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The nucleocapsid protein (N) of SARS-CoV-2 plays a pivotal role during the viral life cycle. It is involved in RNA transcription and accounts for packaging of the large genome into virus particles. N manages the enigmatic balance of bulk RNA-coating versus precise RNA-binding to designated cis-regulatory elements. Numerous studies report the involvement of its disordered segments in non-selective RNA-recognition, but how N organizes the inevitable recognition of specific motifs remains unanswered. We here use NMR spectroscopy to systematically analyze the interactions of N’s N-terminal RNA-binding domain (NTD) with individual cis RNA elements clustering in the SARS-CoV-2 regulatory 5’-genomic end. Supported by broad solution-based biophysical data, we unravel the NTD RNA-binding preferences in the natural genome context. We show that the domain’s flexible regions read the intrinsic signature of preferred RNA elements for selective and stable complex formation within the large pool of available motifs. SARS-CoV-2 genome turnover is mediated by its N protein, but precise parameters driving the necessary RNA specificity have remained enigmatic. Here, Korn et al. reveal N’s N-terminal domain to distinguish regulatory viral RNA motifs with a preference for transiently folded elements of functional impact.

The SARS-CoV-2 nucleocapsid protein is indispensable for viral RNA genome processing. Although the N-terminal domain (NTD) is suggested to mediate specific RNA-interactions, high-resolution structures with viral RNA are still lacking. Available hybrid structures of the NTD with ssRNA and dsRNA provide valuable insights; however, the precise mechanism of complex formation remains elusive. Similarly, the molecular impact of nucleocapsid NTD mutations that have emerged since 2019 has not yet been fully explored. Using crystallography and solution NMR, we investigate how NTD mutations influence structural integrity and RNA-binding. We find that both features rely on a core network of residues conserved in Betacoronaviruses , crucial for protein stability and communication among flexible loop-regions that facilitate RNA-recognition. Our comprehensive structural analysis demonstrates that contacts within this network guide selective RNA-interactions. We propose that the core network renders the NTD evolutionarily robust in stability and plasticity for its versatile RNA processing roles. The authors use solution-state NMR and X-ray crystallography to study mutations in the SARS-CoV-2 Nucleocapsid NTD, uncovering a network essential for protein integrity and the selectively of RNA binding.

How multidomain RNA-binding proteins recognize their specific target sequences, based on a combinatorial code, represents a fundamental unsolved question and has not been studied systematically so far. Here we focus on a prototypical multidomain RNA-binding protein, IMP3 (also called IGF2BP3), which contains six RNA-binding domains (RBDs): four KH and two RRM domains. We establish an integrative systematic strategy, combining single-domain-resolved SELEX-seq, motif-spacing analyses, in vivo iCLIP, functional validation assays, and structural biology. This approach identifies the RNA-binding specificity and RNP topology of IMP3, involving all six RBDs and a cluster of up to five distinct and appropriately spaced CA-rich and GGC-core RNA elements, covering a >100 nucleotide-long target RNA region. Our generally applicable approach explains both specificity and flexibility of IMP3-RNA recognition, allows the prediction of IMP3 targets, and provides a paradigm for the function of multivalent interactions with multidomain RNA-binding proteins in gene regulation. Multidomain RNA-binding proteins recognize specific target sequences through mechanisms that are not well understood. Here the authors present an integrated approach to define the RNA-binding specificity and RNP topology and apply it to the analysis of the prototypical multidomain RNA-binding protein IMP3.

Background Horizontal gene transfer (HGT) is a major driver of microbial evolution, yet the influence of host cellular context on the integration and functionality of transferred genes remains underexplored. In this study, we investigate how host background impacts the horizontal acquisition of post-translational modification (PTM) machinery. Here, we use heterologous expression of the highly conserved and frequently horizontally transferred translational elongation factor P (EF-P) from diverse species in Escherichia coli as a model. EF-P has a heterogenous relationship with PTMs; three characterized variants each undergo distinct PTM pathways, while others function effectively without any modification. Results We demonstrate that EF-P from Deinococcus radiodurans , Geoalkalibacter ferrihydriticus , and Nitrosomonas communis can complement an EF-P knockout in E. coli without requiring any PTM, suggesting they may represent new examples of unmodified EF-P. We also found that the EF-P from the Thermotogota Mesotoga prima is post-translationally modified in an off-target reaction by the rhamnosylation enzyme EarP, thus interfering with its functionality. Conversely, we saw that rhamnosylation by EarP does not impact the function of the EF-P-like protein EfpL. Conclusions Our findings highlight that PTM systems introduced via HGT can have varied effects on host proteins. We found that different EF-P variants are impacted in different ways by off-target rhamnosylation. While some of these off-target reactions may present opportunities to develop novel, catalytically active PTMs, others are detrimental to the function of the modified EF-P. Our results emphasize the complexity of gene integration and functional compatibility in foreign genomic contexts.

