PUS7 deficiency in human patients causes profound neurodevelopmental phenotype by dysregulating protein translation

Abstract

Protein translation is a highly regulated process involving the interaction of numerous genes on every component of the protein translation machinery. Upregulated protein translation is a hallmark of cancer and is implicated in autism spectrum disorder, but the risks of developing each disease do not appear to be correlated with one another. In this study we identified two siblings from the NIH Undiagnosed Diseases Program with loss of function variants in PUS7, a gene previously implicated in the regulation of total protein translation. These patients exhibited a neurodevelopmental phenotype including autism spectrum disorder in the proband. Both patients also had features of Lesch-Nyhan syndrome, including hyperuricemia and self-injurious behavior, but without pathogenic variants in HPRT1. Patient fibroblasts demonstrated upregulation of protein synthesis, including elevated MYC protein, but did not exhibit increased rates of cell proliferation. Interestingly, the dysregulation of protein translation also resulted in mildly decreased levels of HPRT1 protein suggesting an association between dysregulated protein translation and the LNS-like phenotypic findings. These findings strengthen the correlation between neurodevelopmental disease, particularly autism spectrum disorders, and the rate of protein translation.

Keywords: Neurodevelopmental disease; PUS7; Protein translation.

Copyright © 2022. Published by Elsevier Inc.

Figures

Figure 1.. Clinical presentation of PUS7 deficient patients

A) Both siblings showed mild facial dysmorphology. B) Family pedigree with results of segregation analysis for PUS7 variants. C) Table of significant clinical findings.

Figure 2.. Loss of function variants in PUS7 increases total protein translation

A) RT-PCR of RNA extracted from skin fibroblasts demonstrates the presence of a smaller splice form of PUS7 not detected in control. B) Sanger sequencing of RT-PCR products across the site of T387M shows that the majority of stable PUS7 mRNA is derived from the paternal allele. C) qRT-PCR of total RNA extracted from skin fibroblasts of the two patients, two age matched controls, and both parents demonstrates decreased PUS7 mRNA in individuals carrying the c.398+1 G>T splice variant (N = 9 for each sample); bar plots represent mean ± SEM. D) Western blots of total protein lysates purified from patient fibroblasts demonstrate that patients have decreased PUS7 protein quantity than controls and increased rate of protein translation relative to controls as measured by puromycin incorporation. E) Quantification of SUnSET assays conducted in fibroblasts (N = 4 for each sample); bar plots represent mean ± SEM. F) Combined analysis of SUnSET assay comparing control to patient fibroblast demonstrates statistically significant elevation of puromycin signal in patient cells (p = 2.1×10−3); bars represent mean value.

Figure 3.. Dysregulation of protein translation does not affect cell proliferation because regulation of gene transcription remains active

A) Western blot of whole cell lysates from patient and control fibroblasts demonstrates increased expression of MYC and decreased expression of HPRT1 in patient cells relative to controls without alterations to 4EBP1 or phosphorylated 4EBP1. B) Quantification of MYC and HPRT1 protein in control and patient fibroblasts (N = 3 for each sample) demonstrates elevation of MYC (p = 4.9×10−4) and decrease of HPRT1 protein (p = 3.5×10−3); bars represent mean value of each sample. C) Measurements of proliferation of patient and control fibroblasts shows no difference in cell growth (N = 3 for each measurement); error bars represent ± SEM. D) Western blots of whole cell lysates extracted from fibroblasts grown at different densities show that MYC expression is elevated in patient cells but decreases with increasing cell density. Phosphorylated 4EBP1 increases with increasing cell density E) qRT-PCR of demonstrates similar levels of MYC and HPRT1 mRNA between patient and control fibroblasts cells (N = 6); bars represent mean values ± SEM.

Figure 4.. PUS7 activity is sufficient to drive molecular findings in HeLa cells

A) Sanger sequencing of genomic DNA extracted from CRISPR induced PUS7 knockout in HeLa cells demonstrates cells are homozygous for a 34 bp deletion at the 3’ end of exon 10 extending into intron 10. B) RT-PCR from total RNA extracted from PUS7 KO cells demonstrating KO cells have no detectable PUS7 mRNA. C) Western blot of whole cell lysates from KO cells demonstrating that cells do not generate detectable stable protein and that protein translation is increased in KO cells. D) Immunocytochemistry of HeLa and KO cells demonstrates lack of PUS7 protein in KO cells. Transient transfection with plasmid expressing PUS7 cDNA demonstrates that expressed protein localizes to the nucleus. Top row: DAPI only, Middle row: anti-PUS7 IHC, Bottom row: merge E) Western blot of whole cell lysates purified from KO cells transiently transfected with PUS7 expression plasmid demonstrates significant overexpression compared to parental HeLa cells. Plasmid expressing PUS7 cDNA is able to decrease puromycin incorporation and decrease MYC protein levels relative to untransfected KO cells.