a low-abundance RNA recently identified. Following these optimization steps, ChIRP-MS can serve as an effective screening method for identifying candidate proteins interacting with any target RNA of interest.

Long Intergenic Non-Coding RNAs of Human Chromosome 18: Focus on Cancers.

Malignant neoplasms are characterized by high molecular heterogeneity due to multilevel deregulation of gene expression and cellular functions. It is known that non-coding RNAs, including long intergenic non-coding RNAs (lincRNAs), can play significant roles in cancer biology. The current review focuses on a systematical analysis of genomic, transcriptomic, epigenomic, interactomic, and literature data on 65 lincRNAs of human chromosome 18 in the context of pan-cancer studies. The entire group of lincRNAs can be conditionally divided into 4 subgroups depending on experimental evidence on direct or indirect involvement in cancers and the biological associations with cancers, which we found during the data-mining process: the most studied (5 lincRNAs), moderately or poorly studied (11 lincRNAs), and understudied (31 lincRNAs). For the remaining 18 lincRNAs, data for analysis were fragmentary or missing. Among the key findings were the following: Of the lincRNAs of human chromosome 18, 40% have tissue-specific expression patterns, 22% of lincRNAs are known to have gene fusions, 40% of lincRNAs are prone to gene amplifications and/or deletions in cancers at a frequency greater than 3%, and 23% of lincRNAs are differentially expressed across cancer types, whereas 7% have subtype-specific expression patterns. LincRNAs' interactomes consist of 'master' microRNAs and 47 proteins (including cancer-associated proteins and microRNAs) that can interact with 3 or more lincRNAs. Functional enrichment analysis of a set of highly co-expressed genes retrieved for 17 lincRNAs in different cancer types indicated the potential associations of these lincRNAs with cellular signaling pathways. Six lincRNAs encoded small open-reading frame (smORF) proteins with emerging roles in cancers, and microRNAs as well as proteins with known functions in molecular carcinogenesis can bind to coding regions of smORFs. We identified seven transcriptomic signatures with potential prognostic value, consisting of two to seven different lincRNAs only. Taken together, the literature, biomedical, and molecular biology data analyzed indicated that only five of all lincRNAs of human chromosome 18 are cancer-associated, while eleven other lincRNAs have the tendency to be associated with cancers.

Extraribosomal Functions of Bacterial Ribosomal Proteins-An Update, 2023.

Ribosomal proteins (r-proteins) are abundant, highly conserved, and multifaceted cellular proteins in all domains of life. Most r-proteins have RNA-binding properties and can form protein-protein contacts. Bacterial r-proteins govern the co-transcriptional rRNA folding during ribosome assembly and participate in the formation of the ribosome functional sites, such as the mRNA-binding site, tRNA-binding sites, the peptidyl transferase center, and the protein exit tunnel. In addition to their primary role in a cell as integral components of the protein synthesis machinery, many r-proteins can function beyond the ribosome (the phenomenon known as moonlighting), acting either as individual regulatory proteins or in complexes with various cellular components. The extraribosomal activities of r-proteins have been studied over the decades. In the past decade, our understanding of r-protein functions has advanced significantly due to intensive studies on ribosomes and gene expression mechanisms not only in model bacteria like Escherichia coli or Bacillus subtilis but also in little-explored bacterial species from various phyla. The aim of this review is to update information on the multiple functions of r-proteins in bacteria.

Cryo-EM structures of Mycobacterium tuberculosis polynucleotide phosphorylase suggest a potential mechanism for its RNA substrate degradation.

