Transfer RNA post-transcriptional processing, turnover, and subcellular dynamics in the yeast Saccharomyces cerevisiae

Abstract

Transfer RNAs (tRNAs) are essential for protein synthesis. In eukaryotes, tRNA biosynthesis employs a specialized RNA polymerase that generates initial transcripts that must be subsequently altered via a multitude of post-transcriptional steps before the tRNAs beome mature molecules that function in protein synthesis. Genetic, genomic, biochemical, and cell biological approaches possible in the powerful Saccharomyces cerevisiae system have led to exciting advances in our understandings of tRNA post-transcriptional processing as well as to novel insights into tRNA turnover and tRNA subcellular dynamics. tRNA processing steps include removal of transcribed leader and trailer sequences, addition of CCA to the 3' mature sequence and, for tRNA(His), addition of a 5' G. About 20% of yeast tRNAs are encoded by intron-containing genes. The three-step splicing process to remove the introns surprisingly occurs in the cytoplasm in yeast and each of the splicing enzymes appears to moonlight in functions in addition to tRNA splicing. There are 25 different nucleoside modifications that are added post-transcriptionally, creating tRNAs in which ∼15% of the residues are nucleosides other than A, G, U, or C. These modified nucleosides serve numerous important functions including tRNA discrimination, translation fidelity, and tRNA quality control. Mature tRNAs are very stable, but nevertheless yeast cells possess multiple pathways to degrade inappropriately processed or folded tRNAs. Mature tRNAs are also dynamic in cells, moving from the cytoplasm to the nucleus and back again to the cytoplasm; the mechanism and function of this retrograde process is poorly understood. Here, the state of knowledge for tRNA post-transcriptional processing, turnover, and subcellular dynamics is addressed, highlighting the questions that remain.

Figure 1

Steps in tRNA processing involving nucleotide deletion or addition for tRNAs encoded by intron-containing and intron-lacking genes. tRNAs are depicted as linear series of circles that are color coded. Purple circles depict transcribed leader and trailer sequences at the 5′ and 3′ ends, respectively; generally, pre-tRNA leader and trailers contain ∼12 nucleotides. Yellow circles depict intron sequences that vary, depending upon the tRNA species, from 14 to 60 nucleotides. Blue and red-colored nucleosides depict the mature exons, where red indicates the anticodon located at nucleotides 34–36. Green circles depict the post-transcriptionally added CCA nucleotides that are required for aminoacylation. G₋₁ added to the 5′ end of tRNA^His is not shown.

Figure 2

Pre-tRNA splicing in budding yeast. The same color codes are used as for Figure 1. Introns (yellow circles) are located after nucleotide 37 and they are removed in a three-step process—endonucleolytic removal of the intron, ligation, and removal of the residual 2′ phosphate at the splice junction—as detailed in the text.

Figure 3

tRNA subcellular dynamics. tRNAs are drawn in their second cloverleaf structure. The color coding of nucleotides is the same as for Figure 1 and Figure 2 except some nucleosides that occur in the nucleus are orange, whereas representative nucleoside additions that occur in the cytoplasm are brown. Pre-tRNAs are transcribed in the nucleus where leader and trailer sequences (purple) are removed prior to CCA (green) addition. End matured, partially modified intron-containing pre-tRNAs are exported to the cytoplasm by Los1 and at least one unknown exporter. Those pre-tRNAs encoded by genes lacking introns are likely exported by both Los1 and Msn5. Splicing and additional modifications occur after export to the cytoplasm. Cytoplasmic tRNAs constitutively return to the nucleus via retrograde transport. Mtr10 functions in tRNA retrograde import but it is unknown whether its role is direct or indirect. Imported tRNAs accumulate in the nucleus if cells are deprived of nutrients; otherwise they are reexported to the cytoplasm by Los1, Msn5, and at least one unidentified exporter. See text for details.

Figure 4

Cell biology of tRNA modifications. Solid black circle indicates a modification known to occur on initial pre-tRNAs. Several modifications occur in the nucleus; magenta circles indicate those modifications that require the substrate to contain an intron, whereas orange circles indicate modifications that do not appear to require intron-containing tRNAs as substrate. Numerous other modifications occur in the cytoplasm; those that require that the intron first be spliced are brown, whereas those with no known substrate specificity or are restricted to tRNAs encoded by intronless genes are colored khaki. Open circles are catalyzed by enzymes whose subcellular locations are unknown. Different tRNA species possess different subsets of modifications; particular nucleosides that can possess numerous different modifications are indicated; half-colored circles indicate that the modifying enzymes have varying substrate specificity and/or subcellular location. Note that modification of G₃₇ by Trm5 that requires tRNAs to be spliced occurs in the nucleoplasm after retrograde nuclear import.

Figure 5

tRNA turnover pathways in S. cerevisiae. Initial tRNA transcripts with 5′ and 3′ extensions (purple circles) are substrates for 3′ to 5′ exonucleolytic degradation by the nuclear exosome if the transcripts are missing a particular modification, m¹A₅₈ (open circle on initial tRNA transcript) or if 3′ processing is aberrant (not shown). The unmodified/aberrant tRNAs first receive A residues at the 3′ end (yellow circles) by the activity of the TRAMP complex and then the tRNAs are degraded by the nuclear exosome (exosome pac-man). Aberrant tRNAs can also be degraded by the rapid tRNA turnover pathway (RTD) in either the nucleus or the cytoplasm. The RTD pathway acts upon tRNAs missing particular multiple modifications or tRNAs that are otherwise unstructured (see text). As an example, tRNAs missing multiple modifications (open circles) due to trm4Δ trm8Δ mutations are subject to 5′ to 3′ degradation by the exonucleases in either the nucleus (RAT1 pac-man) or in the cytoplasm (Xrn1 pac-man). Solid circles are those modifications affected by mutations of TRM6 or TRM4 and TRM8. Orange circles indicate modifications acquired in the nucleus, whereas brown circles indicate modifications acquired in the cytoplasm. CCA nucleotides are indicated by green circles and the anticodon by red circles. Also indicated is pAp, an intermediate of methionine biosynthesis that inhibits Xrn1 and Rat1, thereby connecting tRNA turnover to amino acid biosynthesis.