Figures and data in A tRNA processing enzyme is a key regulator of the mitochondrial unfolded protein response

Figures
Additional files

8 figures and 3 additional files

Figures

Figure 1

Download asset Open asset

*hoe-1* is required for maximal UPR^mt activation.

(A) Fluorescence images of UPR^mt reporter (*hsp-6p::GFP*) activation in L4 *nuo-6(qm200*) animals on *control* and *hoe-1 RNAi*. Scale bar 200 μm. (B) Fluorescence intensity quantification of *hsp-6p::GFP* in individual L4 *nuo-6(qm200*) animals on control and *hoe-1 RNAi* normalized to *hsp-6p::GFP* in a wildtype background on *control RNAi* (n = 8 and 15 respectively, mean and SD shown, unpaired t-test). (C) Fluorescence images of UPR^mt reporter (*hsp-6p::GFP*) activation in L3/L4 wildtype and *hoe-1* null (*hoe-1(-/-*)) animals on *control*, *cco-1*, and *spg-7 RNAi*. Scale bar 200 μm. (D) Fluorescence intensity quantification of *hsp-6p::GFP* in individual L3/L4 wildtype and *hoe-1(-/-*) animals on *control* and *cco-1 RNAi* (n = 8,12,6 and 13 respectively, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test). (E) Fluorescence intensity quantification of *hsp-6p::GFP* in individual L3/L4 wildtype and *hoe-1(-/-*) animals on *control* and *spg-7 RNAi* (n = 7,15,6 and 18 respectively, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test). (F) Fluorescence images of UPR^mt reporter (*hsp-6p::GFP*) activation in L3/L4 *nuo-6(qm200*) animals with (*hoe-1(+/+*)) and without (*hoe-1(-/-*)) *hoe-1*. Scale bar 200 μm. (G) Fluorescence intensity quantification of *hsp-6p::GFP* in individual L3/L4 *nuo-6(qm200*) animals with (*hoe-1(+/+*)) and without (*hoe-1(-/-*)) *hoe-1* normalized to *hsp-6p::GFP* in a wildtype background (n = 22 for each condition, mean and SD shown, unpaired t-test).

Figure 2 with 5 supplements

Download asset Open asset

Nuclear HOE-1 is required for maximal UPR^mt activation.

(A) Fluorescence images of a terminal intestinal cell in a wildtype animal expressing HOE-1::GFP (green) stained with TMRE (magenta) to visualize mitochondria. GFP and TMRE co-localization shown in white in merged image. Arrow indicates nuclei. Scale bar 20 μm. Representative line segment analysis of individual mitochondrion. (B) Schematic of HOE-1 protein showing the mitochondrial targeting sequence (MTS) and nuclear localization signals (NLS). ΔMTS allele created by replacing START codon with an alanine (M1A). Transcription begins at M74 for nuclear localized HOE-1. ΔNLS allele created by compromising the most N-terminal NLS (⁶³⁶KRPR > AAPA). (C) Fluorescence images of UPR^mt reporter (*hsp-6p::GFP*) in L4 wildtype and *hoe-1(ΔMTS*) animals on *control* and *spg-7 RNAi*. Scale bar 200 μm. (D) Fluorescence intensity quantification of *hsp-6p::GFP* in individual L4 wildtype and *hoe-1(ΔMTS*) animals on *control* and *spg-7 RNAi* (n = 15,20,17, and 19 respectively, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test). (E) Fluorescence images of UPR^mt reporter (*hsp-6p::GFP*) in L4 wildtype and *hoe-1(ΔNLS*) animals on *control* and *spg-7 RNAi*. Scale bar 200 μm. (F) Fluorescence intensity quantification of *hsp-6p::GFP* in individual L4 wildtype and *hoe-1(ΔNLS*) animals on *control* and *spg-7 RNAi* (n = 15 for each condition, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test). (G) Fluorescence images of UPR^mt reporter in L4 *nuo-6(qm200*) animals in wildtype and *hoe-1(ΔNLS*) backgrounds. Scale bar 200 μm. (H) Fluorescence intensity of *hsp-6p::GFP* in individual L4 *nuo-6(qm200*) animals in wildtype and *hoe-1(ΔNLS*) backgrounds (n = 30 for each condition, mean and SD shown, unpaired t-test). (I) mRNA transcript quantification of *hsp-6* in L4 wildtype and *hoe-1(ΔNLS*) animals on *control* and *spg-7 RNAi* normalized to *ama-1* (n = 4 for each condition, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test).

Figure 2—figure supplement 1

Download asset Open asset

*hoe-1::GFP* does not compromise growth or development and is sufficient to rescue the developmental arrest of *hoe-1(-/-*) animals.

Bright-field images of wildtype, *hoe-1::GFP*, *hoe-1(-/-*), and *hoe-1(-/-)/hoe-1::GFP* trans-heterozygous animals 72 hr post-embryo. Scale bar 200 μm.

Figure 2—figure supplement 2

Download asset Open asset

*hoe-1(ΔMTS*) allele attenuates HOE-1 mitochondrial localization.

