Most eukaryotic organisms employ a telomerase complex for the maintenance of chromosome ends. The core of this complex is composed of telomerase reverse transcriptase (TERT) and telomerase RNA (TR) subunits. The TERT reverse transcriptase (RT) domain synthesises telomeric DNA using the TR template sequence. The other TERT domains contribute to this process in different ways. In particular, the TERT RNA-binding domain (TRBD) interacts with specific TR motif(s). Using a yeast 3-hybrid system, we show the critical role of Arabidopsis thaliana (At) TRBD and embryophyta-conserved KRxR motif in the unstructured linker preceding the TRBD domain for binding to the recently identified AtTR subunit. We also show the essential role of the predicted P4 stem and pseudoknot AtTR structures and provide evidence for the binding of AtTRBD to pseudoknot and KRxR motif stabilising interaction with the P4 stem structure. Our results thus provide the first insight into the core part of the plant telomerase complex.

Throughout their life cycle, messenger RNAs (mRNAs) associate with proteins to form ribonucleoproteins (mRNPs). Each mRNA is part of multiple successive mRNP complexes that participate in their biogenesis, cellular localization, translation and decay. The dynamic composition of mRNP complexes and their structural remodelling play crucial roles in the control of gene expression. Studying the endogenous composition of different mRNP complexes is a major challenge. In this chapter, we describe the variety of protein-centric immunoprecipitation methods available for the identification of mRNP complexes and the requirements for their experimental settings.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive single-stranded RNA virus, engages in complex interactions with host cell proteins throughout its life cycle. While these interactions enable the host to recognize and inhibit viral replication, they also facilitate essential viral processes such as transcription, translation, and replication. Many aspects of these virus-host interactions remain poorly understood. Here, we employed the catRAPID algorithm and utilized the RNA-protein interaction detection coupled with mass spectrometry technology to predict and validate the host proteins that specifically bind to the highly structured 5\' and 3\' terminal regions of the SARS-CoV-2 RNA. Among the interactions identified, we prioritized pseudouridine synthase PUS7, which binds to both ends of the viral RNA. Using nanopore direct RNA sequencing, we discovered that the viral RNA undergoes extensive post-transcriptional modifications. Modified consensus regions for PUS7 were identified at both terminal regions of the SARS-CoV-2 RNA, including one in the viral transcription regulatory sequence leader. Collectively, our findings offer insights into host protein interactions with the SARS-CoV-2 UTRs and highlight the likely significance of pseudouridine synthases and other post-transcriptional modifications in the viral life cycle. This new knowledge enhances our understanding of virus-host dynamics and could inform the development of targeted therapeutic strategies.

OBJECTIVE: The Csr/Rsm system (carbon storage regulator or repressor of stationary phase metabolites) is a global post-transcriptional regulatory system that coordinates and responds to environmental cues and signals, facilitating the transition between active growth and stationary phase. Another key determinant of bacterial lifestyle decisions is the management of the cellular gene expression machinery. Here, we investigate the connection between these two processes in Escherichia coli. Disrupted regulation of the transcription and translation machinery impacts many cellular functions, including gene expression, growth, fitness, and stress resistance. Elucidating the role of the Csr system in controlling the activity of RNAP and ribosomes advances our understanding of mechanisms controlling bacterial growth. A more complete understanding of these processes could lead to the improvement of therapeutic strategies for recalcitrant infections.

