Eukaryotic initiation factor EIF-3.G augments mRNA translation efficiency to regulate neuronal activity
- Section of Neurobiology, Division of Biological Sciences, University of California San Diego, United States
Figures
Figure 1 with 3 supplements
eif-3.G(C130Y) suppresses acr-2(gf) convulsion behavior in the cholinergic motor neurons.
(A) Illustration of the genomic locus of eif-3.G: Peif-3.G denotes the promoter, blue boxes are exons for coding sequences and gray for 3′UTR. Arrowhead indicates guanine to adenine change in ju807; and short line below represents a 19 bp deletion in ju1327, designated eif-3.G(0), that would shift the reading frame at aa109, resulting in a premature stop (asterisk) after addition of 84aa of no known homology. (B) Illustration of EIF-3.G: shaded blue represents EIF-3.I binding region, ZF for Zinc Finger, RRM for RNA Recognition Motif. Below is a multi-species alignment of the zinc finger domain with bold residues as the CCHC motif and gray for conserved residues. ju807 causes a C130Y substitution (black arrow). C127Y (red arrow, ju1840) was generated with CRISPR editing. C. elegans (C. e.; NP_001263666.1), S. cerevisiae (S. c.; NP_010717.1), D. melanogaster (D. m.; NP_570011.1), X. laevis (X. l.; NP_001087888.1), and H. sapiens (H.s.; AAC78728.1). (C) Quantification of convulsion frequencies of animals of indicated genotypes, with the strains (left to right) as: N2, MT6241, CZ21759, CZ28495, CZ21759, CZ22977. Ex[eif-3.G(C130Y)] transgenes (juEx7015/juEx7016) expressed full-length genomic DNA cloned from eif-3.G(ju807). (D) Illustration of eif-3.G expression constructs: top shows the transgene expressing genomic eif-3.G(+ for wild type and C130Y for ju807) with the endogenous eif-3.G promoter and 3′UTR, and coding exons in blue; bottom shows cell-type expression of eif-3.G cDNA driven by tissue-specific promoters (Pmyo-3- body muscle, Punc-25- GABAergic motor neurons, Punc-17β - cholinergic motor neurons). (E) Quantification of convulsion frequencies shows that convulsion behavior of eif-3.G(C130Y); acr-2(gf) double mutants is rescued by transgenes that overexpress eif-3.G(+) genomic DNA or an eif-3.G(+) cDNA in the ACh-MNs, but not in the GABAergic motor neurons or body muscle. Strains (left to right)- N2, CZ21759, CZ23125/ CZ23126, CZ22980/ CZ22981, CZ23791/ CZ23880, CZ22982/ CZ22983, CZ27881/ CZ27882. (F) Quantification of convulsion frequencies in animals of the indicated genotypes (left to right)- N2, CZ22917, MT6241, CZ21759, CZ28495, CZ21759, CZ21759, CZ23310, CZ26828. Data in (D-F) are shown as mean ± SEM and sample size is indicated within or above each bar. Statistics: (***) p<0.001, (ns) not significant by one-way ANOVA with Bonferroni’s post hoc test.
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Figure 1—source data 1
Source data for Figure 1C.
Quantification of convulsions per 60 s in strains of the indicated genotypes.
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Figure 1—source data 2
Source data for Figure 1E.
Quantification of convulsions per 60 s in the indicated strains.
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Figure 1—source data 3
Source data for Figure 1F.
Quantification of convulsions per 60 s in strains. Strain name or genotype is indicated in top row.
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Figure 1—figure supplement 1
EIF-3.G is highly conserved and expressed ubiquitously.
(A) Clustal-W sequence alignment of C. elegans EIF-3.G (NP_001263666.1) with the S. cerevisiae (TIF-35; 32% identity; NP_010717.1) and human (eIF3g, 35% identity; AAC78728.1) orthologs. Residues identical to C. elegans EIF-3.G are shaded gray. The EIF-3.I binding region, zinc finger, RRM and RNP motifs are indicated below the corresponding sequences. The frame-shift caused by the ju1327 deletion adds 85aa’s starting from the position marked by the purple arrow. The C130Y mutation (red asterisk), R-F-F residues changed to alanine in our RFF/AAA transgene construct (red boxes), and location of the Q191* mutation used to generate EIF-3.G(∆RRM) (blue arrow head) are shown. (B) Representative maximum intensity z-stack confocal images of L4 stage animals expressing GFP::EIF-3.G(WT) or GFP::EIF-3.G(C130Y) under the Peif-3.G promoter. GFP is visualized throughout C. elegans tissues and excluded from the gonads due to germline transgene silencing. The bright punctae in the animal midbody are autofluorescent gut granules. Scale bar = 100 µm.
Figure 1—figure supplement 2
Motor neuron development is normal in eif-3.G(C130Y) animals.
(A) Representative confocal z-stack projection images of axonal commissures projecting from motor neurons of wild type or eif-3.G(C130Y) single mutant L4 animals (head to the right). The quantification of axon commissure numbers in strains is shown on the right. (B) Single plane confocal images of L4 animals (head to the left) expressing GFP::SNB-1, which appear as puncta along the ventral nerve cord. Puncta quantification, shown at right, was performed in a 30 µm region anterior of the vulva (red box) in each animal. For (A) and (B), the number of animals used in quantification data is indicated in each bar and error bars represent ± SEM. Scales bar = 10 µm.
