The Rapid tRNA Decay Pathway Monitors the Structural Integrity of Mature tRNAs

Abstract

tRNAs are highly modified and highly stable, and subject to several quality control steps during and after biosynthesis. Specific mature tRNA species lacking certain modifications are degraded in yeast by the Rapid tRNA Decay (RTD) pathway, mediated by the 5′-3′ exonucleases Rat1 and Xrn1. However, it is unclear how substrate tRNAs are recognized, since only a subset of hypomodified species are degraded. We used a genetic system to identify determinants for the RTD pathway in cells lacking ac4C and Um, by examining variants of the substrate tRNASer(CGA), or the non-substrates tRNASer(IGA) and tRNASer(GCU). Nearly all tRNASer(CGA) variants with predicted stabilizing substitutions in the acceptor or T-stem restore mature tRNA levels, while anticodon stem substitutions have no effect. Conversely, non-substrate tRNAs and fully modified tRNAs can be subject to RTD if appropriately destabilized. Analysis shows that RTD substrates have a less rigid structure than non-substrates, have a more exposed 5′ end, and are more sensitive to Xrn1. These findings suggest that the RTD pathway monitors the structural integrity of the acceptor and T-stems of mature tRNAs, imposing a lower threshold for their stability.