The nuclear and cytoplasmic activities of RNA polymerase III, and an evolving transcriptome for surveillance.

A 1969 report that described biochemical and activity properties of the three eukaryotic RNA polymerases revealed Pol III as highly distinguishable, even before its transcripts were identified. Now known to be the most complex, Pol III contains several stably-associated subunits referred to as built-in transcription factors (BITFs) that enable highly efficient RNA synthesis by a unique termination-associated recycling process. In vertebrates, subunit RPC7(α/ß) can be of two forms, encoded by POLR3G or POLR3GL, with differential activity. Here we review promoter-dependent transcription by Pol III as an evolutionary perspective of eukaryotic tRNA expression. Pol III also provides nonconventional functions reportedly by promoter-independent transcription, one of which is RNA synthesis from DNA 3'-ends during repair. Another is synthesis of 5'ppp-RNA signaling molecules from cytoplasmic viral DNA in a pathway of interferon activation that is dysfunctional in immunocompromised patients with mutations in Pol III subunits. These unconventional functions are also reviewed, including evidence that link them to the BITF subunits. We also review data on a fraction of the human Pol III transcriptome that evolved to include vault RNAs and snaRs with activities related to differentiation, and in innate immune and tumor surveillance. The Pol III of higher eukaryotes does considerably more than housekeeping.

Dynamic queuosine changes in tRNA couple nutrient levels to codon choice in Trypanosoma brucei.

Every type of nucleic acid in cells undergoes programmed chemical post-transcriptional modification. Generally, modification enzymes use substrates derived from intracellular metabolism, one exception is queuine (q)/queuosine (Q), which eukaryotes obtain from their environment; made by bacteria and ultimately taken into eukaryotic cells via currently unknown transport systems. Here, we use a combination of molecular, cell biology and biophysical approaches to show that in Trypanosoma brucei tRNA Q levels change dynamically in response to concentration variations of a sub-set of amino acids in the growth media. Most significant were variations in tyrosine, which at low levels lead to increased Q content for all the natural tRNAs substrates of tRNA-guanine transglycosylase (TGT). Such increase results from longer nuclear dwell time aided by retrograde transport following cytoplasmic splicing. In turn high tyrosine levels lead to rapid decrease in Q content. Importantly, the dynamic changes in Q content of tRNAs have negligible effects on global translation or growth rate but, at least, in the case of tRNATyr it affected codon choice. These observations have implications for the occurrence of other tunable modifications important for 'normal' growth, while connecting the intracellular localization of modification enzymes, metabolites and tRNAs to codon selection and implicitly translational output.

The role of intracellular compartmentalization on tRNA processing and modification.

A signature of most eukaryotic cells is the presence of intricate membrane systems. Intracellular organization presumably evolved to provide order, and add layers for regulation of intracellular processes; compartmentalization also forcibly led to the appearance of sophisticated transport systems. With nucleus-encoded tRNAs, it led to the uncoupling of tRNA synthesis from many of the maturation steps it undergoes. It is now clear that tRNAs are actively transported across intracellular membranes and at any point, in any compartment, they can be post-transcriptionally modified; modification enzymes themselves may localize to any of the genome-containing compartments. In the following pages, we describe a number of well-known examples of how intracellular compartmentalization of tRNA processing and modification activities impact the function and fate of tRNAs. We raise the possibility that rates of intracellular transport may influence the level of modification and as such increase the diversity of differentially modified tRNAs in cells.

Retrograde nuclear transport from the cytoplasm is required for tRNATyr maturation in T. brucei.