Fluorescence images of a terminal intestinal cell in a *hoe-1(ΔMTS*) day 1 adult animal expressing HOE-1::GFP (green) stained with TMRE (magenta) to visualize mitochondria. GFP and TMRE co-localization shown in white in merged image. Arrow indicates nuclei. Scale bar 20 μm. Representative line segment analysis of individual mitochondrion.

Figure 2—figure supplement 3

Download asset Open asset

*hoe-1(ΔMTS*) does not attenuate *cco-1 RNAi-*induced UPR^mt.

(A) Fluorescence images of UPR^mt reporter (*hsp-6p::GFP*) in L4 wildtype and *hoe-*1(*ΔMTS*) animals on *control* and *cco-1 RNAi*. Scale bar 200 μm. (B) Fluorescence intensity quantification of *hsp-6p::GFP* in individual L4 wildtype and *hoe-1(ΔMTS*) animals on control and *cco-1 RNAi* (n = 12,14,16, and 18 respectively, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test).

Figure 2—figure supplement 4

Download asset Open asset

*hoe-1(ΔNLS*) allele attenuates nuclear HOE-1 localization.

(A) Fluorescence images of a terminal intestinal cell individual a *hoe-1(ΔNLS*) day 1 adult animal expressing HOE-1::GFP(green) stained with TMRE (magenta) to visualize mitochondria. GFP and TMRE co-localization shown in white in merged image. Nuclei are traced with dashed white line. Scale bar 20 μm (B) Fluorescence intensity quantification of HOE-1::GFP in intestinal nuclei of wildtype, *hoe-1(ΔMTS*), *hoe-1(ΔNLS*) and *hoe-1(ΔNES*) backgrounds (n = 57, 52, 60, and 73 respectively, mean and SD shown, ordinary one-way ANOVA with Dunnett’s multiple comparisons test). (C) Fluorescence intensity quantification of HOE-1::GFP in intestinal mitochondria of wildtype, *hoe-1(ΔNLS*) and *hoe-1(ΔNES*) backgrounds (n = 57, 53, and 60 respectively, mean and SD shown, ordinary one-way ANOVA with Dunnett’s multiple comparisons test).

Figure 2—figure supplement 5

Download asset Open asset

UPR^mt-responsive gene *cyp-14A1.4* is downregulated under mitochondrial stress conditions in *hoe-1(ΔNLS*) animals relative to wildtype.

mRNA transcript quantification of *cyp-14A1.4* in L4 wildtype and *hoe-1(ΔNLS*) animals on *control* and *spg-7 RNAi* normalized to *ama-1* (n = 4 for each condition, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test).

Figure 3 with 5 supplements

Download asset Open asset

Nuclear export defective HOE-1 is sufficient to specifically activate UPR^mt.

(A) Fluorescence images of UPR^mt reporter (*hsp-6p::GFP*) activation in day 2 adult wildtype, *nuo-6(qm200*), *atfs-1(et15*), and *hoe-1(ΔNES*) animals. Scale bar 200 μm. (B) Fluorescence intensity quantification of *hsp-6p::GFP* in individual day 2 adult wildtype, *nuo-6(qm200*), *atfs-1(et15*), and *hoe-1(ΔNES*) animals (n = 10 for each condition, mean and SD shown, ordinary one-way ANOVA with Tukey’s multiple comparisons test). (**C–E**) mRNA transcript quantification of *hsp-6*, *clec-47*, and *cyp-14A1.4*, respectively, in day 2 adult wildtype and *hoe-1(ΔNES*) animals normalized to *ama-1* mRNA levels (n = 4 for each condition, mean and SD shown, unpaired t-test). (F) Fluorescence images of UPR^mt reporter (*hsp-6p::GFP*) activation in day 2 adult *hoe-1(ΔNES*) animals on *control* and *atfs-1 RNAi*. Scale bar 200 μm. (G) Fluorescence intensity quantification of *hsp-6p::GFP* in individual day 2 adult wildtype and *hoe-1(ΔNES*) animals on *control* and *atfs-1 RNAi* (n = 10 for each condition, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test). (H) Fluorescence images of UPR^ER reporter (*hsp-4p::GFP*) activation in day 2 adult wildtype and *hoe-1(ΔNES*) animals. Scale bar 200 μm. (I) Fluorescence intensity quantification of *hsp-4p::GFP* in individual day 2 adult wildtype and *hoe-1(ΔNES*) animals (n = 10 for each condition, mean and SD shown, unpaired t-test). (J) Fluorescence images of intestinal-specific basal protein reporter (*ges-1p::GFPcyto*) activation in day 2 adult wildtype and *hoe-1(ΔNES*) animals. Scale bar 200 μm. (K) Fluorescence intensity quantification of *ges-1p::GFPcyto* in individual day 2 adult wildtype and *hoe-1(ΔNES*) animals (n = 10 for each condition, mean and SD shown, unpaired t-test).

Figure 3—figure supplement 1

Download asset Open asset

Nuclear export defective HOE-1 has increased nuclear accumulation relative to wildtype.

