Fig. 5.

7-deazaguanine substitutions in 5ʹtiRNA^Ala prevent bioactivity. a In vitro translation assay in rabbit reticulocyte lysate using NanoLuc reporter to monitor translation efficiency. Substitution of two guanines in 5ʹtiRNA^Ala reduces its ability to repress translation. b Indicated RNAs were transfected into U2OS cells and stress granule formation was monitored through immunofluorescence. 5ʹtiRNA^Ala (WT) readily induces the formation of SGs, while 5ʹtiRNA^Ala(7daG) fails to. c RNA affinity purifications using indicated RNAs from U2OS lysates. Loss of RG4 forming ability in 5ʹtiRNA^Ala (7daG) correlates with loss of binding to YB-1, a protein required for tiRNA mediated SG formation. 5ʹtiRNA^Ala(7daG) also loses the ability to bind to Fxr1 and DHX36, previously identified RG4 binding proteins. In contrast, Vigilin, GRSF1, and TDP43 bind 5ʹtiRNA^Ala regardless of its ability to form RG4. d Inhibition of RG4 formation in 5ʹtiRNA^Ala through incorporation of 7-deazaguanine abrogates its ability to displace eIF4F from m⁷GTP agarose