Figure 4. The efficiency of Nsp1-mediated cleavage of RNAs containing oligoPy regions immediately before or after the critical G nucleotide.

(A-B) Upper panels – sequences of 34nt.-long 5’UTRs comprising CAA repeats interrupted by G14 (red) and containing oligoPy regions immediately before (A) or after (B) the G nucleotide. Lower panels - Nsp1-mediated cleavage of mRNAs with 5' UTRs shown in the upper panels in the presence of the indicated translation components, assayed by primer extension. Cleavage sites are shown on the right and marked on the 5’ UTRs in the upper panels (red arrows). (C) Upper panel - Schematic representation of the CrPV IRES with the proximal cognate coding region, annotated to show IRES domains 1–3, pseudoknots PKI, PKII and PKIII, the P-site codon, the positions of toeprints of IRES-bound 40S subunits (black arrows), the critical G nucleotide (red), and the site of Nsp1-mediated cleavage (red arrow). Coding regions of mutants with introduced substitutions (blue) are shown directly below the proximal cognate coding region. Lower panel – Nsp1-mediated cleavage of wt and mutant CrPV IRES mRNAs shown in the upper panel in the presence of 40S subunits and eIF3g, assayed by primer extension. Toe-prints corresponding to 40S/IRES binary complexes and the cleavage site are indicated on the right. (B-C) Lanes C, T, A, and G show the corresponding sequence derived using the same primer as for primer extension. Separation of lanes by a white line in panel C indicates that these parts were juxtaposed from the same gel after different exposures.