(A) Schematic of HOE-1 protein showing the mitochondrial targeting sequence (MTS), nuclear localization signals (NLS) and nuclear export signal (NES). *hoe-1(ΔNES*) mutant generated by changing the strong hydrophobic residues of NES to alanines (⁷³¹VAELFELTI⁷³⁹>⁷³¹AAEAAEATA⁷³⁹) (B) Fluorescence images of a terminal intestinal cell in a *hoe-1(ΔNES*) day 1 adult animal expressing HOE-1::GFP (green) stained with TMRE (magenta) to visualize mitochondria. GFP and TMRE co-localization shown in white in merged image. Arrow indicates nuclei. Scale bar 20 μm. Quantification of nuclear and mitochondrial HOE-1::GFP levels in *hoe-1(ΔNES*) animals shown in Figure 2—figure supplement 4.

Figure 3—figure supplement 2

Download asset Open asset

Nuclear export defective HOE-1 activates UPR^mt.

(A) Fluorescence images of UPR^mt reporter (*hsp-60p::GFP*) activation in day 2 adult wildtype and *hoe-1(ΔNES*) animals. Scale bar 200 μm. (B) Fluorescence intensity quantification of *hsp-60p::GFP* in individual day 2 adult wildtype and *hoe-1(ΔNES*) animals (n = 24 for each condition, mean and SD shown, unpaired t-test).

Figure 3—figure supplement 3

Download asset Open asset

Compromised nuclear import of HOE-1 completely attenuates *hoe-1(ΔNES*)-induced UPR^mt.

(A) Fluorescence images of UPR^mt reporter (*hsp-6p::GFP*) activation in day 2 adult wildtype, *hoe-1(ΔNLS*), *hoe-1(ΔNES*), and *hoe-1(ΔNLS+ΔNES*) animals. Scale bar 200 μm. (B) Fluorescence intensity quantification of *hsp-6p::GFP* in individual day 2 adult wildtype, *hoe-1(ΔNLS*), *hoe-1(ΔNES*), and *hoe-1(ΔNLS+ΔNES*) animals (n = 24 for each condition, mean and SD shown, ordinary one-way ANOVA with Tukey’s multiple comparisons test).

Figure 3—figure supplement 4

Download asset Open asset

Compromised mitochondrial import of HOE-1 exacerbates *hoe-1(ΔNES)-*induced UPR^mt.

(A) Fluorescence images of UPR^mt reporter (*hsp-6p::GFP*) activation in day 2 adult wildtype, *hoe-1(ΔMTS*), *hoe-1(ΔNES*), and *hoe-1(ΔMTS+ΔNES*) animals. Scale bar 200 μm. (B) Fluorescence intensity quantification of *hsp-6p::GFP* in individual day 2 adult wildtype, *hoe-1(ΔMTS*), *hoe-1(ΔNES*), and *hoe-1(ΔMTS+ΔNES*) animals (n = 24 for each condition, mean and SD shown, ordinary one-way ANOVA with Tukey’s multiple comparisons test).

Figure 3—figure supplement 5

Download asset Open asset

Nuclear export defective HOE-1 activates UPR^mt in the intestine cell autonomously.

(A) Fluorescence images of UPR^mt reporter (*hsp-6p::GFP*) activation in day 2 adult wildtype, *hoe-1(ΔNES::degron*), *hoe-1(ΔNES::degron*) with intestinal-specific AID (*ges-1p::TIR1*), and *hoe-1(ΔNES::degron*) with neuronal-specific AID (*rgef-1p::TIR1*) animals on vehicle and 1 mM auxin. (B) Fluorescence intensity quantification of *hsp-6p::GFP* in individual day 2 adult wildtype, *hoe-1(ΔNES::degron*), *hoe-1(ΔNES::degron*) with intestinal-specific AID (*ges-1p::TIR1*), and *hoe-1(ΔNES::degron*) with neuronal-specific AID (*rgef-1p::TIR1*) animals on vehicle and 1 mM auxin (n = 24 for each condition, mean and SD shown, ordinary one-way ANOVA with Tukey’s multiple comparisons test). Note that the degron tagged *hoe-1(ΔNES*) allele has modestly diminished UPR^mt activation relative to the untagged *hoe-1(ΔNES*) allele.

Figure 4

Download asset Open asset

Nuclear export defective HOE-1 activates UPR^mt, correlating with reduced mitochondrial membrane potential.

(A) Fluorescence images of TMRE stained day 1 adult wildtype, *hoe-1(ΔNES*), and *hoe-1(ΔNLS*) individuals. Scale bar 20 μm. (B) Fluorescence intensity quantification of TMRE staining in individual day 1 adult wildtype, *hoe-1(ΔNES*), and *hoe-1(ΔNLS*) animals (n = 57, 60, and 63 respectively, mean and SD shown, ordinary one-way ANOVA with Tukey’s multiple comparisons test). (C) Fluorescence images of TMRE stained day 1 adult wildtype and *hoe-1(ΔNES*) animals on *control* and *atfs-1 RNAi*. Scale bar 20 μm. (D) Fluorescence intensity quantification of TMRE staining in individual day 1 adult wildtype and *hoe-1(ΔNES*) animals on *control* and *atfs-1* RNAi (n = 65, 62, 65, and 61 respectively, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test).