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Figure 1—figure supplement 2—source data 1
Source data for Figure 1—figure supplement 2A.
Quantification of axonal commissures in strains of the indicated genotypes.
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Figure 1—figure supplement 2—source data 2
Source data for Figure 1—figure supplement 2B.
Quantification of synaptic puncta in strains of the indicated genotypes.
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Figure 1—figure supplement 3
EIF-3.G(C130Y) modulation of convulsion behavior does not involve reduced EIF-3 complex dosage or ACR-2 expression.
(A) Quantification of convulsion frequencies in animals of the indicated genotypes; the corresponding strains (left to right) are: MT6241, CZ27434, CZ27435, CZ21759, CZ27433, CZ27436. Error bars are ± SEM and n = 15 per sample. (ns) not significant by one-way ANOVA with Bonferroni’s post-hoc test. (B) Representative images of L4 animals expressing ACR-2(WT)::GFP in the ventral nerve chord region anterior to the vulva. Quantification of fluorescence in animals (n = 8) is shown on the right. Scale bar = 10 µm. (ns) not significant by two-tailed t-test.
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Figure 1—figure supplement 3—source data 1
Source data for Figure 1—figure supplement 3A.
Quantification of convulsions per 60 s in strains of the indicated genotypes.
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Figure 1—figure supplement 3—source data 2
Source data for Figure 1—figure supplement 3B.
Quantification of relative fluorescence intensity in the indicated strains.
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Figure 2
eif-3.G(C130Y) involves a selective function of EIF-3.G on translational control.
(A) Quantification of convulsion frequency in animals expressing GFP::EIF-3.G(WT) or GFP::EIF-3.G(C130Y) under Peif-3.G in the indicated genetic backgrounds; and the strains (left to right) are: MT6241, CZ24729, CZ24652, CZ28497, CZ21759, CZ28107. Error bars represent ± SEM with n = 15 per sample. (***) P< 0.001, (ns) not significant, by one-way ANOVA with Bonferroni’s post-hoc test. (B) EIF-3.G(WT) and EIF-3.G(C130Y) show comparable expression in ACh-MNs. Left are representative single-plane confocal images of EIF-3.G(WT)::GFP or EIF-3.G(C130Y)::GFP driven by the Peif-3.G promoter as single-copy transgenes in L4 animals (head to the left). Red circles mark the soma of VA10, VB11, and DB7 ACh-MN, based on co-expressing a Pacr-2-mcherry marker. Scale bar = 4 µm. Right: Mean GFP fluorescence intensities (AU) in ACh-MN soma in animals of the indicated genotypes (n = 8). Each data point represents the mean intensity from VA10, VB11, and DB7 neurons in the same animal and normalized to the mean GFP::EIF-3.G intensity in a wildtype background. Error bars represent ± SEM; (ns) not significant by one-way ANOVA with Sidak’s multiple comparisons test. (C) Representative polysome profile traces from total mRNA-protein extracts of wild type and eif-3.G(C130Y) single mutant animals. Vertical lines (marked by *) within traces indicate the boundaries of fraction collection. (D) Polysome::monosome (P/M) ratios calculated based on the area under the respective curves for polysomal and monosome (80S) fractions using two replicates of polysome profiles from total extracts of indicated genotypes. (ns) not significant by one-way ANOVA with Bonferroni’s post-hoc test.
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Figure 2—source data 1
Source data for Figure 2A.
Quantification of convulsions per 60 s in the indicated strains.
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Figure 2—source data 2
Source data for Figure 2B.
Quantification of relative fluorescence intensity in the indicated strains.
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Figure 2—source data 3
Source data for Figure 2C.
Quantification of polysome to monosome ratios in wildtype(N2) and strains of the indicated genotypes.
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Figure 3
eif-3.G(C130Y) requires the RNA-binding domain (RRM) to suppress acr-2(gf) behaviors.
Top illustration of the EIF-3.G protein showing the EIF-3.I binding region (blue), zinc finger (ZF), RRM (dark grey), Q191* mutation in the EIF-3.G(∆RRM) transgene, RNP motifs (purple), and the RFF residues (bold dark blue) changed to alanine in the eif-3.G(RFF/AAA) construct. Below is an illustration of C. elegans EIF-3.I pointing to the position of E252R within the fourth WD40 domain. Bottom graph is quantification of convulsion frequency in acr-2(gf) animals expressing eif-3.G and eif-3.I variants in the nervous system (Prgef-1). The strains (left to right) are: MT6241, CZ23203/ CZ23204, CZ28152/ CZ28153, CZ23304/ CZ23305, CZ28152/ CZ28153, CZ28057/ CZ28058, CZ28064/ CZ28065. Bars represent mean convulsion frequency ± SEM and sample sizes are indicated within or above bars. (***) p< 0.001, (ns) not significant, by one-way ANOVA with Bonferroni’s post-hoc test.
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Figure 3—source data 1
Source data for Figure 3.
Quantification of convulsions per 60 s in the indicated strains.
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Figure 4 with 2 supplements
Both EIF-3.G(WT) and EIF-3.G(C130Y) associate with mRNA 5′UTRs in the cholinergic motor neurons.
