Key words: rapid tRNA decay/nuclear surveillance/S. cerevisiae/SUP4oc/tS(CGA)/intron/splicing 2 ABSTRACT During tRNA maturation in yeast, aberrant pre-tRNAs are targeted for 3'-5' degradation by the nuclear surveillance pathway, and mature tRNAs are targeted for 5'-3' degradation by the rapid tRNA decay (RTD) pathway, due to lack of certain body modifications or to destabilizing mutations. Here we show that the RTD pathway also targets pre-tRNAs through an unknown, but distinct, mechanism that occurs after nuclear export. Anticodon stem RTD variants of both tRNA Tyr and tRNA Ser(CGA) are substrates for pre-tRNA RTD, triggered by the accumulation of end-matured unspliced pre-tRNA due to altered secondary structure of the region comprising the anticodon stem-loop and the intron.Furthermore, increased nuclear availability of a pre-tRNA RTD substrate can provoke decay by nuclear surveillance. We interpret these results in terms of a model of opportunistic tRNA decay, wherein tRNAs are degraded due to a combination of structural instability and increased availability to decay pathways.

Posttranscriptional tRNA modifications are essential for proper gene expression, and defects in the enzymes that perform tRNA modifications are associated with numerous human disorders. Throughout eukaryotes, 2′-O-methylation of residues 32 and 34 of the anticodon loop of tRNA is important for proper translation, and in humans, lack of these modifications results in non-syndromic X-linked intellectual disability. In yeast, the methyltransferase Trm7 forms a complex with Trm732 to 2′-O-methylate tRNA residue 32 and with Trm734 to 2′-O-methylate tRNA residue 34. Trm732 and Trm734 are required for the methylation activity of Trm7, but the role of these auxiliary proteins is not clear. Additionally, Trm732 and Trm734 homologs are implicated in biological processes not directly related to translation, suggesting that these proteins may have additional cellular functions. To identify critical amino acids in Trm732, we generated variants and tested their ability to function in yeast cells. We identified a conserved RRSAGLP motif in the conserved DUF2428 domain of Trm732 that is required for tRNA modification activity by both yeast Trm732 and its human homolog THADA. The identification of Trm732 variants that lack tRNA modification activity will help to determine if other biological functions ascribed to Trm732 and THADA are directly due to tRNA modification, or to secondary effects due to other functions of these proteins.