The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5′ end, the ribosomal frameshift segment and the 3′-untranslated region (3′-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.

Satellite DNA (satDNA) constitutes an important fraction of repetitive DNA in eukaryotic genomes, but it is barely known in most species. The high-throughput analysis of satDNA in the grasshopper Pyrgomorpha conica revealed 87 satDNA variants grouped into 76 different families, representing 9.4% of the genome. Fluorescent in situ hybridization (FISH) analysis of the 38 most abundant satDNA families revealed four different patterns of chromosome distribution. Homology search between the 76 satDNA families showed the existence of 15 superfamilies, each including two or more families, with the most abundant superfamily representing more than 80% of all satDNA found in this species. This also revealed the presence of two types of higher-order repeats (HORs), one showing internal homologous subrepeats, as conventional HORs, and an additional type showing non-homologous internal subrepeats, the latter arising by the combination of a given satDNA family with a non-annotated sequence, or with telomeric DNA. Interestingly, the heterologous subrepeats included in these HORs showed higher divergence within the HOR than outside it, suggesting that heterologous HORs show poor homogenization, in high contrast with conventional (homologous) HORs. Finally, heterologous HORs can show high differences in divergence between their constituent subrepeats, suggesting the possibility of regional homogenization.

The 3 ′ ′ ′ ′ ′ X domain of hepatitis C virus has been reported to control viral replication and translation by modulating the exposure of a nucleotide segment involved in a distal base-pairing interaction with an upstream 5BSL3.2 domain. To study the mechanism of this molecular switch, we have analyzed the structure of 3 ′ ′ ′ ′ ′ X mutants that favor one of the two previously proposed conformations comprising either two or three stem-loops. Only the two-stem conformation was found to be stable and to allow the establishment of the distal contact with 5BSL3.2, and also the formation of 3 ′ ′ ′ ′ ′ X domain homodimers by means of a universally conserved palindromic sequence. Nucleotide changes disturbing the two-stem conformation resulted in poorer replication and translation levels, explaining the high degree of conservation detected for this sequence. The switch function attributed to the 3 ′ ′ ′ ′ ′ X domain does not occur as a result of a transition between two-and three-stem conformations, but likely through the sequestration of the 5BSL3.2-binding sequence by formation of 3 ′ ′ ′ ′ ′ X homodimers.