Figure 5 with 1 supplement

Download asset Open asset

Nuclear export defective HOE-1 animals have increased nuclear accumulation of UPR^mt transcription factors ATFS-1 and DVE-1.

(A), Fluorescence images of ATFS-1::mCherry in the terminal intestine of day 2 adult wildtype *hoe-1(ΔNES*), and *nuo-6(qm200*) individuals (tip of the tail is in the bottom of each panel). Intestinal nuclei outlined with dashed white line. Scale bar 20 μm. (B) Fluorescence intensity quantification of nuclear ATFS-1::mCherry in wildtype, *hoe-1(ΔNES*), and *nuo-6(qm200*) individuals (n = 65, 74, and 72 respectively, mean and SD shown, ordinary one-way ANOVA with Tukey’s multiple comparisons test). (C) Fluorescence intensity quantification of total cellular ATFS-1::mCherry in wildtype, *hoe-1(ΔNES*), and *nuo-6(qm200*) individuals (n = 61, 62, and 67 respectively, mean and SD shown, ordinary one-way ANOVA with Tukey’s multiple comparisons test). (D) mRNA transcript quantification of *atfs-1* in day 2 adult wildtype, *nuo-6(qm200*), and *hoe-1(ΔNES*) animals normalized to *ama-1* (n = 4 for each condition, mean and SD shown, ordinary one-way ANOVA with Tukey’s multiple comparisons test). (E) Fluorescence images of *dve-1p::DVE-1::GFP* in day 2 adult wildtype and *hoe-1(ΔNES*) animals. Scale bar 200 μm. (F) Number of intestinal cell nuclei with DVE-1::GFP puncta above brightness threshold of 25 in day 2 adult wildtype and *hoe-1(ΔNES*) animals (n = 33 and 41 respectively, unpaired t-test). (G) Western blot for DVE-1::GFP and actin from day 1 adult wildtype and *hoe-1(ΔNES*) animals. (H) Quantification of DVE-1::GFP western blot band intensity from day 1 adult wildtype and *hoe-1(ΔNES*) animals normalized to total protein (n = 4 for each condition, mean and SD shown, unpaired t-test).

Figure 5—source data 1 Blots for wildtype and hoe-1(ΔNES) animals with DVE-1::GFP (Figure 4G and H). All panels are the same membrane. (A) Image of stain-free blot for total protein from day 1 adult wildtype and hoe-1(ΔNES) animals. Four biological replicates of each condition: Lane #1 BR Spectra Protein Ladder – ladder bands in kDa denoted, Lane #2–5 wildtype and #6–9 hoe-1(ΔNES). (B) Chemiluminescence image of blot for DVE-1::GFP using GFP primary antibody. (C) Composite image of chemiluminescence and colorimetric images of blot for DVE-1::GFP to show bands relative to ladder. (D) Chemiluminescence image of blot for actin using β-actin primary antibody. (E) Composite image of chemiluminescence and colorimetric images of blot for actin to show bands relative to ladder.: https://cdn.elifesciences.org/articles/71634/elife-71634-fig5-data1-v3.zip
Download elife-71634-fig5-data1-v3.zip

Figure 5—figure supplement 1

Download asset Open asset

Nuclear export defective HOE-1 does not elevate extra-nuclear ATFS-1::mCherry levels.

Fluorescence intensity quantification of extra-nuclear ATFS-1::mCherry in wildtype, *hoe-1(ΔNES*), and *nuo-6(qm200*) individuals (n = 61, 62, and 67 respectively, mean and SD shown, ordinary one-way ANOVA with Tukey’s multiple comparisons test).

Figure 6 with 3 supplements

Download asset Open asset

Nuclear export defective HOE-1 activates UPR^mt via altered tRNA processing.

(A) Fluorescence images of UPR^mt reporter (*hsp-6p::GFP*) activation in day 2 adult wildtype and *hoe-1(ΔNES*) animals on *control* and *popl-1 RNAi*. Scale bar 200 μm. (B) Fluorescence intensity quantification of *hsp-6p::GFP* in individual day 2 adult wildtype and *hoe-1(ΔNES*) animals on *control* and *popl-1 RNAi* (n = 24 for each condition, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test). (C) Fluorescence images of UPR^mt reporter (*hsp-6p::GFP*) activation in day 2 adult wildtype and *nuo-6(qm200*) animals on *control* and *popl-1 RNAi*. Scale bar 200 μm. (D) Fluorescence intensity quantification of *hsp-6p::GFP* in individual day 2 adult wildtype and *nuo-6(qm200*) animals on *control* and *popl-1 RNAi* (n = 24 for each condition, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test). (E) Fluorescence images of intestinal-specific basal protein reporter (*ges-1p::GFPcyto*) activation in day 2 adult wildtype animals on *control* and *popl-1 RNAi*. Scale bar 200 μm. (F) Fluorescence intensity quantification of *ges-1p::GFPcyto* in individual day 2 adult wildtype animals on *control* and *popl-1 RNAi* (n = 24 for each condition, mean and SD shown, unpaired t-test). (G) Fluorescence images of UPR^mt reporter (*hsp-6p::GFP*) activation in day 2 adult wildtype and *hoe-1(ΔNES*) animals on *control* and *xpo-3 RNAi*. Scale bar 200 μm. (H) Fluorescence intensity quantification of *hsp-6p::GFP* in individual day 2 adult wildtype and *hoe-1(ΔNES*) animals on *control* and *xpo-3 RNAi* (n = 24 for each condition, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test). (I) Fluorescence images of UPR^mt reporter (*hsp-6p::GFP*) activation in day 2 adult wildtype and *nuo-6(qm200*) animals on *control* and *xpo-3 RNAi*. Scale bar 200 μm. (J) Fluorescence intensity quantification of *hsp-6p::GFP* in individual day 2 adult wildtype and *nuo-6(qm200*) animals on *control* and *xpo-3 RNAi* (n = 24 for each condition, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test). (K) Fluorescence images of intestinal-specific basal protein reporter (*ges-1p::GFPcyto*) activation in day 2 adult wildtype animals on *control* and *xpo-3 RNAi*. Scale bar 200 μm. (L) Fluorescence intensity quantification of *ges-1p::GFPcyto* in individual day 2 adult wildtype animals on *control* and *xpo-3 RNAi* (n = 24 for each condition, mean and SD shown, unpaired t-test).

