RNA G-quadruplex structures control ribosomal protein production

Four-stranded G-quadruplex (G4) structures form from guanine-rich tracts, but the extent of their formation in cellular RNA and details of their role in RNA biology remain poorly defined. Herein, we first delineate the presence of endogenous RNA G4s in the human cytoplasmic transcriptome via the binding sites of G4-interacting proteins, DDX3X (previously published), DHX36 and GRSF1. We demonstrate that a sub-population of these RNA G4s are reliably detected as folded structures in cross-linked cellular lysates using the G4 structure-specific antibody BG4. The 5′ UTRs of protein coding mRNAs show significant enrichment in folded RNA G4s, particularly those for ribosomal proteins. Mutational disruption of G4s in ribosomal protein UTRs alleviates translation in vitro, whereas in cells, depletion of G4-resolving helicases or treatment with G4-stabilising small molecules inhibit the translation of ribosomal protein mRNAs. Our findings point to a common mode for translational co-regulation mediated by G4 structures. The results reveal a potential avenue for therapeutic intervention in diseases with dysregulated translation, such as cancer.

Emergence of order in condensates composed of multi-valent, multi-domain proteins

Many RNA-binding proteins (RBPs) that assemble into membraneless organelles, have a common architecture including disordered prion-like domain (PLD) and folded RNA-binding domain (RBD). An enrichment of PLD within the condensed phase gives rise to formation, on longer time scales, amyloid-like fibrils (aging). In this study, we employ coarse-grained Langevin dynamics simulations to explore the physical basis for the structural diversity in condensed phases of multi-domain RBPs. We discovered a highly cooperative first order transition between disordered (liquid-like) structures and an ordered (solid-like) phase whereby chains of PLD organize in fibrils with high nematic orientational order. Cooperativity of this liquid-solid transition makes fibril formation highly malleable to mutations or post-translational modifications. An interplay between homo-domain (PLD-PLD) and hetero-domain (PLD-RBD) interactions results in variety of structures with distinct spatial architectures. Our results provide a mechanistic understanding of how multi-domain RBPs could form assemblies with distinct structural and, potentially, material properties.

HspB8 prevents aberrant phase transitions of FUS by chaperoning its folded RNA binding domain

Aberrant liquid-to-solid phase transitions of biomolecular condensates have been linked to various neurodegenerative diseases. However, the underlying molecular interactions that drive aging remain enigmatic. Here, we develop quantitative time-resolved crosslinking mass spectrometry to monitor protein interactions and dynamics inside condensates formed by the protein fused in sarcoma (FUS). We identify misfolding of the RNA recognition motif (RRM) of FUS as a key driver of condensate ageing. We demonstrate that the small heat shock protein HspB8 partitions into FUS condensates via its intrinsically disordered domain and prevents condensate hardening via condensate-specific interactions that are mediated by its α-crystallin domain (αCD). These αCD-mediated interactions are altered in a disease-associated mutant of HspB8, which abrogates the ability of HspB8 to prevent condensate hardening. We propose that stabilizing aggregation-prone folded RNA-binding domains inside condensates by molecular chaperones may be a general mechanism to prevent aberrant phase transitions.

HspB8 prevents aberrant phase transitions of FUS by chaperoning its folded RNA binding domain

Aberrant liquid-to-solid phase transitions of biomolecular condensates have been linked to various neurodegenerative diseases. However, the underlying molecular interactions that drive aging remain enigmatic. Here, we develop quantitative time-resolved crosslinking mass spectrometry to monitor protein interactions and dynamics inside condensates formed by the protein fused in sarcoma (FUS). We identify misfolding of the RNA recognition motif (RRM) of FUS as a key driver of condensate ageing. We demonstrate that the small heat shock protein HspB8 partitions into FUS condensates via its intrinsically disordered domain and prevents condensate hardening via condensate-specific interactions that are mediated by its α-crystallin domain (αCD). These αCD-mediated interactions are altered in a disease-associated mutant of HspB8, which abrogates the ability of HspB8 to prevent condensate hardening. We propose that stabilizing aggregation-prone folded RNA-binding domains inside condensates by molecular chaperones may be a general mechanism to prevent aberrant phase transitions.

