Fig. 3

Deletion of RG4 structures in 5ʹtiRNA^Ala through ionic equilibration abolishes activity. a Model of tetrameric 5ʹtiRNA^Alacoordinated by a central G-quadruplex core containing 5 stacked G-quartets. b The 5ʹTOG motif of 5ʹtiRNA^Ala was mutated to varying extents. Indicated 5ʹtiRNA^Ala mutants were run on a 15% denaturing acrylamide gel and post-stained with SYBR gold or NMM to detect total RNA or RG4, respectively. The propensity to form RG4 structures correlated with the bioactivity of each tiRNA (Supplementary Table 2). c 5ʹtiRNA^Ala was equilibrated overnight in indicated salt or left untreated (NT). In vitro translation assays were carried out in rabbit reticulocyte lysate using polyadenylated NanoLuc RNA as a reporter. To each, 1 μl of 100 pmol salt equilibrated 5ʹtiRNA^Ala or 1 μl of indicated salt was added prior to initiating translation reaction. Depletion of RG4 structure of 5ʹtiRNA^Ala through equilibration in Li⁺ ions abolished the ability of 5ʹtiRNA^Ala to repress translation. d eIF4F was assembled onto m⁷GTP agarose and challenged with indicated RNAs equilibrated in NaCl or LiCl. Depletion of RG4 structures through equilibration in Li⁺ ions abrogated the ability of the tiRNA to displace eIF4G or eIF4E from m⁷GTP cap. e RNPs were purified from U2OS lysates using biotinylated RNAs equilibrated in indicated salt solutions. Depletion of RG4 structures reduced 5ʹtiRNA^Ala’s association with YB-1, a key regulator of 5ʹtiRNA^Ala induced stress granules, to background levels. The interaction of 5ʹtiRNA^Ala with FUS/TLS or TIAR was unaltered through ionic equilibration