(A) Pie charts displaying the proportion of EIF-3.G(WT) and EIF-3.G(C130Y) footprints located within each gene feature. (B) seCLIP read density track of EIF-3.G(WT) and EIF-3.G(C130Y) footprints on the 5′UTR of egl-30, compared to the EIF-3.G(∆RRM) control. (C) Scatter plot comparing the signal intensity, in reads per million (RPM), of all 231 5′UTR proximal footprints detected in EIF-3.G(WT) or EIF-3.G(C130Y). (D) Plots show the cumulative coverage of all 5′UTR proximal (top) or 3′UTR (bottom) footprints of EIF-3.G(WT) or EIF-3.G(C130Y) relative to the start codon (top) or stop codon (bottom) position. Coverage is presented as reads per million (RPM). (E–F) Box plots comparing length and GC-content of all 5′UTR sequences of EIF-3.G target mRNAs with annotations (n = 179) to all 5′UTRs in the acr-2(gf) cholinergic neuronal transcriptome (n = 4573). Boxes are 5–95 percentile with outliers aligned in red. Statistics: (***) p< 0.001, (**) p< 0.01 by two-tailed Mann-Whitney test.
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Figure 4—source data 1
Source data for Figure 4A.
Number of read clusters representing footprints of EIF-3.G(WT) or EIF-3.G(C130Y) mapping to 5′UTR proximal or 3′UTR regions.
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Figure 4—source data 2
Source data for Figure 4C.
seCLIP reads for EIF-3.G(WT) and EIF-3.G(C130Y) 5’UTR proximal footprints represented as log2(reads per million).
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Figure 4—source data 3
Source data for Figure 4D.
Cumulative EIF-3.G(WT) and EIF-3.G(C130Y) footprint coverage per base distance from the start codon (5’UTR proximal footprints) or stop codon (3’UTR footprints) represented as reads per million.
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Figure 4—source data 4
Source data for Figure 4E.
Length of 5’UTRs in mRNAs expressed in the ACh-MN transcriptome and EIF-3.G targets.
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Figure 4—source data 5
Source data for Figure 4F.
Percent GC of 5’UTRs in mRNAs expressed in the ACh-MN transcriptome and EIF-3.G targets.
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Figure 4—figure supplement 1
EIF-3.G transgenes are expressed and produce similar results from replicate seCLIP experiments.
(A) Western blotting from strains expressing each indicated 3xFLAG-tagged EIF-3.G transgene in the ACh-MNs under the Punc-17β promoter. Quantification of expression intensity relative to the actin control is shown to the right. Statistics: (*) P< 0.05, (ns) not significant, one-way Anova with Bonferroni’s post hoc test. (B) Scatter plots comparing the cumulative RPM (reads per million mapped reads) mapped in genes detected between seCLIP replicates. Each dot represents a unique gene and RPM values are the total reads for all clusters mapped within the respective gene. p-Values are derived from a two-tailed Pearson’s correlation (r2). The number of genes per dataset (n) and linear fit (red line) are shown.
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Figure 4—figure supplement 1—source data 1
Source data for Figure 4—figure supplement 1A.
Quantification of band intensities from western blots using the indicated antibodies in each strain.
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Figure 4—figure supplement 1—source data 2
Source data for Figure 4—figure supplement 1B.
Number of EIF-3.G(WT), EIF-3.G(C130Y), or EIF-3.G(∆RRM) seCLIP reads mapping to each indicated gene.
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Figure 4—figure supplement 2
EIF-3.G associates with long and GC-rich 5′UTRs.
(A) Pie charts show the proportion of trans-splicing (ts) among genes with 5′UTR proximal EIF-3.G footprints according to Allen et al. Unannotated genes were absent from the Allen et al. dataset. (B) Box plots compare the lengths of non-trans-spliced (blue) or trans-spliced (red) 5′UTR sequences of EIF-3.G target mRNAs (non-trans-spliced n = 57, trans-spliced n = 133) to all 5′UTRs in the C. elegans transcriptome (WS271; non-trans-spliced n = 5,970, trans-spliced n = 6,674). Boxes are 10–90 percentile with outliers in red. (C) Heat map shows 5′UTR %GC along the first 150 nts from the start codon in 10 nt bins of EIF-3.G target genes. Only genes with a 5′UTR of at least 150 nts (n = 49) are shown. Genes are ordered by one-minus Pearson hierarchical clustering. Asterisks point to example 5′UTRs with relative GC-rich sequence near the start codon (red) or closer to the distal 5′ end (blue). (D) and (E) Box plots comparing (D) length or (E) GC-content of 5′UTR sequences of HEK293 eIF3g target mRNAs (n = 255; Lee et al., 2016) versus all human transcriptome 5′UTRs (hg38; n = 19,914). Boxes are 5–95 percentile with outliers in red. For (B, D, and E) Statistics: (***) p< 0.001, (**) p< 0.01, (ns) not significant, two-tailed Mann-Whitney test.
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Figure 4—figure supplement 2—source data 1
Source data for Figure 4—figure supplement 2A.
Number of EIF-3.G target genes exhibiting trans-splicing.
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Figure 4—figure supplement 2—source data 2
Source data for Figure 4—figure supplement 2B.
Length of 5’UTRs among trans-spliced or non-trans-spliced mRNAs expressed in the C. elegans transcriptome or EIF-3.G target mRNAs.
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Figure 4—figure supplement 2—source data 3
Source data for Figure 4—figure supplement 2C.
Percent GC in 10nt bins up to 150nt from the start codon for each indicated gene.