Structural Variants in SARS-CoV-2 Occur at Template-Switching Hotspots

ABSTRACTThe evolutionary dynamics of SARS-CoV-2 have been carefully monitored since the COVID-19 pandemic began in December 2019, however, analysis has focused primarily on single nucleotide polymorphisms and largely ignored the role of structural variants (SVs) as well as recombination in SARS-CoV-2 evolution. Using sequences from the GISAID database, we catalogue over 100 insertions and deletions in the SARS-CoV-2 consensus sequences. We hypothesize that these indels are artifacts of imperfect homologous recombination between SARS-CoV-2 replicates, and provide four independent pieces of evidence. (1) The SVs from the GISAID consensus sequences are clustered at specific regions of the genome. (2) These regions are also enriched for 5’ and 3’ breakpoints in the transcription regulatory site (TRS) independent transcriptome, presumably sites of RNA-dependent RNA polymerase (RdRp) template-switching. (3) Within raw reads, these structural variant hotspots have cases of both high intra-host heterogeneity and intra-host homogeneity, suggesting that these structural variants are both consequences of de novo recombination events within a host and artifacts of previous recombination. (4) Within the RNA secondary structure, the indels occur in “arms” of the predicted folded RNA, suggesting that secondary structure may be a mechanism for TRS-independent template-switching in SARS-CoV-2 or other coronaviruses. These insights into the relationship between structural variation and recombination in SARS-CoV-2 can improve our reconstructions of the SARS-CoV-2 evolutionary history as well as our understanding of the process of RdRp template-switching in RNA viruses.

Comprehensive in-vivo secondary structure of the SARS-CoV-2 genome reveals novel regulatory motifs and mechanisms

SummarySARS-CoV-2 is the positive-sense RNA virus that causes COVID-19, a disease that has triggered a major human health and economic crisis. The genome of SARS-CoV-2 is unique among viral RNAs in its vast potential to form stable RNA structures and yet, as much as 97% of its 30 kilobases have not been structurally explored in the context of a viral infection. Our limited knowledge of SARS-CoV-2 genomic architecture is a fundamental limitation to both our mechanistic understanding of coronavirus life cycle and the development of COVID-19 RNA-based therapeutics. Here, we apply a novel long amplicon strategy to determine for the first time the secondary structure of the SARS-CoV-2 RNA genome probed in infected cells. In addition to the conserved structural motifs at the viral termini, we report new structural features like a conformationally flexible programmed ribosomal frameshifting pseudoknot, and a host of novel RNA structures, each of which highlights the importance of studying viral structures in their native genomic context. Our in-depth structural analysis reveals extensive networks of well-folded RNA structures throughout Orf1ab and reveals new aspects of SARS-CoV-2 genome architecture that distinguish it from other single-stranded, positive-sense RNA viruses. Evolutionary analysis of RNA structures in SARS-CoV-2 shows that several features of its genomic structure are conserved across beta coronaviruses and we pinpoint individual regions of well-folded RNA structure that merit downstream functional analysis. The native, complete secondary structure of SAR-CoV-2 presented here is a roadmap that will facilitate focused studies on mechanisms of replication, translation and packaging, and guide the identification of new RNA drug targets against COVID-19.

Structural Diversity and Phylogenetic Distribution of Valyl tRNA-like Structures in Viruses

ABSTRACTViruses commonly use specifically folded RNA elements that interact with both host and viral proteins to perform functions important for diverse viral processes. Examples are found at the 3′ termini of certain positive-sense ssRNA virus genomes where they partially mimic tRNAs, including being aminoacylated by host cell enzymes. Valine-accepting tRNA-like structures (TLSVal) are an example that share some clear homology to canonical tRNAs but have several important structural differences. Although many examples of TLSVal have been identified, we lacked a full understanding of their structural diversity and phylogenetic distribution. To address this, we undertook an in-depth bioinformatic and biochemical investigation of these RNAs, guided by recent high-resolution structures of a TLSVal. We cataloged many new examples in plant-infecting viruses but also in unrelated insect-specific viruses. Using biochemical and structural approaches, we verified the secondary structure of representative TLSVal substrates and tested their ability to be valylated, finding structural heterogeneity within this class. In a few cases, large stem-loop structures are inserted within distinct variable regions located in an area of the TLS distal to known host cell factor binding sites. In addition, we identified one virus whose TLS has switched its anticodon away from valine; the implications of this remain unclear. These results refine our understanding of the structural and functional mechanistic details of tRNA mimicry and how this may be used in viral infection.

The crystal structure of a Polerovirus exoribonuclease-resistant RNA shows how diverse sequences are integrated into a conserved fold

ABSTRACTExonuclease-resistant RNAs (xrRNAs) are discrete elements that block the progression of 5’ to 3’ exonucleases using specifically folded RNA structures. A recently discovered class of xrRNA is widespread in several genera of plant-infecting viruses, within both noncoding and protein-coding subgenomic RNAs. The structure of one such xrRNA from a dianthovirus revealed three-dimensional details of the resistant fold but did not answer all questions regarding the conservation and diversity of this xrRNA class. Here, we present the crystal structure of a representative polerovirus xrRNA that contains sequence elements that diverge from the previously solved structure. This new structure rationalizes previously unexplained sequence conservation patterns and shows interactions not present in the first structure. Together, the structures of these xrRNAs from dianthovirus and polerovirus genera support the idea that these plant virus xrRNAs fold through a defined pathway that includes a programmed intermediate conformation. This work deepens our knowledge of the structure-function relationship of xrRNAs and shows how evolution can craft similar RNA folds from divergent sequences.