Biology subjects

Pestova, T.

Publications and source records attributed to Pestova, T..

5 recordsLinked to original sources

bioRxiv2026

Mechanism and key RNA determinants of SARS-CoV-2 Nsp1-induced endonucleolytic cleavage of mRNA

SARS-CoV-2 nonstructural protein 1 (Nsp1) binds to 40S ribosomal subunits and induces host protein synthesis shut off by inhibiting translation initiation and triggering endonucleolytic cleavage of cellular mRNAs. Irrespective of the mode of initiation, Nsp1-mediated cleavage is induced by the cooperative action of the N-terminal domain of Nsp1, the RRM domain of eIF3g and 40S subunits. Using in vitro reconstitution, we determined that cleavage occurs by transesterification following intramolecular nucleophilic attack of the 2OH of the ribose on the adjacent phosphodiester bond yielding 5OH and 2,3 -cyclic phosphate termini. Cleavage requires a guanosine [~]10-22 nucleotides from the 5 end of mRNA, occurs within a narrow window upstream of this G, is most efficient between nucleotides at positions -6/-7 and -7/-8 relative to G, and shows a preference for Pu at positions -7 or -8 which provides the 2OH for the nucleophilic attack. Zero-length UV cross-linking of Nsp1 to nucleotides at positions -1 and -2 suggests that the critical guanosine may be recognized by Nsp1. Resistance to Nsp1-mediated cleavage of SARS-CoV-2 mRNA was ensured both by the relatively long distance between its G23G24 and the 5end and by the preceding oligoPy stretch lacking purines at positions -7 or -8 upstream of G23G24.

Preprint↗

bioRxiv2025

The translation initiation factor DHX29 appears to pull on mRNA in a direction opposite to scanning.

The DExH-box helicase DHX29 plays a critical role in mammalian translation initiation. It is required for the scanning of mRNAs with complex 5UTRs. Despite its importance, the detailed mechanism of DHX29s action has remained debated. Using structural models derived from AlphaFold and cryo-EM structure of DHX29 bound to the ribosomal 43S pre-initiation complex, we provide a revised structural framework that clarifies the interplay between DHX29, the 40S ribosomal subunit, and eIF3. Our findings suggest that the 40S subunit regulates DHX29s NTPase activity through an activation mechanism resembling the G-patch protein regulation of DEAH helicases. Moreover, our model supports a 3 to 5 translocase mechanism, in which DHX29 transiently pulls the mRNA opposite to the scanning direction, destabilizing stable stem-loops trapped in the mRNA channel and allowing scanning to resume. This structural analysis refines our understanding of DHX29s function and provides new hypotheses regarding its role in mRNA unwinding during scanning and start codon selection.

biophysics↗

bioRxiv2025

The mechanism of ribosomal recruitment during translation initiation on Type 2 IRESs

The encephalomyocarditis virus (EMCV) IRES and other Type 2 IRESs comprise domains H-L and specifically interact with eIF4G/eIF4A through their essential JK domain. However, the JK domain is not sufficient for IRES function, which also requires the preceding domain I of unknown function. To identify interactions that drive ribosomal recruitment of eIF4G/eIF4A-bound Type 2 IRESs, we determined the cryo-EM structure of 48S initiation complexes formed on the EMCV IRES. It revealed that the apical domain I cloverleaf contacts ribosomal proteins uS13 and uS19 via its Id subdomain and that the essential GNRA tetraloop in subdomain Ic interacts directly with the T{psi}C domain of initiator tRNA. Functional assays supported the exceptional role of these interactions for initiation on this IRES. The strong conservation of primary and secondary structures of the apex of domain I among Type 2 IRESs suggests that the reported interactions are a common essential feature of them all.

molecular biology↗

bioRxiv2024

Inconsistencies in the published rabbit ribosomal rRNAs: a proposal for uniformity in sequence and site numbering

Examination of all publicly available Oryctolagus cuniculus (rabbit) ribosome cryo-EM structures reveals numerous confusing inconsistencies. First, there are a plethora of single nucleotide differences among the various rabbit 28S and 18S rRNA structures. Second, two nucleotides are absent from the NCBI Reference Sequence for the 18S rRNA gene. Moving forward, we propose using the Broad Institutes rabbit whole genome shotgun sequence and numbering to reduce modeling ambiguity and improve consistency between ribosome models.

biophysics↗

bioRxiv2024

Genetic mechanisms underlying the structural elaboration and dissemination of viral internal ribosomal entry sites

Viral internal ribosomal entry sites (IRESs) form several classes that use distinct mechanisms to mediate end-independent initiation of translation. The origin of viral IRESs is a longstanding question. The simplest IRESs comprise tandem pseudoknots and occur in the intergenic region (IGR) of Dicistroviridae genomes (order Picornavirales). Larger IGR IRESs contain additional elements that determine specific properties such as binding to the head of the ribosoma l 40S subunit. Metagenomic analyses reported here identified novel groups of structurally distinct IGR-like IRESs. The smallest of these ([~]120nt long) comprise three pseudoknots and bind directly to the ribosomal P site. Others are up to 260nt long: insertions occurred at specific loci, possibly reflecting non-templated nucleotide insertion during replication. Various groups can be arranged in order, differing by the cumulative addition of single structural elements, suggesting an accretion mechanism for the structural elaboration of IRESs. Identification of chimeric IRESs implicates recombinational exchange of domains as a second mechanism for the diversification of IRES structure. Recombination likely also accounts for the presence of IGR-like IRESs at the 5-end of some dicistrovirus-like genomes (e.g. Hangzhou dicistrovirus 3) and in the RNA genomes of Tombusviridae (order Tolivirales), Marnaviridae (order Picornavirales), and the Ripiresk picorna-like clade (order Picornavirales).

molecular biology↗