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Figure 4—figure supplement 2—source data 4
Source data for Figure 4—figure supplement 2D.
Length of 5’UTRs among mRNAs expressed in the human(hg38) transcriptome or human eIF3 target mRNAs.
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Figure 4—figure supplement 2—source data 5
Source data for Figure 4—figure supplement 2E.
Percent GC in 5’UTRs among mRNAs expressed in the human(hg38) transcriptome or human eIF3 target mRNAs.
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Figure 5
Gene network analyses of EIF-3.G target mRNAs show enrichment in activity-dependent expression.
(A) Cytoscape network of EIF-3.G target genes with enriched GO terms (neuropeptide signaling, response to stress, and protein translation and protein metabolism) or KEGG pathways (calcium and synaptic signaling, metabolic components, MAPK-signaling, and mRNA surveillance). Enrichment p-values are derived from statistical analysis of our EIF-3.G targets (n = 225) in the PANTHER database (Mi et al., 2019). (B) EIF-3.G target genes exhibiting significant transcript level changes in acr-2(gf) versus wild-type animals as determined from transcriptome sequencing of cholinergic neurons by McCulloch et al. PT and PM refers to protein translation and protein metabolism. Differential expression was assessed using DeSeq2 (Love et al., 2014) with significance thresholds of (*) p<0.05 and (**) p<0.01.
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Figure 5—source data 1
Source data for Figure 5B.
Foldchange in transcript expression in the cholinergic neuronal transcriptome of acr-2(gf) and wild-type animals.
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Figure 6 with 1 supplement
EIF-3.G(C130Y) impairs HLH-30 expression in ACh-MNs of acr-2(gf) animals.
(A) Gene models of hlh-30 isoforms a (pink), b (blue), and d (green), with presumptive promoters for each isoform depicted as right-pointing arrows and the 5′UTR of isoform d in green to the right of its promoter. (B) seCLIP read density tracks of footprints on the 5′ end of hlh-30 isoform b and d (left) and the 5′ end of hlh-30 isoform a (right) in each indicated EIF-3.G dataset. Purple arrows show footprints on the 5′UTR of hlh-30 isoform d. (C) Top: Illustration of the wgIs433 fosmid locus with hlh-30 coding exons in black and 5′UTR of isoform d in green to the right of the promoter. Bottom: Representative single-plane confocal images of the fosmid translational reporter wgIs433[hlh-30::EGFP::3xFLAG] in ACh-MNs in animals of indicated genotypes. Quantification of GFP intensity is shown on the right (n = eight for each genotype). Animals are oriented with anterior to the left. Scale bar = 4 µm. Red dashes indicate labeled ACh-MN soma. Each data point is the average fluorescence intensity quantified from the three ACh-MN soma per animal and normalized to the mean intensity obtained from wgIs433 in the wild type background. Statistics: (***) p< 0.001, (ns) not significant, one-way Anova with Bonferroni’s post hoc test.
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Figure 6—source data 1
Source data for Figure 6C.
Quantification of relative fluorescence intensity in strains of the indicated genotypes.
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Figure 6—figure supplement 1
EIF-3.G (C130Y) has no effect on translation of hlh-30.a in the ACh-MNs.
Representative single-plane confocal images of hlh-30a isoform-specific reporter (sqIs17, with the expression construct illustrated above) in ACh-MNs in animals of the indicated genotypes. Scale bar = 4 µm. Average GFP intensities quantified from the soma of ACh-MNs in animals (n = 8) expressing hlh-30a::GFP are shown to the right. Data are normalized to the mean hlh-30a::GFP fluorescence in a wild-type background. Red dashes indicate positions of labeled ACh-MN soma. Statistics: (***) p< 0.001, (ns) not significant, one-way Anova with Bonferroni’s post hoc test.
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Figure 6—figure supplement 1—source data 1
Source data for Figure 6—figure supplement 1.
Quantification of relative fluorescence intensity in strains of the indicated genotypes.
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Figure 7 with 1 supplement
Regulation of NCS-2 expression by EIF-3.G depends on its GC-rich 5′UTR.
(A) Illustration of the ncs-2 genomic region. Dark blue represents 5′UTR, green boxes are coding exons, and gray is the 3′UTR. The inset below shows the read density track of seCLIP footprints on the 5′ region of ncs-2 mRNA. (B) Top: Schematic of the NCS-2(cDNA)::GFP translation reporter, including its 5′UTR (dark blue), driven by the 4 kb promoter Pncs-2. The 5′UTR sequences are GC rich (purple). Bottom: Representative single-plane confocal images of NCS-2::GFP in ventral nerve chord processes in young adult animals of the indicated genotypes. GFP intensity quantification is shown to the right. (C) Top: The ncs-2(5′UTR mutant)::GFP translational reporter has the 5′UTR of eif-3.G (red boxed sequence) replacing the ncs-2 5′UTR, driven by Pncs-2. Bottom: Representative single-plane confocal images of ventral nerve chord processes expressing the NCS-2(5′UTR mutant)::GFP translation reporter in young adult animals of the indicated genotypes. GFP intensity quantification is shown to the right. For (B) and (C), data points are normalized to the average fluorescence intensity of the respective translation reporter in the wild-type background. ROIs used for fluorescence quantification are boxed. Scale bar = 15 µm. Statistics: (**) P< 0.01, (ns) not significant by one-way Anova with Bonferroni’s post hoc test.
