tRNA biology charges to the front

Abstract

tRNA biology has come of age, revealing an unprecedented level of understanding and many unexpected discoveries along the way. This review highlights new findings on the diverse pathways of tRNA maturation, and on the formation and function of a number of modifications. Topics of special focus include the regulation of tRNA biosynthesis, quality control tRNA turnover mechanisms, widespread tRNA cleavage pathways activated in response to stress and other growth conditions, emerging evidence of signaling pathways involving tRNA and cleavage fragments, and the sophisticated intracellular tRNA trafficking that occurs during and after biosynthesis.

Figure 1.

A schematic of modifications found in cytoplasmic tRNA in S. cerevisiae. tRNA is shown in its usual secondary structure form, with circles representing nucleotides and lines representing base pairs. (Green circles) Residues that are unmodified in all yeast tRNA species; (pink circles) residues that are modified in some or all tRNA species; (white circles) additional residues (20a and 20b) that are present in some, but not all, tRNAs and are sometimes modified; (red circles) anticodon residues, which are modified in some tRNAs; (light-blue circles) the CCA end. Conventional abbreviations are used; see the Modomics database (http://modomics.genesilico.pl). (Ψ) Pseudouridine; (Am) 2′-O-methyladenosine; (Cm) 2′-O-methylcytidine; (m¹G) 1-methylguanosine; (m²G) 2-methylguanosine; (ac⁴C) 4-acetylcytidine; (D) dihydrouridine; (Gm) 2′-O-methylguanosine; (m^2,2G) N2,N2-dimethylguanosine; (m³C) 3-methylcytidine; (I) inosine; (m⁵C) 5-methylcytidine; (mcm⁵U) 5-methoxycarbonylmethyluridine; (mcm⁵s²U) 5-methoxycarbonylmethyl-2-thiouridine; (ncm⁵U) 5-carbamoylmethyluridine; (ncm⁵Um) 5-carbamoylmethyl-2′-O-methyluridine; (m¹I) 1-methylinosine; (i⁶A) N6-isopentenyl adenosine; (yW) wybutosine; (t⁶A) N6-threonylcarbamoyladenosine; (Um) 2′-O-methyluridine; (m⁷G) 7-methylguanosine; (rT) ribothymidine; [Ar(p)] 2′-O-ribosyladenosine (phosphate). The pictured molecule starts at position −1 and is numbered consecutively from the next base (+1) to 76 (with the insertion of two residues [20a and 20b]). Several tRNA species have a longer variable arm starting after residue 44, and some tRNAs have different numbers of residues in the D-loop and the variable arm, but the anticodon is always numbered residues 34, 35, and 36, and the CCA end is always numbered residues 74, 75, and 76.

Figure 2.

tRNA splicing and ligation pathways. tRNA is shown in its usual secondary structure, with the antidocodon indicated by red circles, and the intron after residue 37 indicated by blue circles. The endonuclease (comprised of Sen2, Sen15, Sen34, and Sen54 in yeast) excises the intron by cleaving the pre-tRNA at each exon/intron border, leaving tRNA half-molecules with a 2′–3′ cyclic phosphate (indicated by a triangle with a white circle containing the phosphate) and a 5′-OH group at their ends. In yeast and plants, the ligase (Trl1 in yeast) RNA 5′ kinase activity phosphorylates the 5′-OH end of the 3′ half-molecule (black circle), and the ligase cyclic phosphodiesterase activity opens the 2′–3′ cyclic phosphate to a 2′ phosphate. Then ligase joins the half-molecules (after activation of the 5′ phosphate, which is not shown), using the 5′ phosphate (black circle) as the junction phosphate, and leaving the 2′ phosphate at the splice junction (white circle). This 2′ phosphate is subsequently transferred to NAD by the 2′ phosphotransferase (Tpt1 in yeast). The yeast-like ligation pathway is also found in vertebrates, but, in vertebrates and some archaea, the predominant vertebrate ligase directly joins the phosphate of the 2′–3′ cyclic phosphate (white circle) to the 3′ half-molecule.

Figure 3.

Formation of mature tRNA from split tRNA genes. In N. equitans, split tRNA genes are transcribed from distant chromsomal loci (indicated by filled and open circles) as half-molecules, each containing additional nucleotides at their 5′ or 3′ end (blue circles). The additional sequences at the 3′ end of the 5′ half-molecule occur after nucleotide 37, the usual position of an intron, and pair with the additional 5′ sequences of the 3′ half-molecule, forming a hybrid intron. Splicing by the endonuclease, followed by ligation and CCA addition, results in formation of the mature tRNA.

Figure 4.

Two different tRNA degradation pathways in yeast. pre-tRNA transcribed in the nucleus is processed (black arrows) in the nucleus and the cytoplasm (steps 1) to remove the 5′ leader and 3′ trailer (purple circles), to add CCA to the 3′ end (blue circles), to remove the intron if present (not shown), and to add modifications [pink circles, as for tRNA^Val(AAC)], ultimately emerging in the cytoplasm for translation (step 2). If m¹A₅₈ is not added to pre-tRNA_i^Met (absence of m¹A indicated by black circle), this pre-tRNA is degraded by the nucelar surveillance pathway (step 3, red arrow) in which the pre-tRNA is first polyadenylated by the TRAMP complex, and then degraded from the 3′ end by the nuclear exosome. If m⁷G₄₆ and m⁵C₄₉ are not added to tRNA^Val(AAC) (black circles), the hypomodified mature tRNA is at least partially functional, but is degraded by the RTD pathway (red arrows), by Xrn1 in the cytoplasm (step 4), or by Rat1 in the nucleus (step 6), possibly after nuclear import (step 5). (Step 7) The elevated presence of pAp in met22 mutants inhibits the RTD pathway by inhibiting both Xrn1 and Rat1.

Figure 5.

The cell biology of tRNA biosynthesis and nuclear–cytoplasmic trafficking for intron-containing tRNAs in the yeast S. cerevisiae. tRNA transcription and 5′ end-processing occur in the nucleolus. Following 3′ end-processing, CCA addition, and various modification steps in the nucleoplasm and at the INM, intron-containing pre-tRNAs are exported to the cytoplasm via the Los1 exportin and at least one unknown pathway. After pre-tRNA splicing on the cytoplasmic surface of mitochondria, additional modifications in the cytoplasm, and aminoacylation, mature charged tRNAs can participate in protein synthesis. Cytoplasmic tRNAs are constitutively imported into nuclei, directly or indirectly, via Mtr10. Re-export of nuclear tRNAs to the cytoplasm is mediated by Los1 and Msn5 and is regulated by nutrient status; likely, Msn5-dependent re-export requires that the tRNA be appropriately structured and aminoacylated in the nucleus. (Green and red circles) Parts of the tRNA that are maintained in the mature structure; (red circles) anticodon; (purple circles) transcribed 5′ leader and 3′ trailer sequences; (dark-blue circles) intron sequence; (light-blue circles) CCA end; (yellow, orange, and pink circles) various modifications made in the nucleoplasm, at the INM, and in the cytoplasm, respectively; (aa) amino acid. Processing steps are labeled, as are the β-importin members that function in the nucleus–cytoplasm import and export steps.