Figure 6—figure supplement 1

Download asset Open asset

Nuclear export defective HOE-1 induced UPR^mt is dependent upon the catalytic activity of HOE-1.

(A) Fluorescence images of UPR^mt reporter (*hsp-6p::GFP*) activation and corresponding bright-field images of wildtype, catalytically-dead *hoe-1* (*hoe-1(D624A*)) mutant, catalytically-dead nuclear export defective *hoe-1* (*hoe-1(D624A+ΔNES*)) mutant, *hoe-1(ΔNES*), and *hoe-1(ΔNES)/hoe-1(D624A+ΔNES*) trans-heterozygous mutant animals 96 hr post-embryo. Scale bar 200 μm. (B) Fluorescence intensity quantification of *hsp-6p::GFP* in individual wildtype, *hoe-1(D624A*), *hoe-1(D624A+ΔNES*), *hoe-1(ΔNES*), and *hoe-1(ΔNES)/hoe-1(D624A+ΔNES*) trans-heterozygous animals 96 hr post-embryo (n = 24 for each condition, mean and SD shown, ordinary one-way ANOVA with Tukey’s multiple comparisons test).

Figure 6—figure supplement 2

Download asset Open asset

RNAi against RNA polymerase III subunit, *rpc-1*, preferentially attenuates *hoe-1(ΔNES*)-induced UPR^mt.

(A) Fluorescence images of UPR^mt reporter (*hsp-6p::GFP*) activation in wildtype and *hoe-1(ΔNES*) day 2 adult animals on *control* and *rpc-1 RNAi*. Scale bar 200 μm. (B) Fluorescence intensity quantification of *hsp-6p::GFP* in individual wildtype and *hoe-1(ΔNES*) day 2 adult animals on *control* and *rpc-1 RNAi* (n = 24 for each condition, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test). (C) Fluorescence images of UPR^mt reporter (*hsp-6p::GFP*) activation in wildtype and *nuo-6(qm200*) day 2 adult animals on *control* and *rpc-1 RNAi*. Scale bar 200 μm. (D) Fluorescence intensity quantification of *hsp-6p::GFP* in individual wildtype and *nuo-6(qm200*) day 2 adult animals on *control* and *rpc-1 RNAi* (n = 24 for each condition, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test).

Figure 6—figure supplement 3

Download asset Open asset

RNAi against tRNA nucleotidyl transferase, *hpo-31*, and tRNA ligase, *rtcb-1*, mildly attenuate both *hoe-1(ΔNES*)- and *nuo-6(qm200*)-induced UPR^mt.

(A) Fluorescence images of UPR^mt reporter (*hsp-6p::GFP*) activation in wildtype and *hoe-1(ΔNES*) day 2 adult animals on *control*, *hpo-31*, and *rtcb-1 RNAi*. Scale bar 200 μm. (B) Fluorescence intensity quantification of *hsp-6p::GFP* in individual wildtype and *hoe-1(ΔNES*) day 2 adult animals on *control* and *hpo-31 RNAi* (n = 24 for each condition, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test). (C) Fluorescence intensity quantification of *hsp-6p::GFP* in individual wildtype and *hoe-1(ΔNES*) day 2 adult animals on *control* and *rtcb-1 RNAi* (n = 24 for each condition, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test). Note that the same *control* RNAi animals were used for analysis in both panels B and C as experiments were conducted simultaneously. (D) Fluorescence images of UPR^mt reporter (*hsp-6p::GFP*) activation in wildtype and *nuo-6(qm200*) day 2 adult animals on *control*, and *rtcb-1 RNAi*. Scale bar 200 μm. (E) Fluorescence intensity quantification of *hsp-6p::GFP* in individual wildtype and *nuo-6(qm200*) day 2 adult animals on *control* and *rtcb-1 RNAi* (n = 24 for each condition, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test).

Figure 7

Download asset Open asset

Nuclear export defective HOE-1 induced UPR^mt is not *gcn-2* or *eIF2α* dependent.