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Figure 7—source data 1
Source data for Figure 7B.
Quantification of relative fluorescence intensity in strains of the indicated genotypes.
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Figure 7—source data 2
Source data for Figure 7C.
Quantification of relative fluorescence intensity in the indicated strains.
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Figure 7—figure supplement 1
EIF-3.G(C130Y) reduces NCS-2 expression in the ACh-MNs of acr-2(gf) animals dependent on its conserved 5′UTR.
(A) Plot shows conservation of nucleotides (by phyloP135way scores) in the 5′UTR sequences of ncs-2 (blue) and eif-3.G (red), with higher scores indicating greater conservation. Genomic position (x-axis) is relative to the start codon (AUG). The horizontal dashed line marks PhyloP score = 0. (B) Top: Schematic of the Pncs-2::GFP translation reporter containing the endogenous GC-rich (purple) ncs-2 5′UTR and the first 4aa of NCS-2 followed by GFP and driven by the Pncs-2 promoter. Bottom: Representative single-plane confocal images of Pncs-2::GFP expression in the VA10, VB11, DB7 soma in animals of the indicated genotypes. GFP intensity quantification are shown to the right (n = 8). (C) Top: The Pncs-2(5′UTR mutant)::GFP translation reporter contains the 5′UTR of eif-3.G (red, GC content is purple), followed by the ncs-2 CDS 5′ end. Bottom: Representative single-plane confocal images of Pncs-2(5′UTR mutant)::GFP in VA10, VB11, DB7 soma in the indicated genetic backgrounds. GFP intensity quantification are shown to the right (n = 8). For GFP quantification in panels (B) and (C), data points are normalized to the mean intensity of the respective reporter in the wild type background. Red dashes indicate positions of labeled ACh-MN soma. Scale bar = 15 µm. Statistics: (***) p< 0.001, (ns) not significant by one-way Anova with Bonferroni’s post hoc test.
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Figure 7—figure supplement 1—source data 1
Quantification of relative fluorescence intensity in the indicated strains.
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Figure 7—figure supplement 1—source data 2
Quantification of relative fluorescence intensity in the indicated strains.
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Videos
Video 1
N2 [Wild type] C. elegans movement on solid nematode growth media.
Video 2
MT6241 [acr-2(gf)] C. elegans movement on solid nematode growth media.
Video 3
CZ21759 [eif-3.G(C130Y); acr-2(gf)] C. elegans movement on solid nematode growth media.
Video 4
CZ22197 [eif-3.G(C130Y)] C. elegans movement on solid nematode growth media.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | anti-FLAG (Rabbit) | Sigma-Aldrich | Cat# F7425, RRID:AB_439687 | WB (1:2000) |
| Antibody | anti-Actin clone C4 (Mouse monoclonal) | MP Biomedicals | Cat# 08691002, RRID:AB_2335304 | WB (1:2000) |
| Antibody | Anti-FLAG M2 Magnetic Beads | Sigma-Aldrich | Cat# M8823, RRID:AB_2637089 | IP |
| Recombinant protein reagent | Cas9-NLS (purified protein) | UC Berkely QB3 | ||
| Genetic reagent (C. elegans) | + | CGC | RRID:CGC_N2 | |
| Genetic reagent (C. elegans) | acr-2(n2420) X | Jospin et al., 2009 | MT6241 | |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II | This work | CZ22197 | Figure 1F |
| Genetic reagent (C. elegans) | eif-3.G(ju1840) II | This work | CZ28494 | Figure 1C |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; acr-2(n2420) X | This work | CZ21759 | Figure 1C |
| Genetic reagent (C. elegans) | eif-3.G(ju1840) II; acr-2(n2420) X | This work | CZ28495 | Figure 1C |
| Genetic reagent (C. elegans) | eif-3.G(ju1327) / mnC1 II | This work | CZ22974 | Figure 1F |
| Genetic reagent (C. elegans) | acr-2(n2420) X; juEx7015 | This work | CZ22976 | Figure 1C |
| Genetic reagent (C. elegans) | acr-2(n2420) X; juEx7016 | This work | CZ22977 | Figure 1C |
| Genetic reagent (C. elegans) | eif-3.G(ju807) I;acr-2(n2420) X; juEx7045 | This work | CZ23125 | Figure 1E |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; acr-2(n2420) X; juEx7046 | This work | CZ23126 | Figure 1E |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; acr-2(n2420) X; juEx7019 | This work | CZ22980 | Figure 1E |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; acr-2(n2420) X; juEx7020 | This work | CZ22981 | Figure 1E |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; acr-2(n2420) X; juEx7439 | This work | CZ23791 | Figure 1E |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; acr-2(n2420) X; juEx7440 | This work | CZ23880 | Figure 1E |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; acr-2(n2420) X; juEx7021 | This work | CZ22982 | Figure 1E |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; acr-2(n2420) X; juEx7022 | This work | CZ22983 | Figure 1E |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; acr-2(n2420) X; juEx8062 | This work | CZ27881 | Figure 1E |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; acr-2(n2420) X; juEx8063 | This work | CZ27882 | Figure 1E |