Positive-strand RNA viruses use long open reading frames to express large polyproteins that are processed into individual proteins by viral proteases. Polyprotein processing is highly regulated and yields intermediate species with different functions than the fully processed proteins, increasing the biochemical diversity of the compact viral genome while also presenting challenges in that proteins must remain stably folded in multiple contexts. We have used circular dichroism spectroscopy and single molecule microscopy to examine the solution structure and self-association of the poliovirus P3 region protein composed of membrane binding 3A, RNA priming 3B (VPg), 3Cpro protease, and 3Dpol RNA-dependent RNA polymerase proteins. Our data indicate that co-folding interactions within the 3ABC segment stabilize the conformational state of the 3C protease region, and this stabilization requires the full-length 3A and 3B proteins. Enzymatic activity assays show that 3ABC is also an active protease, and it cleaves peptide substrates at rates comparable to 3Cpro. The cleavage of a larger polyprotein substrate is stimulated by the addition of RNA, and 3ABCpro becomes 20-fold more active than 3Cpro in the presence of stoichiometric amounts of viral cre RNA. The data suggest that co-folding within the 3ABC region results in a protease that can be highly activated toward certain cleavage sites by localization to specific RNA elements within the viral replication center, providing a mechanism for regulating viral polyprotein processing.

Splicing of pre-mRNAs critically contributes to gene regulation and proteome expansion in eukaryotes, but our understanding of the recognition and pairing of splice sites during spliceosome assembly lacks detail. Here, we identify the multidomain RNA-binding protein FUBP1 as a key splicing factor that binds to a hitherto unknown cis-regulatory motif. By collecting NMR, structural, and in vivo interaction data, we demonstrate that FUBP1 stabilizes U2AF2 and SF1, key components at the 3\' splice site, through multivalent binding interfaces located within its disordered regions. Transcriptional profiling and kinetic modeling reveal that FUBP1 is required for efficient splicing of long introns, which is impaired in cancer patients harboring FUBP1 mutations. Notably, FUBP1 interacts with numerous U1 snRNP-associated proteins, suggesting a unique role for FUBP1 in splice site bridging for long introns. We propose a compelling model for 3\' splice site recognition of long introns, which represent 80% of all human introns.

Protein-RNA interactions play vital roles in plethora of biological processes such as regulation of gene expression, protein synthesis, mRNA processing and biogenesis. Identification of RNA-binding residues (RBRs) in proteins is essential to understand RNA-mediated protein functioning, to perform site-directed mutagenesis and to develop novel targeted drug therapies. Moreover, the extensive gap between sequence and structural data restricts the identification of binding sites in unsolved structures. However, efficient use of computational methods demanding only sequence to identify binding residues can bridge this huge sequence-structure gap. In this study, we have extensively studied protein-RNA interface in known RNA-binding proteins (RBPs). We find that the interface is highly enriched in basic and polar residues with Gly being the most common interface neighbor. We investigated several amino acid features and developed a method to predict putative RBRs from amino acid sequence. We have implemented balanced random forest (BRF) classifier with local residue features of protein sequences for prediction. With 5-fold cross-validations, the sequence pattern derived dipeptide composition based BRF model (DCP-BRF) resulted in an accuracy of 87.9%, specificity of 88.8%, sensitivity of 82.2%, Mathew\'s correlation coefficient of 0.60 and AUC of 0.93, performing better than few existing methods. We further validated our prediction model on known human RBPs through RBR prediction and could map ~54% of them. Further, knowledge of binding site preferences obtained from computational predictions combined with experimental validations of potential RNA binding sites can enhance our understanding of protein-RNA interactions. This may serve to accelerate investigations on functional roles of many novel RBPs.

RNA-binding proteins (RBPs) govern the lifespan of nearly all transcripts and play key roles in adaptive responses in microbes. A robust approach to examine protein-RNA interactions involves irradiating cells with UV light to form covalent adducts between RBPs and their cognate RNAs. Combined with RNA or protein purification, these procedures can provide global RBP censuses or transcriptomic maps for all target sequences of a single protein in living cells. The recent development of novel methods has quickly populated the RBP landscape in microorganisms. Here, we provide an overview of prominent UV cross-linking techniques which have been applied to investigate RNA interactomes in microbes. By assessing their advantages and caveats, this technical evaluation intends to guide the selection of appropriate methods and experimental design as well as to encourage the use of complementary UV-dependent techniques to inspect RNA-binding activity.

