Biology subjects

Kieft, J. S.

Publications and source records attributed to Kieft, J. S..

25 records · Page 2Linked to original sources

bioRxiv2020

A new subclass of exoribonuclease resistant RNA found in multiple Flaviviridae genera

Viruses have developed innovative strategies to exploit the cellular machinery and overcome the host antiviral defenses, often using specifically structured RNA elements. Examples are found in flaviviruses; during flaviviral infection, pathogenic subgenomic flaviviral RNAs (sfRNAs) accumulate in the cell. These sfRNAs are formed when a host cell 5 to 3 exoribonuclease degrades the viral genomic RNA but is blocked by an exoribonuclease resistant RNA structure (xrRNA) located in the viral genomes 3untranslated region (UTR). Although known to exist in several Flaviviridae genera the full distribution and diversity of xRNAs in this virus family was unknown. Using the recent high-resolution structure of an xrRNA from the divergent flavivirus Tamana bat virus (TABV) as a reference, we used bioinformatic searches to identify xrRNA in the Pegivirus, Pestivirus, and Hepacivirus genera. We biochemically and structurally characterized several examples, determining that they are genuine xrRNAs with a conserved fold. These new xrRNAs look superficially similar to the previously described xrRNAs but possess structural differences making them distinct from previous classes of xrRNAs. Our findings thus require adjustments of previous xrRNA classification schemes and expand on the previously known distribution of the xrRNA in Flaviviridae, indicating their widespread distribution and illustrating their importance. IMPORTANCEThe Flaviviridae comprise one of the largest families of positive sense single stranded (+ssRNA) and it is divided into the Flavivirus, Pestivirus, Pegivirus, and Hepacivirus genera. The genus Flavivirus contains many medically relevant viruses such as Zika Virus, Dengue Virus, and Powassan Virus. In these, a part of the viruss RNA twists up into a very special three-dimensional shape called an xrRNA that blocks the ability of the cell to "chew up" the viral RNA. Hence, part of the virus RNA remains intact, and this protected part is important for viral infection. This was known to occur in Flaviviruses but whether it existed in the other members of the family was not known. In this study, we not only identified a new subclass of xrRNA found in Flavivirus but also in the remaining three genera. The fact that this process of viral RNA maturation exists throughout the entire Flaviviridae family makes it clear that this is an important but underappreciated part of the infection strategy of these diverse human pathogens.

microbiology↗

bioRxiv2020

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

Viruses 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.

biochemistry↗

bioRxiv2020

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

Exonuclease-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.

biochemistry↗

bioRxiv2020

RNA matchmaking remodels lncRNA structure and promotes PRC2 activity

Human Polycomb Repressive Complex 2 (PRC2) catalysis of histone H3 lysine 27 methylation at certain loci depends on long noncoding RNAs (lncRNAs). Yet, in apparent contradiction, RNA is a potent catalytic inhibitor of PRC2. Here we show that intermolecular RNA-RNA interactions between the lncRNA HOTAIR and its target genes can relieve RNA inhibition of PRC2. RNA matchmaking is promoted by heterogenous nuclear ribonucleoprotein (hnRNP) B1, which uses multiple protein domains to bind regions of HOTAIR via multi-valent protein-RNA interactions. Chemical probing demonstrates that RNA matchmaking changes HOTAIR RNA structure. Genome-wide HOTAIR/PRC2 activity occurs at genes whose transcripts can make favorable RNA-RNA interactions with HOTAIR. We demonstrate that RNA-RNA matches of HOTAIR with target gene RNAs can relieve the inhibitory effect of a single lncRNA for PRC2 activity. Our work highlights an intrinsic switch that allows PRC2 activity in specific RNA contexts, which could explain how many lncRNAs work with PRC2.

biochemistry↗

bioRxiv2018

Pervasiveness of exoribonuclease-resistant RNAs in plant viruses suggests new roles for these conserved RNA structures

Exoribonuclease-resistant RNAs (xrRNAs) are discrete folded RNA elements that block the processive degradation of RNA by exoribonucleases. xrRNAs found in the 3' untranslated regions (UTRs) of animal-infecting flaviviruses and in all three members of the plant-infecting Dianthovirus adopt a complex ring-like fold that blocks the exoribonuclease; this ability gives rise to viral non-coding subgenomic RNAs. The degree to which these folded RNA elements exist in other viruses and in diverse contexts has been unclear. Using computational tools and biochemical assays, we discovered that xrRNA elements are widely found in viruses belonging to the Tombusviridae and Luteoviridae families of plant-infecting RNA viruses, demonstrating their importance and widespread utility. Unexpectedly, many xrRNAs are located in intergenic regions rather than in the 3UTR and some are associated with the 5' ends of subgenomic RNAs with protein-coding potential, suggesting that xrRNAs with similar scaffolds are involved in the maturation or maintenance of diverse subgenomic RNAs, not just the ones generated from the 3'UTR.

microbiology↗

bioRxiv2018

A folded viral noncoding RNA blocks host cell exoribonucleases through programmed remodeling of RNA structure

Folded RNA elements that block processive 5'[->]3' cellular exoribonucleases (xrRNAs) to produce biologically active viral non-coding RNAs were discovered in flaviviruses, potentially revealing a new mode of RNA maturation. However, it was unknown if this RNA structure-dependent mechanism exists elsewhere and if so, whether a singular RNA fold is required. Here, we demonstrate the existence of authentic RNA structure-dependent xrRNAs in dianthoviruses, plant-infecting viruses unrelated to animal-infecting flaviviruses. These novel xrRNAs have no sequence similarity to known xrRNAs, thus we used a combination of biochemistry and virology to characterize their sequence requirements and mechanism of stopping exoribonucleases. By solving the structure of a dianthovirus xrRNAs by x-ray crystallography, we reveal a complex fold that is very different from the flavivirus xrRNAs. However, both versions of xrRNAs contain a unique topological feature that is created by a different set of intramolecular contacts; this may be a defining structural feature of xrRNAs. Remarkably, the dianthovirus xrRNA can use co-degradational remodeling, exploiting the exoribonucleases degradation-linked helicase activity to help form their resistant structure; such a mechanism has not previously been reported. Convergent evolution has created RNA structure-dependent exoribonuclease resistance in different contexts, which establishes it as a general RNA maturation mechanism and defines xrRNAs as an authentic functional class of RNAs.

biochemistry↗

bioRxiv2017

Computational Design of Asymmetric Three-dimensional RNA Structures and Machines.

The emerging field of RNA nanotechnology seeks to create nanoscale 3D machines by repurposing natural RNA modules, but successes have been limited to symmetric assemblies of single repeating motifs. We present RNAMake, a suite that automates design of RNA molecules with complex 3D folds. We first challenged RNAMake with the paradigmatic problem of aligning a tetraloop and sequence-distal receptor, previously only solved via symmetry. Single-nucleotide-resolution chemical mapping, native gel electrophoresis, and solution x-ray scattering confirmed that 11 of the 16 miniTTR designs successfully achieved clothespin-like folds. A 2.55 [A] diffraction-resolution crystal structure of one design verified formation of the target asymmetric nanostructure, with large sections achieving near-atomic accuracy (< 2.0 [A]). Finally, RNAMake designed asymmetric segments to tether the 16S and 23S rRNAs together into a synthetic singlestranded ribosome that remains uncleaved by ribonucleases and supports life in Escherichia coli, a challenge previously requiring several rounds of trial-and-error.

bioengineering↗