| Genetic reagent (C. elegans) | eif-3.G(ju1327) /mnC1 II; acr-2(n2420) X | This work | CZ23310 | Figure 1F |
| Genetic reagent (C. elegans) | eif-3.G(ju807) / eif-3.G(ju1327) II | This work | CZ25714 | Figure 1F |
| Genetic reagent (C. elegans) | eif-3.G(ju807) / eif-3.G(ju1327) II; acr-2(n2420) X | This work | CZ26828 | Figure 1F |
| Genetic reagent (C. elegans) | juSi320 IV | This work | CZ24063 | Figure 2B; Figure 1—figure supplement 1B |
| Genetic reagent (C. elegans) | eif-3.G(ju1327) /mnC1 II; juSi320 IV | This work | CZ24079 | Figure 2A |
| Genetic reagent (C. elegans) | juSi320 IV; acr-2(n2420) X | This work | CZ24729 | Figure 2A |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; juSi320 IV; acr-2(n2420) X | This work | CZ28107 | Figure 2A |
| Genetic reagent (C. elegans) | juSi331 IV | This work | CZ24651 | Figure 2B; Figure 1—figure supplement 1B |
| Genetic reagent (C. elegans) | juSi331 IV; acr-2(n2420) X | This work | CZ24652 | Figure 2A |
| Genetic reagent (C. elegans) | eif-3.G(ju1327) / mnC1 II; juSi331 IV; acr-2(n2420) X | This work | CZ28497 | Figure 2A |
| Genetic reagent (C. elegans) | juIs14 IV | Wang et al., 2017 | CZ631 | |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; juIs14 IV | This work | CZ24161 | Figure 1—figure supplement 2 |
| Genetic reagent (C. elegans) | juIs14 IV; acr-2(n2420) X | McCulloch et al., 2020 | CZ5808 | |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; juIs14 IV; acr-2(n2420) X | This work | CZ8905 | Figure 1—figure supplement 2A |
| Genetic reagent (C. elegans) | nuIs94 | Hallam et al., 2000 | KP2229 | |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; nuIs94 | This work | CZ24021 | Figure 1—figure supplement 2B |
| Genetic reagent (C. elegans) | acr-2(n2420) X; nuIs94 | This work | CZ5815 | Figure 1—figure supplement 2B |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; acr-2(n2420)X; nuIs94 | This work | CZ24021 | Figure 1—figure supplement 2B |
| Genetic reagent (C. elegans) | eif-3.E(ok2607) I / hT2 I,III; acr-2(n2420) X | This work | CZ27434 | Figure 1—figure supplement 3A |
| Genetic reagent (C. elegans) | eif-3.E(ok2607) I / hT2 I, III; eif-3.G(ju807) II; acr-2(n2420) X | This work | CZ27433 | Figure 1—figure supplement 3A |
| Genetic reagent (C. elegans) | eif-3.H(ok1353) I / hT2 I, III; acr-2(n2420) X | This work | CZ27435 | Figure 1—figure supplement 3A |
| Genetic reagent (C. elegans) | eif-3.H(ok1353) I / hT2 I, III; eif-3.G(ju807) II; acr-2(n2420) X | This work | CZ27436 | Figure 1—figure supplement 3A |
| Genetic reagent (C. elegans) | oxSi39 IV | Qi et al., 2013 | CZ12338 | |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; oxSi39 IV | This work | CZ23854 | Figure 1—figure supplement 3B |
| Genetic reagent (C. elegans) | acr-2(n2420) X; juEx7056 | This work | CZ23203 | Figure 3 |
| Genetic reagent (C. elegans) | acr-2(n2420) X; juEx7057 | This work | CZ23204 | Figure 3 |
| Genetic reagent (C. elegans) | acr-2(n2420) X; juEx8100 | This work | CZ28152 | Figure 3 |
| Genetic reagent (C. elegans) | acr-2(n2420) X; juEx8101 | This work | CZ28153 | Figure 3 |
| Genetic reagent (C. elegans) | juEx7113 | This work | CZ26777 | Figure 3 |
| Genetic reagent (C. elegans) | acr-2(n2420) X; juEx7114 | This work | CZ23304 | Figure 3 |
| Genetic reagent (C. elegans) | acr-2(n2420) X; juEx7115 | This work | CZ23305 | Figure 3 |
| Genetic reagent (C. elegans) | acr-2(n2420) X; juEx8095 | This work | CZ28066 | Figure 3 |
| Genetic reagent (C. elegans) | acr-2(n2420) X; juEx8096 | This work | CZ28067 | Figure 3 |
| Genetic reagent (C. elegans) | acr-2(n2420) X; juEx8087 | This work | CZ28057 | Figure 3 |
| Genetic reagent (C. elegans) | acr-2(n2420) X; juEx8088 | This work | CZ28058 | Figure 3 |
| Genetic reagent (C. elegans) | acr-2(n2420) X; juEx8089 | This work | CZ28064 | Figure 3 |
| Genetic reagent (C. elegans) | acr-2(n2420) X; juEx8090 | This work | CZ28065 | Figure 3 |
| Genetic reagent (C. elegans) | unc-119(tm4063) III; wgIs433 | Sarov et al., 2006 | OP433 | |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; unc-119(tm4063)III; wgIs433 | This work | CZ28145 | Figure 6C |
