Pus1p-dependent tRNA Pseudouridinylation Becomes Essential When tRNA Biogenesis Is Compromised in Yeast

Yeast Pus1p catalyzes the formation of pseudouridine (ψ) at specific sites of several tRNAs, but its function is not essential for cell viability. We show here that Pus1p becomes essential when another tRNA: pseudouridine synthase, Pus4p, or the essential minor tRNA for glutamine are mutated. Strikingly, this mutant tRNA, which carries a mismatch in the TψC arm, displays a nuclear export defect. Furthermore, nuclear export of at least one wild-type tRNA species becomes defective in the absence of Pus1p. Our data, thus, show that the modifications formed by Pus1p are essential when other aspects of tRNA biogenesis or function are compromised and suggest that impairment of nuclear tRNA export in the absence of Pus1p might contribute to this phenotype

Formation and nuclear export of tRNA, rRNA and mRNA is regulated by the ubiquitin ligase Rsp5p

The yeast ubiquitin-protein ligase Rsp5p regulates processes as diverse as polII transcription and endocytosis. Here, we identify Rsp5p in a screen for tRNA export (tex) mutants. The tex23-1/rsp5-3 mutant, which is complemented by RSP5, not only shows a strong nuclear accumulation of tRNAs at the restrictive temperature, but also is severely impaired in the nuclear export of mRNAs and 60S pre-ribosomal subunits. In contrast, nuclear localization sequence (NLS)-mediated nuclear protein import is unaffected in this mutant. Strikingly, the nuclear RNA export defects seen in the rsp5-3 strain are accompanied by a dramatic inhibition of both rRNA and tRNA processing, a combination of phenotypes that has not been reported for any previously characterized mutation in yeast. These data implicate ubiquitination as a mechanism coordinating the major nuclear RNA biogenesis pathways

XRN2 Autoregulation and Control of Polycistronic Gene Expresssion in <i>Caenorhabditis elegans</i>

<div><p>XRN2 is a conserved 5’→3’ exoribonuclease that complexes with proteins that contain XRN2-binding domains (XTBDs). In <i>Caenorhabditis elegans</i> (<i>C</i>. <i>elegans</i>), the XTBD-protein PAXT-1 stabilizes XRN2 to retain its activity. XRN2 activity is also promoted by 3'(2'),5'-bisphosphate nucleotidase 1 (BPNT1) through hydrolysis of an endogenous XRN inhibitor 3’-phosphoadenosine-5'-phosphate (PAP). Here, we find through unbiased screening that loss of <i>bpnt-1</i> function suppresses lethality caused by <i>paxt-1</i> deletion. This unexpected finding is explained by XRN2 autoregulation, which occurs through repression of a cryptic promoter activity and destabilization of the <i>xrn-2</i> transcript. De-repression appears to be triggered such that more robust XRN2 perturbation, by elimination of both PAXT-1 and BPNT1, is less detrimental to worm viability than absence of PAXT-1 alone. Indeed, we find that two distinct XRN2 repression mechanisms are alleviated at different thresholds of XRN2 inactivation. Like more than 15% of <i>C</i>. <i>elegans</i> genes, <i>xrn-2</i> occurs in an operon, and we identify additional operons under its control, consistent with a broader function of XRN2 in polycistronic gene regulation. Regulation occurs through intercistronic regions that link genes in an operon, but a part of the mechanisms may allow XRN2 to operate on monocistronic genes in organisms lacking operons.</p></div

BPNT1 depletion induces XRN2 de-repression.

<p>(A) <i>rpl-43</i>_<i>Prom</i>::<i>rpl-43</i>_<i>Body</i>::<i>rpl-43</i>_<i>ICR</i> reporter animals in wild-type (<i>bpnt-1(+)</i>) or <i>bpnt-1(xe22)</i> genetic background were cultured from L1 to L4 for 40 hours at 20°C and observed. GFP signal was detected in hypodermal (top), intestinal (middle, arrows) and vulval (bottom) cells. Positions of vulvae are indicated by square brackets. Corresponding DIC images of mid-L4 stage vulvae are shown (bottom). Scale bar: 100 μm. (B) Diagram of an inferred BPNT1-XRN2 regulatory network. (C) <i>rpl-43</i>_<i>Prom</i>::<i>rpl-43</i>_<i>Body</i>::<i>rpl-43</i>_<i>ICR</i> reporter animals were exposed to mock or <i>xrn-2</i> RNAi from L1 to L4 at 20°C and observed. GFP signal was detected in hypodermal (top), intestinal (middle, arrows) and vulval (bottom) cells. Images are shown as described in (A).</p

<i>rpl-43</i>_<i>ICR</i> is required while the operon promoter and the first gene are replaceable for XRN2 autoregulation.

