Figure 1. Mechanism of Nsp1-induced RNA cleavage.

(A) A critical requirement for G at a specific distance from the 5’ end of mRNA for Nsp1-mediated cleavage. Upper panel - schematic representation of mRNAs containing unstructured 34nt.-long 5’UTRs comprising CAA repeats interrupted by a single G (red) at different positions from the 5’ end. Lower panel – Nsp1-mediated cleavage of mRNAs shown in the upper panel in the presence of the indicated translation components, assayed by primer extension. Cleavage sites are shown on the right and marked on 5’ UTR sequences in the upper panel (red arrows). (B) Factor requirements of Nsp1-mediated cleavage of 30nt.-long unstructured RNAs corresponding to the 5’-terminal regions of mRNAs shown in A (upper panel). After incubation of 5'[³²P]-labeled RNAs with Nsp1 and the indicated translation components, cleavage products were visualized directly by electrophoresis (red arrows). (C) The length of RNA downstream of G required for Nsp1-induced cleavage. 14–30nt.-long 5'[³²P]-labeled (CAA)n RNAs containing G14 were incubated with Nsp1, eIF3g and 40S subunits after which cleavage products were visualized by electrophoresis (red arrow). (D) Determination of the cleavage site and the location of the phosphate group after cleavage. 5'[³²P]-labeled 30nt.-long (CAA)n RNAs containing G12 or G14 were cleaved by incubation with Nsp1, eIF3g and 40S subunits. Cleavage products were either treated with T4 PNK or left untreated and visualized by electrophoresis (green and red arrows, respectively). To estimate the size of the cleavage products, 4–8nt.-long 5'[³²P]-labeled oligo RNA markers containing 3’OH were employed. (E) Nsp1-induced cleavage of 5'[³²P]-labeled 30nt.-long (CAA)n RNAs with G14 containing the indicated 2’-derivatized nucleotides at positions −7 or −8 relative to G14. Cleavage products were visualized by electrophoresis (red arrows). (F) Comparison of Nsp1-induced cleavage products of 30nt.-long (CAA)n RNAs with G12 or G14 and of RNAs containing the indicated 2’-derivatized nucleotides at position −7 relative to G14. Cleavage products of the corresponding 5'[³²P]-labeled RNAs were either treated with T4 PNK or left untreated and visualized by electrophoresis (green and red arrows, respectively).