Organophosphate hydrolases (OPH), hitherto known to hydrolyze the third ester bond of organophosphate (OP) insecticides and nerve agents, have recently been shown to interact with outer membrane transport components, namely, TonB and ExbB/ExbD. In an OPH negative background, Sphingopyxis wildii cells failed to transport ferric enterobactin and showed retarded growth under iron-limiting conditions. We now show the OPH-encoding organophosphate degradation (opd) gene from Sphingobium fuliginis ATCC 27551 to be part of the iron regulon. A fur-box motif found to be overlapping with the transcription start site (TSS) of the opd gene coordinates with an iron responsive element (IRE) RNA motif identified in the 5\' coding region of the opd mRNA to tightly regulate opd gene expression. The fur-box motif serves as a target for the Fur repressor in the presence of iron. A decrease in iron concentration leads to the derepression of opd. IRE RNA inhibits the translation of opd mRNA and serves as a target for apo-aconitase (IRP). The IRP recruited by the IRE RNA abrogates IRE-mediated translational inhibition. Our findings establish a novel, multilayered, iron-responsive regulation that is crucial for OPH function in the transport of siderophore-mediated iron uptake. IMPORTANCE Sphingobium fuliginis, a soil-dwelling microbe isolated from agricultural soils, was shown to degrade a variety of insecticides and pesticides. These synthetic chemicals function as potent neurotoxins, and they belong to a class of chemicals termed organophosphates. S. fuliginis codes for OPH, an enzyme that has been shown to be involved in the metabolism of several organophosphates and their derivatives. Interestingly, OPH has also been shown to facilitate siderophore-mediated iron uptake in S. fuliginis and in another Sphingomonad, namely, Sphingopyxis wildii, implying that this organophosphate-metabolizing protein has a role in iron homeostasis, as well. Our research dissects the underlying molecular mechanisms linking iron to the expression of OPH, prompting a reconsideration of the role of OPH in Sphingomonads and a reevaluation of the evolutionary origins of the OPH proteins from soil bacteria.

Enveloped RNA viruses are rare among plant viruses. Fimoviridae is a newly founded family of plant viruses within the Bunyavirales order that inflicts diverse crop losses worldwide. The fig mosaic virus (FMV), the representative member of the Fimoviridae family, was shown to be a causative agent for the fig mosaic disease. Like all bunyaviruses, FMV has a segmented, negative-sense, single-stranded RNA (ssRNA) genome that is encapsulated by the viral nucleoprotein (N). Here, we present high-resolution crystal structures of FMV N in its RNA-free and RNA-bound forms, revealing a \"paper fortune teller\" structural transition between the two states. The tightly packed tetramer of FNV N is similar to the structures of other N proteins of different members of the Bunyavirales order. In its RNA-bound form, the tetramer reorganizes to adopt a more open state that allows the accommodation of the RNA. Despite the low sequence similarity to N proteins of animal-infecting bunyaviruses, there is a striking structural resemblance between FMV N and nucleoproteins from members of the Peribunyaviridae, an animal-infecting family of viruses. This structural homology implies that enveloped plant viruses and animal-infecting viruses might have a common ancestor from which they diverged. IMPORTANCE Most insect-born viruses circulate within the Animalia kingdom, whereas plant-infecting RNA viruses are cross-kingdom pathogens. Many plant-infecting viruses cause devastating crop damage that leads to food security endangerment. The evolutionary crossroads of interkingdom circulation and infection are poorly understood. Thus, we took the structural approach to understand the similarities and differences between interkingdom-infecting viruses and viruses that circulate within one kingdom of life. Using our structures of FMV N in its free form and in complex with a single-stranded RNA (ssRNA), we dissected the mechanism by which FMV N binds to the RNA and revealed the conformational changes associated with the binding. The resemblance of our structure to N proteins from members of the Peribunyaviridae family and their recently published ribonucleoprotein (RNP) pseudoatomic resolution assembly model suggests that the FMV genome is similarly encapsulated. Thus, our finding unveils yet another bridge by which plant- and animal-infecting viruses are interconnected.