| Genetic reagent (C. elegans) | acr-2(n2420) X; unc-119(tm4063) III; wgIs433 | This work | CZ27913 | Figure 6C |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; unc-119(tm4063) III; acr-2(n2420) X; wgIs433 | This work | CZ27914 | Figure 6C |
| Genetic reagent (C. elegans) | sqIs17 | Dittman and Kaplan, 2006 | MAH240 | |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; sqIs17 | This work | CZ28334 | Figure 6—figure supplement 1 |
| Genetic reagent (C. elegans) | acr-2(n2420) X; sqIs17 | This work | CZ28212 | Figure 6—figure supplement 1 |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; acr-2(n2420) X; sqIs17 | This work | CZ28218 | Figure 6—figure supplement 1 |
| Genetic reagent (C. elegans) | unc-13(s69) I; wgIs433 | This work | CZ28491 | Figure 6C |
| Genetic reagent (C. elegans) | unc-13(s69) I; acr-2(n2420) X; wgIs433 | This work | CZ28492 | Figure 6C |
| Genetic reagent (C. elegans) | unc-13(s69) I; eif-3.G(ju807); acr-2(n2420) X; wgIs433 | This work | CZ28493 | Figure 6C |
| Genetic reagent (C. elegans) | juSi260 ncs-2(tm1943) I | Zhou et al., 2017 | CZ22459 | |
| Genetic reagent (C. elegans) | juSi260 ncs-2(tm1943) I; eif-3.G(ju807) II | This work | CZ23225 | Figure 7B |
| Genetic reagent (C. elegans) | juSi260 ncs-2(tm1943) I; acr-2(n2420) X | This work | CZ22345 | Figure 7B |
| Genetic reagent (C. elegans) | juSi260 ncs-2(tm1943) I; eif-3.G(ju807) II; acr-2(n2420) X | This work | CZ28110 | Figure 7B |
| Genetic reagent (C. elegans) | juSi391 ncs-2(tm1943) I | This work | CZ28213 | Figure 7C |
| Genetic reagent (C. elegans) | juSi391 ncs-2(tm1943) I; eif-3.G(ju807) II | This work | CZ28340 | Figure 7C |
| Genetic reagent (C. elegans) | juSi391 ncs-2(tm1943) I;acr-2(n2420) X | This work | CZ28252 | Figure 7C |
| Genetic reagent (C. elegans) | juSi391 ncs-2(tm1943) I;eif-3.G(ju807) II; acr-2(n2420) X | This work | CZ28253 | Figure 7C |
| Genetic reagent (C. elegans) | juSi392 ncs-2(tm1943) I | This work | CZ28277 | Figure 7—figure supplement 1B |
| Genetic reagent (C. elegans) | juSi392 ncs-2(tm1943) I;eif-3.G(ju807) II | This work | CZ28312 | Figure 7—figure supplement 1B |
| Genetic reagent (C. elegans) | juSi392 ncs-2(tm1943) I; acr-2(n2420) X | This work | CZ28291 | Figure 7—figure supplement 1B |
| Genetic reagent (C. elegans) | juSi392 ncs-2(tm1943) I; eif-3.G(ju807) II; acr-2(n2420) X | This work | CZ28292 | Figure 7—figure supplement 1B |
| Genetic reagent (C. elegans) | juSi393 ncs-2(tm1943) I | This work | CZ28278 | Figure 7—figure supplement 1C |
| Genetic reagent (C. elegans) | juSi393 ncs-2(tm1943) I; eif-3.G(ju807) II | This work | CZ28311 | Figure 7—figure supplement 1C |
| Genetic reagent (C. elegans) | juSi393 ncs-2(tm1943) I; acr-2(n2420) X | This work | CZ28293 | Figure 7—figure supplement 1C |
| Genetic reagent (C. elegans) | juSi393 ncs-2(tm1943) I; eif-3.G(ju807) II; acr-2(n2420) X | This work | CZ28294 | Figure 7—figure supplement 1C |
| Genetic reagent (C. elegans) | juEx2045 | --- | CZ9635 | |
| Genetic reagent (C. elegans) | hlh-30(tm1978) IV | CGC | CZ23321 | |
| Genetic reagent (C. elegans) | hlh-30(tm1978) IV; acr-2(n2420) X | This work | CZ28174 | Related to Figure 6C |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; hlh-30(tm1978) IV; acr-2(n2420) X | This work | CZ28175 | Related to Figure 6C |
| Genetic reagent (C. elegans) | eif-3.G(ju1327) II /mnC1; juSi363 IV; acr-2(n2420) X | This work | CZ26759 | Related to Figure 4—figure supplement 1A |
| Genetic reagent (C. elegans) | eif-3.G(ju1327) II / mnC1 II; juSi366 IV; acr-2(n2420) X | This work | CZ26760 | Related to Figure 4—figure supplement 1A |
| Genetic reagent (C. elegans) | juSi364 IV; acr-2(n2420) X | This work | CZ26494 | Figure 4—figure supplement 1A |
| Genetic reagent (C. elegans) | eif-3.G(ju807)II juSi364 IV; acr-2(n2420) X | This work | CZ26243 | Related to Figure 4—figure supplement 1A |
| Genetic reagent (C. elegans) | juSi365 IV | This work | CZ26588 | Related to Figure 4—figure supplement 1A |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; juSi365 IV; acr-2(n2420) X | This work | CZ26565 | Figure 4—figure supplement 1A |
| Genetic reagent (C. elegans) | juSi365 IV; acr-2(n2420) X | This work | CZ26566 | Related to Figure 4—figure supplement 1A |
| Genetic reagent (C. elegans) | juSi368 IV | This work | CZ26656 | Related to Figure 4—figure supplement 1A |
| Genetic reagent (C. elegans) | juSi368 IV; acr-2(n2420) X | This work | CZ26623 | Figure 4—figure supplement 1A |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; juSi368 IV; acr-2(n2420) X | This work | CZ26480 | Related to Figure 4—figure supplement 1A |