(A) Fluorescence images of UPR^mt reporter (*hsp-6p::GFP*) activation in day 2 adult wildtype, *gcn-2(ok871*), *eIF2α(S46A,S49A*), *hoe-1(ΔNES*), *hoe-1(ΔNES);gcn-2(ok871),* and *hoe-1(ΔNES);eIF2α(S46A,S49A*) animals. Scale bar 200 μm. (B) Fluorescence intensity quantification of *hsp-6p::GFP* in individual day 2 adult wildtype, *gcn-2(ok871*), *eIF2α(S46A,S49A*), *hoe-1(ΔNES*), *hoe-1(ΔNES);gcn-2(ok871),* and *hoe-1(ΔNES);eIF2α(S46A,S49A*) animals (n = 24 for each condition, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test).

Figure 8 with 4 supplements

Download asset Open asset

Nuclear HOE-1 levels are elevated during mitochondrial stress in the absence of ATFS-1 but decreased in the presence of ATFS-1.

(A) Fluorescence images of HOE-1::GFP in day 1 adult wildtype and *nuo-6(qm200*) animals on *control* and *atfs-1 RNAi*. Scale bar 200 μm. (B) Fluorescence intensity quantification of intestinal nuclei relative to extranuclear signal in day 1 adult wildtype and *nuo-6(qm200*) animals on *control* and *atfs-1 RNAi* (n = 40 for each condition, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test). (C) Western blot for HOE-1::GFP and actin from day 1 adult wildtype and *nuo-6(qm200*) animals on *control* and *atfs-1 RNAi*. (D) Quantification of HOE-1::GFP western blot band intensity from day 1 adult wildtype and *nuo-6(qm200*) animals on *control* and *atfs-1 RNAi* normalized to total protein (n = 4 for each condition, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test). (E) Fluorescence images of HOE-1::GFP in day 1 adult wildtype and *atfs-1(et15*) animals. Scale bar 200 μm. (F) Fluorescence intensity quantification of intestinal nuclei relative to extranuclear signal in day 1 adult wildtype and *atfs-1(et15*) animals (n = 40 for each condition, mean and SD shown, unpaired t-test). (G) Western blot for HOE-1::GFP and actin from day 1 adult wildtype and *atfs-1(et15*) animals. (H) Quantification of HOE-1::GFP western blot band intensity from day 1 adult wildtype and *atfs-1(et15*) animals normalized to total protein (n = 4 for each condition, mean and SD shown, unpaired t-test). (I) Mitochondrial stress triggers activation of HOE-1 resulting in altered RNA processing that facilitates UPR^mt via ATFS-1. Activation of UPR^mt negatively regulates HOE-1.

Figure 8—source data 1 Blots for wildtype and nuo-6(qm200) animals on control and atfs-1 RNAi (Figure 8C). All panels are the same membrane. (A) Image of stain-free blot for total protein from day 1 adult wildtype and nuo-6(qm200) animals on control and atfs-1 RNAi. Two biological replicates of each condition: Lane # 1&11 BR Spectra Protein Ladder – ladder bands in kDa denoted. Lane # 2&7 wildtype on control RNAi, 3&8 wildtype on atfs-1 RNAi, 4&9 nuo-6(qm200) on control RNAi, and 5&10 nuo-6(qm200) on atfs-1 RNAi. Lane # 6 empty. (B) Chemiluminescence image of blot for HOE-1::GFP using GFP primary antibody. (C) Composite image of chemiluminescence and colorimetric images of blot for HOE-1::GFP to show bands relative to ladder. (D) Chemiluminescence image of blot for actin using β-actin primary antibody. (E) Composite image of chemiluminescence and colorimetric images of blot for actin to show bands relative to ladder.: https://cdn.elifesciences.org/articles/71634/elife-71634-fig8-data1-v3.zip
Download elife-71634-fig8-data1-v3.zip
Figure 8—source data 2 Blots for wildtype and nuo-6(qm200) animals on control and atfs-1 RNAi (Figure 8D). Samples were loaded and ran on two separate membranes simultaneously (Membrane A and Membrane B). All panels in each column are the same membrane. (A) Image of stain-free blots for total protein from day 1 adult wildtype and nuo-6(qm200) animals on control and atfs-1 RNAi. Two biological replicates on each blot of each condition: Lane # 1&10 BR Spectra Protein Ladder – ladder bands in kDa denoted. Lane # 2&6 wildtype on control RNAi, 3&7 wildtype on atfs-1 RNAi, 4&8 nuo-6(qm200) on control RNAi, and 5&9 nuo-6(qm200) on atfs-1 RNAi. (B) Chemiluminescence image of blots for HOE-1::GFP using GFP primary antibody. (C) Composite images of chemiluminescence and colorimetric images of blots for HOE-1::GFP to show bands relative to ladder. (D) Chemiluminescence images of blots for actin using β-actin primary antibody. (E) Composite images of chemiluminescence and colorimetric images of blots for actin to show bands relative to ladder.: https://cdn.elifesciences.org/articles/71634/elife-71634-fig8-data2-v3.zip
Download elife-71634-fig8-data2-v3.zip
Figure 8—source data 3 Blots for wildtype and atfs-1(et15) animals (Figure 8G). All panels are the same membrane. (A) Image of stain-free blot for total protein from day 1 adult wildtype and atfs-1(et15) animals. Two biological replicates of each condition: Lane # 1&6 BR Spectra Protein Ladder – ladder bands in kDa denoted. Lane #2&4 wildtype and #3&5 atfs-1(et15). (B) Chemiluminescence image of blot for HOE-1::GFP using GFP primary antibody. (C) Composite image of chemiluminescence and colorimetric images of blot for HOE-1::GFP to show bands relative to ladder. (D) Chemiluminescence image of blot for actin using β-actin primary antibody. (E) Composite image of chemiluminescence and colorimetric images of blot for actin to show bands relative to ladder.: https://cdn.elifesciences.org/articles/71634/elife-71634-fig8-data3-v3.zip
Download elife-71634-fig8-data3-v3.zip
Figure 8—source data 4 Blots for wildtype and atfs-1(et15) animals (Figure 8H). All panels are the same membrane. (A), Image of stain-free blot for total protein from day 1 adult wildtype and atfs-1(et15) animals. Four biological replicates of each condition: Lane # 1 BR Spectra Protein Ladder – ladder bands in kDa denoted. Lanes #2,4,6,8 wildtype and #3,5,7,9 atfs-1(et15). (B) Chemiluminescence image of blot for HOE-1::GFP using GFP primary antibody. (C) Composite image of chemiluminescence and colorimetric images of blot for HOE-1::GFP to show bands relative to ladder. (D) Chemiluminescence image of blot for actin using β-actin primary antibody. (E) Composite image of chemiluminescence and colorimetric images of blot for actin to show bands relative to ladder.: https://cdn.elifesciences.org/articles/71634/elife-71634-fig8-data4-v3.zip
Download elife-71634-fig8-data4-v3.zip

