The preference signature of the SARS-CoV-2 Nucleocapsid NTD for its 5’-genomic RNA elements

Abstract

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.

A specific phosphorylation-dependent conformational switch of SARS-CoV-2 nucleoprotein inhibits RNA binding

“…We have investigated the RNA binding profile of N234 using a 14 Nt segment (UCUAAACGAACUUU) from the 5' UTR of SARS-CoV-2 and a 30 Nt polyA. 15 N-1 H (CSPs) in N2 measured in N234 (figure 4) resemble those recently published by numerous authors (25,(28)(29)(30)(31)44), involving the RNA binding finger (residues 90-106), and the surface of the base-plate formed by residues 48-66, 148-160 and 166-174. These observations are confirmed by 13 C-1 H methyl chemical shift perturbations (figure 4).…”

“…N is highly disordered as revealed by NMR spectroscopy (17,18), comprising three intrinsically disordered domains (N1, N3 and N5), flanking the RNA-binding domain (N2) and the dimerization domain N4 (figure 1), whose structures have both been determined at atomic resolution (19)(20)(21)(22)(23). RNA binding to N2 has been described (24)(25)(26)(27)(28)(29)(30)(31), including its potential role in liquid-liquid phase separation of N-RNA mixtures (26,27,(32)(33)(34)(35)(36)(37)(38). N4 forms a highly stable dimeric structure involving two domain-swapped b-hairpins.…”

Assembly reactions of SARS-CoV-2 nucleocapsid protein with nucleic acid

“…The assembly pathway of SARS-CoV-2 N-protein to package and condense the large gRNA has remained enigmatic, despite cryo-ET density maps of distinct vRNP particles (1, 2), increasing atomistic insight in the structural and dynamic aspects of NA binding modes of the folded NTD and CTD domains (7,14,20,30,36,37), and a recently described protein-protein interface in the disordered linker (17). In part this is due to the formidable problem of compounded effects of highly multivalent protein-NA and proteinprotein interactions taking place side-by-side, the limited specificity of NA sites, high flexibility of Nprotein imparted by the IDRs (38), and the resulting polymorphic nature of the complexes.…”

“…It consists in two folded domains, the NTD and CTD, which are linked and flanked by large intrinsically disordered regions (IDRs) termed N-arm, linker, and C-arm (6) (Figure 1A). The NTD forms a nucleic acid (NA) binding site that is thought to harbor recognition mechanism of a packaging signal (7). The CTD provides a high-affinity dimerization interface and also binds NA (6) (Figure 1B).…”

Advances, Applications, and Perspectives in Small‐Angle X‐ray Scattering of RNA

“…[117] Some studies of RNPs have integrated SANS with SAXS [118] or NMR data, [119] with a combination of SAXS and NMR, [14] to compare the crystal structures to the solution appearance. [51] Nevertheless, SAXS of RNPs offers a reliable readout of stoichiometries [55] through multiple parameters and can be used to confirm complex formation [51,75] or monitor assembly compaction.…”