| Genetic reagent (C. elegans) | wgIs506 | Sarov et al., 2006 | OP506 | Supplementary file 1 |
| Genetic reagent (C. elegans) | acr-2(n2420) X; wgIs506 | This work | CZ27926 | Supplementary file 1 |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; acr-2(n2420) X; wgIs506 | This work | CZ27927 | Supplementary file 1 |
| Genetic reagent (C. elegans) | dhc-1::GFP(it45) I | Lapierre et al., 2013 | OD2955 | Supplementary file 1 |
| Genetic reagent (C. elegans) | dhc-1::GFP(it45) I; acr-2(n2420) X | This work | CZ27858 | Supplementary file 1 |
| Genetic reagent (C. elegans) | dhc-1::GFP(it45) I; eif-3.G(ju807) II; acr-2(n2420) X | This work | CZ27859 | Supplementary file 1 |
| Genetic reagent (C. elegans) | wgIs432 | Sarov et al., 2006 | OP432 | Supplementary file 1 |
| Genetic reagent (C. elegans) | acr-2(n2420) X; wgIs432 | This work | CZ27915 | Supplementary file 1 |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; acr-2(n2420) X; wgIs432 | This work | CZ28021 | Supplementary file 1 |
| Genetic reagent (C. elegans) | wgIs638 | Sarov et al., 2006 | OP638 | Supplementary file 1 |
| Genetic reagent (C. elegans) | unc-119(tm4063) III;acr-2(n2420) X; wgIs638 | This work | CZ28108 | Supplementary file 1 |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; acr-2(n2420) X; wgIs638 | This work | CZ27916 | Supplementary file 1 |
| Genetic reagent (C. elegans) | let-607(tm1423) I; unc-119(ed3) III; vrIs121 | Sarov et al., 2006 | YL651 | Supplementary file 1 |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; let-607(tm1423) I; unc-119(ed3) III; vrIs121 | This work | CZ28143 | Supplementary file 1 |
| Genetic reagent (C. elegans) | acr-2(n2420) X; let-607(tm1423) I;unc-119(ed3) III; vrIs121 | This work | CZ28119 | Supplementary file 1 |
| Genetic reagent (C. elegans) | eif-3.G(ju807) II; acr-2(n2420) X; let-607(tm1423) I; unc-119(ed3) III; vrIs121 | This work | CZ28111 | Supplementary file 1 |
| Genetic reagent (C. elegans) | juIs172 | CGC | EE86 | |
| Genetic reagent (C. elegans) | egl-30(md186) I;dpy-20(e1282ts) IV; syIs105 | CGC | PS4263 | |
| Genetic reagent (C. elegans) | juEx7964 | McCulloch et al., 2020 | CZ27420 | |
| Genetic reagent (C. elegans) | acr-2(n2420) X; juEx7964 | McCulloch et al., 2020 | CZ27217 | |
| Genetic reagent (C. elegans) | eif-3.G(C130Y) II; acr-2(n2420) X; juEx7964 | This work | CZ28109 | Supplementary file 1 |
| Recombinant DNA reagent (plasmid) | pCZGY2729 | Andrusiak et al., 2019 | RRID:Addgene_135096 | Site-specific insertion using CRISPR/Cas9 editing of C. elegans ChrIV |
| Recombinant DNA reagent (plasmid) | pCZGY2750 | Andrusiak et al., 2019 | RRID:Addgene_135094 | Expresses Cas9 and sgRNA for editing of C. elegans ChrIV |
| Recombinant DNA reagent (plasmid) | pCZGY2727 | This work | Site-specific insertion using CRISPR/Cas9 editing of C. elegans ChrI | |
| Recombinant DNA reagent (plasmid) | pCZGY2748 | This work | Expresses Cas9 and sgRNA for editing of C. elegans ChrI |
Additional files
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Supplementary file 1
Strains used in this study.
- https://cdn.elifesciences.org/articles/68336/elife-68336-supp1-v2.docx
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Supplementary file 2
Genotyping primers used in this study.
- https://cdn.elifesciences.org/articles/68336/elife-68336-supp2-v2.docx
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Supplementary file 3
Constructs and related primers used in this study.
- https://cdn.elifesciences.org/articles/68336/elife-68336-supp3-v2.docx
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Supplementary file 4
Number of mapped reads in seCLIP replicate datasets obtained after sequencing and CLIPPER filtering.
- https://cdn.elifesciences.org/articles/68336/elife-68336-supp4-v2.docx
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Supplementary file 5
Number of read clusters detected in each dataset after subtraction of IgG control background.
- https://cdn.elifesciences.org/articles/68336/elife-68336-supp5-v2.docx
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Supplementary file 6
Number of EIF-3.G footprints detected in each dataset after subtraction of background from both IgG and ∆RRM controls.
- https://cdn.elifesciences.org/articles/68336/elife-68336-supp6-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/68336/elife-68336-transrepform-v2.pdf
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Eukaryotic initiation factor EIF-3.G augments mRNA translation efficiency to regulate neuronal activity
eLife 10:e68336.
https://doi.org/10.7554/eLife.68336