<p>(A) wild-type animals were exposed to mock or <i>xrn-2</i> RNAi from L1 to L4 at 20°C. mRNA levels of <i>ran-4</i> and <i>R05D11</i>.<i>5</i> were quantified by RT-qPCR and normalized to <i>act-1</i> mRNA levels with mock values defined as 1 (n = 3, means ± SEM). Values are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006313#pgen.1006313.s007" target="_blank">S1 Table</a>. (B, D, F) Indicated reporter animals were exposed to mock or <i>xrn-2</i> RNAi from L1 to L4 at 20°C and observed. GFP signal was detected in hypodermal (top), intestinal (middle, arrows) and vulval (bottom) cells. Positions of vulvae are indicated by square brackets. Corresponding DIC images of mid-L4 stage vulvae are shown (bottom). Scale bar: 100 μm. (C, E, G) Indicated reporter animals in <i>bpnt-1(+)</i> or <i>bpnt-1(xe22)</i> genetic background were cultured from L1 to L4 at 20°C and observed. GFP signal was detected in hypodermal (top), intestinal (middle, arrows) and vulval (bottom) cells. Images are shown as described in (B).</p

Effects of <i>xrn-2</i> knockdown or the <i>bpnt-1(xe22)</i> allele on reporter activities.

<p>Effects of <i>xrn-2</i> knockdown or the <i>bpnt-1(xe22)</i> allele on reporter activities.</p

XRN2 controls polycistronic gene expression of a subset of operons.

<p>(A) wild-type animals were exposed to mock or <i>xrn-2</i> RNAi from L1 to L4 at 20°C. RNA was extracted, and poly(A)-RNA expression was examined by deep sequencing. Relative mRNA levels of the first and the second genes in operons were quantified. Fold changes of the second gene mRNA levels upon <i>xrn-2</i> RNAi are plotted against those of the first gene mRNA levels. Operons whose second gene mRNA showed a larger fold change than the first gene mRNA (cut-off as described in “Materials and Methods”) are shown in red. (B) wt animals were exposed to mock or <i>xrn-2</i> RNAi from L1 to L4 at 20°C. mRNA levels were quantified by RT-qPCR and normalized to <i>act-1</i> mRNA levels with control values defined as 1 (n = 3, means ± SEM). Values are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006313#pgen.1006313.s007" target="_blank">S1 Table</a>. (C) <i>ran-4</i>_<i>Prom</i>::<i>ran-4</i>_<i>Body</i>::<i>cri-3</i>_<i>ICR</i> rerporter animals were exposed to mock or <i>xrn-2</i> RNAi from L1 to L4 at 20°C and observed. GFP signal was detected in hypodermal (top), intestinal (middle, arrows) but not in vulval (bottom) cells. DIC images of mid-L4 stage vulvae are shown (bottom). Scale bar: 100 μm. (D) <i>ran-4</i>_<i>Prom</i>::<i>ran-4</i>_<i>Body</i>::<i>cri-3</i>_<i>ICR</i> reporter animals in <i>bpnt-1(+)</i> or <i>bpnt-1(xe22)</i> genetic background were cultured from L1 to L4 at 20°C and observed. GFP signal was detected in hypodermal (top), intestinal (middle, arrows) and vulval (bottom) cells. Positions of vulvae are indicated by square brackets. Corresponding DIC images of mid-L4 stage vulvae are shown (bottom). Scale bar: 100 μm.</p

Mechanistic models for polycistronic gene regulation by XRN2.

<p>XRN2-mediated polycistronic gene regulation occurs through an (A) operon promoter-independent and (B) an operon-promoter-dependent mechanism. (A) Premature termination model. XRN2 degrades uncapped or decapped nascent RNA transcripts from a promoter in the ICR and terminates transcription at promoter proximal site. (B) Competition model. Following 3’ end cleavage of the upstream gene, XRN2 degrades the downstream fragment. Competition with (i) ongoing transcription by RNA pol II, whose activity XRN2 can terminate, or (ii) capping by <i>trans</i>-splicing determines production of <i>xrn-2</i> mRNA. SL indicates SL1 or SL2.</p