As one of the oldest infectious diseases in the world, tuberculosis (TB) is the second most deadly infectious disease after COVID-19. Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), which can attack various organs of the human body. Up to now, drug-resistant TB continues to be a public health threat. Pyrazinamide (PZA) is regarded as a sterilizing drug in the treatment of TB due to its distinct ability to target Mtb persisters. Previously we demonstrated that a D67N mutation in Mycobacterium tuberculosis polynucleotide phosphorylase (MtbPNPase, Rv2783c) confers resistance to PZA and Rv2783c is a potential target for PZA, but the mechanism leading to PZA resistance remains unclear. To gain further insight into the MtbPNPase, we determined the cryo-EM structures of apo Rv2783c, its mutant form and its complex with RNA. Our studies revealed the Rv2783c structure at atomic resolution and identified its enzymatic functional groups essential for its phosphorylase activities. We also investigated the molecular mechanisms underlying the resistance to PZA conferred by the mutation. Our research findings provide structural and functional insights enabling the development of new anti-tuberculosis drugs.

In silico λ-dynamics predicts protein binding specificities to modified RNAs.

RNA modifications shape gene expression through a smorgasbord of chemical changes to canonical RNA bases. Although numbering in the hundreds, only a few RNA modifications are well characterized, in part due to the absence of methods to identify modification sites. Antibodies remain a common tool to identify modified RNA and infer modification sites through straightforward applications. However, specificity issues can result in off-target binding and confound conclusions. This work utilizes in silico λ-dynamics to efficiently estimate binding free energy differences of modification-targeting antibodies between a variety of naturally occurring RNA modifications. Crystal structures of inosine and N6-methyladenosine (m6A) targeting antibodies bound to their modified ribonucleosides were determined and served as structural starting points. λ-Dynamics was utilized to predict RNA modifications that permit or inhibit binding to these antibodies. In vitro RNA-antibody binding assays supported the accuracy of these in silico results. High agreement between experimental and computed binding propensities demonstrated that λ-dynamics can serve as a predictive screen for antibody specificity against libraries of RNA modifications. More importantly, this strategy is an innovative way to elucidate how hundreds of known RNA modifications interact with biological molecules without the limitations imposed by in vitro or in vivo methodologies.

Dissection of protein and RNA regions required for SPEN binding to XIST A-repeat RNA.

XIST noncoding RNA promotes the initiation of X chromosome silencing by recruiting the protein SPEN to one X chromosome in female mammals. The SPEN protein is also called SHARP (SMRT and HDAC-associated repressor protein) and MINT (Msx-2 interacting nuclear target) in humans. SPEN recruits N-CoR2 and HDAC3 to initiate histone deacetylation on the X chromosome, leading to the formation of repressive chromatin marks and silencing gene expression. We dissected the contributions of different RNA and protein regions to the formation of a human XIST-SPEN complex in vitro and identified novel sequence and structure determinants that may contribute to X chromosome silencing initiation. Binding of SPEN to XIST RNA requires RRM 4 of the protein, in contrast to the requirement of RRM 3 and RRM 4 for specific binding to SRA RNA. Measurements of SPEN binding to full-length, dimeric, trimeric, or other truncated versions of the A-repeat region revealed that high-affinity binding of XIST to SPEN in vitro requires a minimum of four A-repeat segments. SPEN binding to XIST A-repeat RNA changes the accessibility of the RNA at specific nucleotide sequences, as indicated by changes in RNA reactivity through chemical structure probing. Based on computational modeling, we found that inter-repeat duplexes formed by multiple A-repeats can present an unpaired adenosine in the context of a double-stranded region of RNA. The presence of this specific combination of sequence and structural motifs correlates with high-affinity SPEN binding in vitro. These data provide new information on the molecular basis of the XIST and SPEN interaction.

Defining Distinct RNA-Protein Interactomes of SARS-CoV-2 Genomic and Subgenomic RNAs.

Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of viral replication. Here, for the first time, we demonstrate the successful isolation of SARS-CoV-2 genomic RNA and three distinct sgRNAs (N, S, and ORF8) from a single population of infected cells and characterize their protein interactomes. Over 500 protein interactors (including 260 previously unknown) were identified as associated with one or more target RNA. These included protein interactors unique to a single RNA pool and others present in multiple pools, highlighting our ability to discriminate between distinct viral RNA interactomes despite high sequence similarity. Individual interactomes indicated viral associations with cell response pathways, including regulation of cytoplasmic ribonucleoprotein granules and posttranscriptional gene silencing. We tested the significance of three protein interactors in these pathways (APOBEC3F, PPP1CC, and MSI2) using siRNA knockdowns, with several knockdowns affecting viral gene expression, most consistently PPP1CC. This study describes a new technology for high-resolution studies of SARS-CoV-2 RNA regulation and reveals a wealth of new viral RNA-associated host factors of potential functional significance to infection.

