Ribosomal frameshifting in response to hypomodified tRNAs in Xenopus oocytes

Abstract

The question of whether hypomodified tRNAs enhanced mammalian retroviral ribosomal frameshifting (RF) was resolved several years ago when Phe tRNA lacking the highly modified wyebutoxine (Y) base at position 37 enhanced RF in retroviruses using UUU at the frameshift site (Carlson et al. Virology 255: 2, 1999). However, the level of RF in this earlier study and subsequent studies (Carlson et al. Virology 279: 130, 2001; Waas et al. JBC 282: 26026, 2007) far exceeded that naturally found with mammalian retroviruses which raised concerns whether Phe tRNA ‐Y only enhanced RF or was actually required for the event to occur. In the present study, we examined Xenopus oocytes as an "in vivo" protein synthesis system to assess whether the important issue of only an enhancement vs. an essential requirement could be resolved. Microinjection of oocytes with a construct encoding UUU at the frameshift site demonstrated that the level of RF was lower than that found naturally in retroviral RF, while co‐injection with Phe tRNA ‐Y enhanced RF and co‐injection with Phe tRNA +Y inhibited RF. Thus, Phe tRNA ‐Y does not only enhance RF, but is an essential requirement. This finding provides a novel avenue of inhibiting retroviral expression by reducing the level of intracellular hypomodified tRNA. This research was supported by the Intramural Research Program of the NIH, NCI, CCR.