Xrn1-resistant RNA motifs are disseminated throughout the RNA virome and are able to block scanning ribosomes.

RNAs that are able to prevent degradation by the 5'-3' exoribonuclease Xrn1 have emerged as crucial structures during infection by an increasing number of RNA viruses. Several plant viruses employ the so-called coremin motif, an Xrn1-resistant RNA that is usually located in 3' untranslated regions. Investigation of its structural and sequence requirements has led to its identification in plant virus families beyond those in which the coremin motif was initially discovered. In this study, we identified coremin-like motifs that deviate from the original in the number of nucleotides present in the loop region of the 5' proximal hairpin. They are present in a number of viral families that previously did not have an Xrn1-resistant RNA identified yet, including the double-stranded RNA virus families Hypoviridae and Chrysoviridae. Through systematic mutational analysis, we demonstrated that a coremin motif carrying a 6-nucleotide loop in the 5' proximal hairpin generally requires a YGNNAD consensus for stalling Xrn1, similar to the previously determined YGAD consensus required for Xrn1 resistance of the original coremin motif. Furthermore, we determined the minimal requirements for the 3' proximal hairpin. Since some putative coremin motifs were found in intergenic regions or coding sequences, we demonstrated their capacity for inhibiting translation through an in vitro ribosomal scanning inhibition assay. Consequently, this study provides a further expansion on the number of viral families with known Xrn1-resistant elements, while adding a novel, potentially regulatory function for this structure.

Investigating the correlation between Xrn1-resistant RNAs and frameshifter pseudoknots.

Xrn1-resistant RNA structures are multifunctional elements employed by an increasing number of RNA viruses. One of such elements is the coremin motif, discovered in plant virus RNAs, of which the structure has been hypothesized to form a yet unelucidated pseudoknot. Recently, the coremin motif was shown to be capable of stalling not only Xrn1, but scanning ribosomes as well. Following that observation, in this study we demonstrate that the coremin motif can promote -1 ribosomal frameshifting, similar to better-characterized viral frameshifting pseudoknots. Since this function was lost in concert with substitutions that were known to disturb Xrn1-resistance, we developed a frameshifting screen for finding novel Xrn1-resistant RNAs by randomizing parts of the coremin motif. This yielded new insights into the coremin motif structure, as Xrn1-resistant variations were identified that more clearly indicate a pseudoknot interaction. In addition, we show that the Xrn1-resistant RNA of Zika virus promotes frameshifting as well, while known -1 programmed ribosomal frameshifting pseudoknots do not stall Xrn1, suggesting that promoting frameshifting is a universal characteristic of Xrn1-resistant RNAs, but that Xrn1-resistance requires more than just a frameshifting pseudoknot.

All genera of Flaviviridae host a conserved Xrn1-resistant RNA motif.

After infection by flaviviruses like Zika and West Nile virus, eukaryotic hosts employ the well-conserved endoribonuclease Xrn1 to degrade the viral genomic RNA. Within the 3' untranslated regions, this enzyme encounters intricate Xrn1-resistant structures. This results in the accumulation of subgenomic flaviviral RNAs, an event that improves viral growth and aggravates viral pathogenicity. Xrn1-resistant RNAs have been established throughout the flaviviral genus, but not yet throughout the entire Flaviviridae family. In this work, we use previously determined characteristics of these structures to identify homologous sequences in many members of the genera pegivirus, hepacivirus and pestivirus. We used structural alignment and mutational analyses to establish that these sequences indeed represent Xrn1-resistant RNA and that they employ the general features of the flaviviral xrRNAs, consisting of a double pseudoknot formed by five base-paired regions stitched together by a crucial triple base interaction. Furthermore, we demonstrate that the pestivirus Bungowannah virus produces subgenomic RNA in vivo. Altogether, these results indicate that viruses make use of a universal Xrn1-resistant RNA throughout the Flaviviridae family.

Xrn1-resistant RNA structures are well-conserved within the genus flavivirus.

Subgenomic RNAs are produced by several RNA viruses through incomplete degradation of their genomic RNA by the exoribonuclease Xrn1, and have been shown to be essential for viral growth and pathogenicity. Within the flavivirus genus of the Flaviviridae family, two distinct classes of Xrn1-resistant RNA motifs have been proposed; one for mosquito-borne and insect-specific flaviviruses, and one for tick-borne flaviviruses and no-known-vector flaviviruses. We investigated tick-borne and no-known-vector flavivirus Xrn1-resistant RNA motifs through systematic in vitro mutational analysis and showed that both classes actually possess very similar structural configurations, including a double pseudoknot and a base-triple at identical, conserved locations. For the no-known-vector flavivirus Modoc virus, we show that in vivo generation of subgenomic flaviviral RNA was affected by mutations targeted at nucleotides involved in the structural features of flaviviral Xrn1-resistant RNA motifs that were defined in this work. Our results suggest that throughout the genus flavivirus Xrn1-resistant RNA motifs adopt the same topologically conserved structure.

Structural features of an Xrn1-resistant plant virus RNA.

Xrn1 is a major 5'-3' exoribonuclease involved in the RNA metabolism of many eukaryotic species. RNA viruses have evolved ways to thwart Xrn1 in order to produce subgenomic non-coding RNA that affects the hosts RNA metabolism. The 3' untranslated region of several beny- and cucumovirus RNAs harbors a so-called 'coremin' motif that is required for Xrn1 stalling. The structural features of this motif have not been studied in detail yet. Here, by using in vitro Xrn1 degradation assays, we tested over 50 different RNA constructs based on the Beet necrotic yellow vein virus sequence to deduce putative structural features responsible for Xrn1 stalling. We demonstrated that the minimal benyvirus stalling site consists of two hairpins of 3 and 4 base pairs respectively. The 5' proximal hairpin requires a YGAD (Y = U/C, D = G/A/U) consensus loop sequence, whereas the 3' proximal hairpin loop sequence is variable. The sequence of the 10-nucleotide spacer that separates the hairpins is highly conserved and potentially involved in tertiary interactions. Similar coremin motifs were identified in plant virus isolates from other families including Betaflexiviridae, Virgaviridae, Potyviridae and Secoviridae (order of the Picornavirales). We conclude that Xrn1-stalling motifs are more widespread among RNA viruses than previously realized.

Post-translational modification of nucleoid-associated proteins: an extra layer of functional modulation in bacteria?

Post-translational modification (PTM) of histones has been investigated in eukaryotes for years, revealing its widespread occurrence and functional importance. Many PTMs affect chromatin folding and gene activity. Only recently the occurrence of such modifications has been recognized in bacteria. However, it is unclear whether PTM of the bacterial counterparts of eukaryotic histones, nucleoid-associated proteins (NAPs), bears a comparable significance. Here, we scrutinize proteome mass spectrometry data for PTMs of the four most abundantly present NAPs in Escherichia coli (H-NS, HU, IHF and FIS). This approach allowed us to identify a total of 101 unique PTMs in the 11 independent proteomic studies covered in this review. Combined with structural and genetic information on these proteins, we describe potential effects of these modifications (perturbed DNA-binding, structural integrity or interaction with other proteins) on their function.