Retrograde transport of tRNAs from the cytoplasm to the nucleus was first described in Saccharomyces cerevisiae and most recently in mammalian systems. Although the function of retrograde transport is not completely clear, it plays a role in the cellular response to changes in nutrient availability. Under low nutrient conditions tRNAs are sent from the cytoplasm to nucleus and presumably remain in storage there until nutrient levels improve. However, in S. cerevisiae tRNA retrograde transport is constitutive and occurs even when nutrient levels are adequate. Constitutive transport is important, at least, for the proper maturation of tRNAPhe, which undergoes cytoplasmic splicing, but requires the action of a nuclear modification enzyme that only acts on a spliced tRNA. A lingering question in retrograde tRNA transport is whether it is relegated to S. cerevisiae and multicellular eukaryotes or alternatively, is a pathway with deeper evolutionary roots. In the early branching eukaryote Trypanosoma brucei, tRNA splicing, like in yeast, occurs in the cytoplasm. In the present report, we have used a combination of cell fractionation and molecular approaches that show the presence of significant amounts of spliced tRNATyr in the nucleus of T. brucei. Notably, the modification enzyme tRNA-guanine transglycosylase (TGT) localizes to the nucleus and, as shown here, is not able to add queuosine (Q) to an intron-containing tRNA. We suggest that retrograde transport is partly the result of the differential intracellular localization of the splicing machinery (cytoplasmic) and a modification enzyme, TGT (nuclear). These findings expand the evolutionary distribution of retrograde transport mechanisms to include early diverging eukaryotes, while highlighting its importance for queuosine biosynthesis.

The leucine-NH4+ uptake regulator Any1 limits growth as part of a general amino acid control response to loss of La protein by fission yeast.

The sla1+ gene of Schizosachharoymces pombe encodes La protein which promotes proper processing of precursor-tRNAs. Deletion of sla1 (sla1Δ) leads to disrupted tRNA processing and sensitivity to target of rapamycin (TOR) inhibition. Consistent with this, media containing NH4+ inhibits leucine uptake and growth of sla1Δ cells. Here, transcriptome analysis reveals that genes upregulated in sla1Δ cells exhibit highly significant overalp with general amino acid control (GAAC) genes in relevant transcriptomes from other studies. Growth in NH4+ media leads to additional induced genes that are part of a core environmental stress response (CESR). The sla1Δ GAAC response adds to evidence linking tRNA homeostasis and broad signaling in S. pombe. We provide evidence that deletion of the Rrp6 subunit of the nuclear exosome selectively dampens a subset of GAAC genes in sla1Δ cells suggesting that nuclear surveillance-mediated signaling occurs in S. pombe. To study the NH4+-effects, we isolated sla1Δ spontaneous revertants (SSR) of the slow growth phenotype and found that GAAC gene expression and rapamycin hypersensitivity were also reversed. Genome sequencing identified a F32V substitution in Any1, a known negative regulator of NH4+-sensitive leucine uptake linked to TOR. We show that 3H-leucine uptake by SSR-any1-F32V cells in NH4+-media is more robust than by sla1Δ cells. Moreover, F32V may alter any1+ function in sla1Δ vs. sla1+ cells in a distinctive way. Thus deletion of La, a tRNA processing factor leads to a GAAC response involving reprogramming of amino acid metabolism, and isolation of the any1-F32V rescuing mutant provides an additional specific link.

Cutting, dicing, healing and sealing: the molecular surgery of tRNA.

All organisms encode transfer RNAs (tRNAs) that are synthesized as precursor molecules bearing extra sequences at their 5' and 3' ends; some tRNAs also contain introns, which are removed by splicing. Despite commonality in what the ultimate goal is (i.e., producing a mature tRNA), mechanistically, tRNA splicing differs between Bacteria and Archaea or Eukarya. The number and position of tRNA introns varies between organisms and even between different tRNAs within the same organism, suggesting a degree of plasticity in both the evolution and persistence of modern tRNA splicing systems. Here we will review recent findings that not only highlight nuances in splicing pathways but also provide potential reasons for the maintenance of introns in tRNA. Recently, connections between defects in the components of the tRNA splicing machinery and medically relevant phenotypes in humans have been reported. These differences will be discussed in terms of the importance of splicing for tRNA function and in a broader context on how tRNA splicing defects can often have unpredictable consequences.