Figure 8—figure supplement 1

Download asset Open asset

*hoe-1* mRNA levels are upregulated under conditions of mitochondrial stress.

(A) Copies of total *hoe-1* mRNA (primer pair 1) versus *hoe-1* mRNA that include the mitochondrial targeting sequence (primer pair 2) in day 1 adult wildtype animals. ddPCR droplet counts shown. Paired samples connected with solid black line. (**B–C**) mRNA transcript quantification of *hoe-1* in day 1 adult wildtype and *nuo-6(qm200*) animals normalized to *ama-1* (n = 4 for each condition, mean and SD shown, unpaired t-test) measured with two separate primer pairs.

Figure 8—figure supplement 2

Download asset Open asset

UPR^mt-inducing *cco-1 and spg-7 RNAi* both attenuate HOE-1 nuclear levels.

(A) Fluorescence images of HOE-1::GFP expressing animals on *control*, *cco-1*, and *spg-7 RNAi*. Scale bar 200 μm. (B) Fluorescence intensity quantification of intestinal nuclei relative to extranuclear signal of HOE-1::GFP on *control*, *cco-1*, and *spg-7 RNAi* (n = 40 for each condition, mean and SD shown, ordinary one-way ANOVA with Dunnett’s multiple comparisons test).

Figure 8—figure supplement 3

Download asset Open asset

Figure 8—figure supplement 4

Download asset Open asset

Constitutive activation of UPR^mt by *atfs-1* gain-of-function (*atfs-1(et15*)) depletes nuclear HOE-1 levels.

(A) Fluorescence images of the intestine of individual day 1 adult wildtype and *atfs-1(et15*) animals expressing HOE-1::GFP (green) stained with TMRE (magenta) to visualize mitochondria. GFP and TMRE co-localization shown in white in merged image. Nuclei are traced with dashed white line. Scale bar 20 μm. (B) Fluorescence intensity quantification of the nuclear to cytosolic ratio of HOE-1::GFP in intestine of wildtype and *atfs-1(et15*) animals (n = 56 and 66 respectively, mean and SD shown, unpaired t-test). (C) Fluorescence intensity quantification of nuclear HOE-1::GFP in intestine of wildtype and *atfs-1(et15*) animals (n = 77 and 81 respectively, mean and SD shown, unpaired t-test). (D) Fluorescence intensity quantification of mitochondrial HOE-1::GFP in intestine of wildtype and *atfs-1(et15*) animals (n = 56 and 66 respectively, mean and SD shown, unpaired t-test).

Additional files

Transparent reporting form: https://cdn.elifesciences.org/articles/71634/elife-71634-transrepform1-v3.pdf
Download elife-71634-transrepform1-v3.pdf
Supplementary file 1 C. elegans strains used in this study.: https://cdn.elifesciences.org/articles/71634/elife-71634-supp1-v3.xlsx
Download elife-71634-supp1-v3.xlsx
Supplementary file 2 Oligonucleotides used in this study.: https://cdn.elifesciences.org/articles/71634/elife-71634-supp2-v3.xlsx
Download elife-71634-supp2-v3.xlsx

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Mendeley

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

James P Held
Gaomin Feng
Benjamin R Saunders
Claudia V Pereira
Kristopher Burkewitz
Maulik R Patel

(2022)

A tRNA processing enzyme is a key regulator of the mitochondrial unfolded protein response

eLife 11:e71634.

https://doi.org/10.7554/eLife.71634

Share this article

Cite this article

hoe-1 is required for maximal UPRmt activation.

