Coordination of tRNA transcription with export at nuclear pore complexes in budding yeast

Abstract

tRNAs are encoded by RNA polymerase III-transcribed genes that reside at seemingly random intervals along the chromosomes of budding yeast. Existing evidence suggests that the genes congregate together at the nucleolus and/or centromeres. In this study, we re-examined spatial and temporal aspects of tRNA gene (tDNA) expression. We show that tDNA transcription fluctuates during cell cycle progression. In M phase, when tRNA synthesis peaks, tDNAs localize at nuclear pore complexes (NPCs). Docking of a tDNA requires the DNA sequence of the contacted gene, nucleoporins Nup60 and Nup2, and cohesin. Characterization of mutants that block NPC localization revealed that docking is a consequence of elevated tDNA transcription. NPC-tDNA contact falters in the absence of the principal exportin of nascent tRNA, Los1, and genetic assays indicate that gating of tDNAs at NPCs favors cytoplasmic accumulation of functional tRNA. Collectively, the data suggest that tDNAs associate with NPCs to coordinate RNA polymerase III transcription with the nuclear export of pre-tRNA. The M-phase specificity of NPC contact reflects a regulatory mechanism that may have evolved, in part, to avoid collisions between DNA replication forks and transcribing RNA polymerase III machinery at NPCs.[Keywords: tRNA gene; RNA polymerase III; nuclear pore complex; cohesin; cell cycle; Los1 exportin] Supplemental material is available for this article. Nuclear pore complexes (NPCs) form large aqueous channels through the nuclear envelope that permit transfer of materials between the nucleus and cytoplasm. In 1985, Blobel (1985 proposed the gene-gating hypothesis, which stipulated that transcriptionally poised genes associate with NPCs. In this way, tethered genes would be situated to export transcripts directly to the cytoplasm. The original gene-gating scheme also envisioned that genome packaging would influence the distribution of NPCs on the nuclear envelope. A contemporary spin on this idea posits that anchorage of genes to NPCs and other structures at the edge of the nucleus orchestrates the three-dimensional structure of the genome (Taddei and Gasser 2012).Substantial evidence for gating of RNA polymerase II genes has been obtained primarily from studies of the budding yeast Saccharomyces cerevisiae. Genome-wide approaches revealed that components of the NPC, known as nucleoporins or Nups, associate with many active genes (Casolari et al. 2004;Schmid et al. 2006). Focused analyses of representative examples showed that the genes moved from the nucleoplasm to the nuclear periphery upon induction in a Nup-dependent manner. That transcription and export might be coupled events emerged from studies showing that mRNA export machinery associated with transcribed genes and that the machinery was required for positioning the genes at NPCs (for review, see Dieppois and Stutz 2010). Whether gating of genes at NPCs affects their expression has been less certain. For example, one study showed that artificially targeting a reporter gene to NPCs increased transcri...

Focal accumulation of preribosomes outside the nucleolus during metaphase–anaphase in budding yeast

“…Likewise, we did not detect any change in the formation of the extranucleolar body upon the loss of either Nup2 or Nup60 (Fig. 4B), two nucleoporins known to be involved in the correct tethering of tRNA genes to NPCs in early mitosis (Chen and Gartenberg 2014). Similar data were obtained in cells deficient in nucleoporins (Nup42, Nup100, Nup116, and Nup159) that directly interact with Crm1 (Fig.…”

“…The potential presence of mechanisms to accelerate the delivery of ribosomes to the growing bud also remains unexplored. Interestingly, tRNA production is subject to spatial regulation during the cell cycle because tRNA genes become tethered to nuclear pore complexes (NPCs) in early mitosis through a Los1 exportin-mediated process (Chen and Gartenberg 2014). This mechanism is thought to expedite the export and accumulation of tRNAs in the cytoplasm.…”

Rbs1, a New Protein Implicated in RNA Polymerase III Biogenesis in Yeast Saccharomyces cerevisiae

“…Pol II must be delivered to specific genes, and the nuclear positioning of a given Pol II-transcribed gene can be important for its expression (7). Finally, Pol III-transcribed tRNA genes occupy distinct subnuclear positions; both the nucleous and nuclear pores are considered in budding yeast (8,9). It is also possible that a nuclear/nucleous structure exists, which functions as a platform to localize these assembly events.…”

Ty3, a Position-specific Retrotransposon in Budding Yeast

“…Although the nucleolus is located on the nuclear envelope in yeast, genes could transiently relocalize to other NPC. In addition to gene gating for RNAP II promoters such as the LTRs, transcriptionally active tDNAs have been shown to localize to NPC (30). Consistent with the latter possibility, chromatin conformation capture analysis of yeast chromosomal DNA indicated that a subpopulation of tDNAs are not associated with rDNA (31).…”

“…RNAP III genes undergo specific types of epigenetic modifications and bind chromatin remodelers and condensins (179)(180)(181)(182)(183)(184)(185)(186)(187)(188)(189)(190). In addition to possible differences in chromatin context, tDNAs also act as replication fork barriers (191), and subpopulations localize to the nucleolus (31,192) and nuclear pores (30). Overall, it seems that we are just beginning to learn about the dynamic activities of RNAP IIItranscribed genes.…”