Elongator and codon bias regulate protein levels in mammalian peripheral neurons

Abstract

Familial dysautonomia (FD) results from mutation in IKBKAP/ELP1, a gene encoding the scaffolding protein for the Elongator complex. This highly conserved complex is required for the translation of codon-biased genes in lower organisms. Here we investigate whether Elongator serves a similar function in mammalian peripheral neurons, the population devastated in FD. Using codon-biased eGFP sensors, and multiplexing of codon usage with transcriptome and proteome analyses of over 6,000 genes, we identify two categories of genes, as well as specific gene identities that depend on Elongator for normal expression. Moreover, we show that multiple genes in the DNA damage repair pathway are codon-biased, and that with Elongator loss, their misregulation is correlated with elevated levels of DNA damage. These findings link Elongator's function in the translation of codon-biased genes with both the developmental and neurodegenerative phenotypes of FD, and also clarify the increased risk of cancer associated with the disease.

Fig. 1

The wobble uridine (U₃₄) of tRNA molecules that recognize both AA- and AG-ending codons for Lys, Gln, and Glu, is modified by the addition of both a thiol (s²) and a methoxy-carbonyl-methyl (mcm⁵). This double modification enhances the translational efficiency of AA-ending codons

Fig. 2

Expression of codon-biased reporter constructs in CKO neurons. a Fluorescent and transmitted images of control and CKO neurons expressing AA- and AG-biased eGFP. b Quantification of corrected total cell fluorescence (CTCF); P-values: AG-biased versus AA-biased: black asterisk, *control = 0.011; *blue asterisk, *CKO = < 0.001. AA-biased control versus AA-biased CKO = 0.89; *pink asterisk, *AG-biased control versus AG-biased CKO = 0.008; error bars denote SEM; scale bar: 6 μm

Fig. 3

AA:AG ratio and transcript size dictate Elongator dependence. a Plotting the number of genes as a function of AA:AG ratio shows that 84.1% of mouse and 74.2% of human coding sequences preferentially use AG-ending codons (AA:AG ratio < 1) for Lys, Gln, and Glu. Each x axis value represents a minimum of one gene. b–e Proteome comparison of normally transcribed DRG genes from E17.5 control and CKO embryos. Transcript size in number of codons is indicated by line color. b, c In the absence of Elongator, the percentage of proteins that are under-expressed (fold change ≤ − 2) increases with increasing AA bias and transcript length b, whereas the percentage expressed normally (fold change < 2 and > − 2) decreases c. d AA abundance in the absence of AA bias does not impact protein levels. e With increasing AG bias and decreasing transcript length, the percentage of proteins that are upregulated (fold change ≥ 2) increases. Dashed lines represent data calculated from fewer than five proteins. For numerical data and individual proteins, see Supplementary Data 2

Fig. 4

Depleted levels of BRCA2 are correlated with elevated levels of DNA damage. a RT-qPCR confirms normal levels of Brca2 transcript (P = 0.06). b–d Quantitative immunohistochemistry confirms decreased levels of BRCA2 protein (P < 0.001). e–g Longer comet tails in the CKO show fragmented DNA (P = 0.009); error bars denote SD in a and SEM in d, g; scale bar: b, c 40 μm; e, f 12 μm

Fig. 5

Multiple H2A histones are upregulated in Elongator CKO embryos. a RT-qPCR confirms normal transcription of Hist2H2aa (P = 0.26) and Hist1H2ab (P = 0.81). b–g Quantitative immunohistochemistry using an anti-HistH2A primary antibody confirms increased levels of H2A type histones (P < 0.0001); error bars denote SD in a and SEM in d; scale bar: 20 μm

Fig. 6

Significantly enriched GO biological processes for genes that are strong candidates for Elongator regulation. Green circles represent downregulated proteins encoded by genes with ≥ 1,005 codons and an AA:AG ratio > 1.3. Red circles represent upregulated proteins encoded by genes with ≤ 300 codons and an AA:AG ratio < 0.3. % Numbers indicate the percentage of proteins within a given GO category that are misregulated. Yellow bars represent the – log10 adjusted P-value. *All genes were transcribed normally