Nuclear HOE-1 is required for maximal UPRmt activation.

hoe-1::GFP does not compromise growth or development and is sufficient to rescue the developmental arrest of hoe-1(-/-) animals.

hoe-1(ΔMTS) allele attenuates HOE-1 mitochondrial localization.

hoe-1(ΔMTS) does not attenuate cco-1 RNAi-induced UPRmt.

hoe-1(ΔNLS) allele attenuates nuclear HOE-1 localization.

UPRmt-responsive gene cyp-14A1.4 is downregulated under mitochondrial stress conditions in hoe-1(ΔNLS) animals relative to wildtype.

Nuclear export defective HOE-1 is sufficient to specifically activate UPRmt.

Nuclear export defective HOE-1 has increased nuclear accumulation relative to wildtype.

Nuclear export defective HOE-1 activates UPRmt.

Compromised nuclear import of HOE-1 completely attenuates hoe-1(ΔNES)-induced UPRmt.

Compromised mitochondrial import of HOE-1 exacerbates hoe-1(ΔNES)-induced UPRmt.

Nuclear export defective HOE-1 activates UPRmt in the intestine cell autonomously.

Nuclear export defective HOE-1 activates UPRmt, correlating with reduced mitochondrial membrane potential.

Nuclear export defective HOE-1 animals have increased nuclear accumulation of UPRmt transcription factors ATFS-1 and DVE-1.

Figure 5—source data 1

Nuclear export defective HOE-1 does not elevate extra-nuclear ATFS-1::mCherry levels.

Nuclear export defective HOE-1 activates UPRmt via altered tRNA processing.

Nuclear export defective HOE-1 induced UPRmt is dependent upon the catalytic activity of HOE-1.

RNAi against RNA polymerase III subunit, rpc-1, preferentially attenuates hoe-1(ΔNES)-induced UPRmt.

RNAi against tRNA nucleotidyl transferase, hpo-31, and tRNA ligase, rtcb-1, mildly attenuate both hoe-1(ΔNES)- and nuo-6(qm200)-induced UPRmt.

Nuclear export defective HOE-1 induced UPRmt is not gcn-2 or eIF2α dependent.

Nuclear HOE-1 levels are elevated during mitochondrial stress in the absence of ATFS-1 but decreased in the presence of ATFS-1.

Figure 8—source data 1

Figure 8—source data 2

Figure 8—source data 3

Figure 8—source data 4

hoe-1 mRNA levels are upregulated under conditions of mitochondrial stress.

UPRmt-inducing cco-1 and spg-7 RNAi both attenuate HOE-1 nuclear levels.

Nuclear HOE-1 levels are elevated during mitochondrial stress in the absence of ATFS-1 but decreased in the presence of ATFS-1.

Constitutive activation of UPRmt by atfs-1 gain-of-function (atfs-1(et15)) depletes nuclear HOE-1 levels.

Transparent reporting form

Supplementary file 1

Supplementary file 2

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

hoe-1 is required for maximal UPR^mt activation.

Nuclear HOE-1 is required for maximal UPR^mt activation.

hoe-1(ΔMTS) does not attenuate cco-1 RNAi-induced UPR^mt.

UPR^mt-responsive gene cyp-14A1.4 is downregulated under mitochondrial stress conditions in hoe-1(ΔNLS) animals relative to wildtype.

Nuclear export defective HOE-1 is sufficient to specifically activate UPR^mt.

Nuclear export defective HOE-1 activates UPR^mt.

Compromised nuclear import of HOE-1 completely attenuates hoe-1(ΔNES)-induced UPR^mt.

Compromised mitochondrial import of HOE-1 exacerbates hoe-1(ΔNES)-induced UPR^mt.

Nuclear export defective HOE-1 activates UPR^mt in the intestine cell autonomously.

Nuclear export defective HOE-1 activates UPR^mt, correlating with reduced mitochondrial membrane potential.

Nuclear export defective HOE-1 animals have increased nuclear accumulation of UPR^mt transcription factors ATFS-1 and DVE-1.

Nuclear export defective HOE-1 activates UPR^mt via altered tRNA processing.

Nuclear export defective HOE-1 induced UPR^mt is dependent upon the catalytic activity of HOE-1.

RNAi against RNA polymerase III subunit, rpc-1, preferentially attenuates hoe-1(ΔNES)-induced UPR^mt.

RNAi against tRNA nucleotidyl transferase, hpo-31, and tRNA ligase, rtcb-1, mildly attenuate both hoe-1(ΔNES)- and nuo-6(qm200)-induced UPR^mt.

Nuclear export defective HOE-1 induced UPR^mt is not gcn-2 or eIF2α dependent.

UPR^mt-inducing cco-1 and spg-7 RNAi both attenuate HOE-1 nuclear levels.

Constitutive activation of UPR^mt by atfs-1 gain-of-function (atfs-1(et15)) depletes nuclear HOE-1 levels.