Measuring Proximity-Mediated Function of mRNA Regulatory Proteins by Engineered Tethering.

A powerful approach for studying the functional consequences of site-specific RNA-protein interactions is to artificially tether a protein to a messenger (or noncoding) RNA through a selective, high-affinity interaction. We share a strategy for evaluating the contribution of protein positioning within an mRNA on gene expression. We introduced an RNA hairpin recognition site for the MS2 coat protein into the untranslated regions or coding sequence of mRNAs expressing a luminescent reporter protein, NanoLuc. Effector proteins fused to the MS2 coat protein could thus be targeted to distinct regions across the mRNA. We illustrate this approach using ZFP36L2, which recruits the CCR4-NOT complex for poly(A) tail deadenylation. Tethering ZFP36L2 to the 3'-UTR decreased NanoLuc expression, as expected, given the known interaction of this adapter protein with adenine uridine-rich elements (AREs). Intriguingly, ZFP36L2 also decreased NanoLuc expression when bound within the coding sequence, revealing that ZFP36L2-and potentially many other mRNA regulatory proteins-can function when targeted to diverse locations within an mRNA. This multi-target tethering strategy enables exploration of the interplay between mRNA-protein proximity and gene expression.

CsrA selectively modulates sRNA-mRNA regulator outcomes.

Post-transcriptional regulation, by small RNAs (sRNAs) as well as the global Carbon Storage Regulator A (CsrA) protein, play critical roles in bacterial metabolic control and stress responses. The CsrA protein affects selective sRNA-mRNA networks, in addition to regulating transcription factors and sigma factors, providing additional avenues of cross talk between other stress-response regulators. Here, we expand the known set of sRNA-CsrA interactions and study their regulatory effects. In vitro binding assays confirm novel CsrA interactions with ten sRNAs, many of which are previously recognized as key regulatory nodes. Of those 10 sRNA, we identify that McaS, FnrS, SgrS, MicL, and Spot42 interact directly with CsrA in vivo. We find that the presence of CsrA impacts the downstream regulation of mRNA targets of the respective sRNA. In vivo evidence supports enhanced CsrA McaS-csgD mRNA repression and showcases CsrA-dependent repression of the fucP mRNA via the Spot42 sRNA. We additionally identify SgrS and FnrS as potential new sRNA sponges of CsrA. Overall, our results further support the expanding impact of the Csr system on cellular physiology via CsrA impact on the regulatory roles of these sRNAs.

Structural basis of RNA-induced autoregulation of the DExH-type RNA helicase maleless.

RNA unwinding by DExH-type helicases underlies most RNA metabolism and function. It remains unresolved if and how the basic unwinding reaction of helicases is regulated by auxiliary domains. We explored the interplay between the RecA and auxiliary domains of the RNA helicase maleless (MLE) from Drosophila using structural and functional studies. We discovered that MLE exists in a dsRNA-bound open conformation and that the auxiliary dsRBD2 domain aligns the substrate RNA with the accessible helicase tunnel. In an ATP-dependent manner, dsRBD2 associates with the helicase module, leading to tunnel closure around ssRNA. Furthermore, our structures provide a rationale for blunt-ended dsRNA unwinding and 3'-5' translocation by MLE. Structure-based MLE mutations confirm the functional relevance of our model for RNA unwinding. Our findings contribute to our understanding of the fundamental mechanics of auxiliary domains in DExH helicase MLE, which serves as a model for its human ortholog and potential therapeutic target, DHX9/RHA.