The nuclear exosome and its essential co-factor, the RNA helicase MTR4, play crucial roles in several RNA degradation pathways. Besides unwinding RNA substrates for exosome-mediated degradation, MTR4 associates with RNA-binding proteins that function as adaptors in different RNA processing and decay pathways. Here, we identify and characterize the interactions of human MTR4 with a ribosome processing adaptor, NVL, and with ZCCHC8, an adaptor involved in the decay of small nuclear RNAs. We show that the unstructured regions of NVL and ZCCHC8 contain short linear motifs that bind the MTR4 arch domain in a mutually exclusive manner. These short sequences diverged from the arch-interacting motif (AIM) of yeast rRNA processing factors. Our results suggest that nuclear exosome adaptors have evolved canonical and non-canonical AIM sequences to target human MTR4 and demonstrate the versatility and specificity with which the MTR4 arch domain can recruit a repertoire of different RNA-binding proteins. The human RNA exosome contains a nuclear co-factor MTR4, which unwinds structural RNAs and recruits adaptors for different RNA processing and decay pathways. Here the authors uncover new variations of the arch-interacting motif (AIM) in NVL and ZCCHC8 and characterise their interaction with MTR4.

Polyproline sequences are deleterious to cells because they stall ribosomes. In bacteria, EF-P plays an important role in overcoming such polyproline sequence-induced ribosome stalling. Additionally, numerous bacteria possess an EF-P paralog called EfpL (also known as YeiP) of unknown function. Here, we functionally and structurally characterize EfpL from Escherichia coli and demonstrate its role in the translational stress response. Through ribosome profiling, we analyze the EfpL arrest motif spectrum and find additional sequences beyond the canonical polyproline motifs that both EF-P and EfpL can resolve. Notably, the two factors can also induce pauses. We further report that EfpL can sense the metabolic state of the cell via lysine acylation. Overall, our work characterizes the role of EfpL in ribosome rescue at proline-containing sequences, and provides evidence that co-occurrence of EF-P and EfpL is an evolutionary driver for higher bacterial growth rates. EF-P plays an important role in overcoming polyproline sequence-induced stalling in bacteria. Here, the authors show that its paralog, EfpL, can also resolve this type of stalling, and also sense cellular metabolic states via lysine acylation.

Roquin controls T-cell activity through interactions with mRNAs of stimulatory receptors. Structural and functional elucidation of its RNA-binding domain reveals how it interacts with constitutive decay elements in the 3' UTR of its targets to regulate their expression. Roquin function in T cells is essential for the prevention of autoimmune disease. Roquin interacts with the 3′ untranslated regions (UTRs) of co-stimulatory receptors and controls T-cell activation and differentiation. Here we show that the N-terminal ROQ domain from mouse roquin adopts an extended winged-helix (WH) fold, which is sufficient for binding to the constitutive decay element (CDE) in the Tnf 3′ UTR. The crystal structure of the ROQ domain in complex with a prototypical CDE RNA stem-loop reveals tight recognition of the RNA stem and its triloop. Surprisingly, roquin uses mainly non-sequence-specific contacts to the RNA, thus suggesting a relaxed CDE consensus and implicating a broader spectrum of target mRNAs than previously anticipated. Consistently with this, NMR and binding experiments with CDE-like stem-loops together with cell-based assays confirm roquin-dependent regulation of relaxed CDE consensus motifs in natural 3′ UTRs.

The RNA-binding proteins Roquin-1 and Roquin-2 redundantly control gene expression and cell-fate decisions. Here, we show that Roquin not only interacts with stem–loop structures, but also with a linear sequence element present in about half of its targets. Comprehensive analysis of a minimal response element of the Nfkbid 3′-UTR shows that six stem–loop structures cooperate to exert robust and profound post-transcriptional regulation. Only binding of multiple Roquin proteins to several stem–loops exerts full repression, which redundantly involved deadenylation and decapping, but also translational inhibition. Globally, most Roquin targets are regulated by mRNA decay, whereas a small subset, including the Nfat5 mRNA, with more binding sites in their 3′-UTRs, are also subject to translational inhibition. These findings provide insights into how the robustness and magnitude of Roquin-mediated regulation is encoded in complex cis -elements. Roquin targets are known to contain two types of sequence-structure motifs, the constitutive and the alternative decay elements (CDE and ADE). Here, the authors describe a linear Roquin binding element (LBE) also involved in target recognition, and show that Roquin binding affects the translation of a subset of targeted mRNAs.

The SARS-CoV-2 nucleocapsid (N) protein is crucial for the highly organized packaging and transcription of the genomic RNA. Studying atomic details of the role of its intrinsically disordered regions (IDRs) in RNA recognition is challenging due to the absence of structure and to the repetitive nature of their primary sequence. IDRs are known to act in concert with the folded domains of N and here we use NMR spectroscopy to identify the priming events of N interacting with a regulatory SARS-CoV-2 RNA element. 13C-detected NMR experiments, acquired simultaneously to 1H detected ones, provide information on the two IDRs flanking the N-terminal RNA binding domain (NTD) within the N-terminal region of the protein (NTR, 1–248). We identify specific tracts of the IDRs that most rapidly sense and engage with RNA, and thus provide an atom-resolved picture of the interplay between the folded and disordered regions of N during RNA interaction.

The RNA-binding protein Roquin is required to prevent autoimmunity. Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs4 or Ox40). A constitutive decay element (CDE) with a characteristic triloop hairpin was previously shown to be recognized by Roquin. Here we use SELEX assays to identify a novel U-rich hexaloop motif, representing an alternative decay element (ADE). Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes. In cells, ADE-like and CDE-like motifs cooperate in the repression of Ox40 by Roquin. Our data reveal an unexpected recognition of hexaloop cis elements for the posttranscriptional regulation of target messenger RNAs by Roquin. Roquin is an RNA-binding protein that prevents autoimmunity by limiting expression of receptors such as Ox40. Here, the authors identify an RNA structure that they describe as an alternative decay element, and they characterise its interaction with Roquin using structural and biochemical techniques.