Adenylosuccinate lyase (ADSL) catalyzes two steps in de novo purine synthesis (DNPS). Mutations in ADSL can result in inborn errors of metabolism characterized by developmental delay and disorder phenotypes, with no effective treatment options. Recently, SAICAR, a metabolic substrate of ADSL, has been found to have alternative roles in the cell, complicating the role of ADSL. crADSL, a CRISPR KO of ADSL in HeLa cells, was constructed to investigate DNPS and ADSL in a human cell line. Here we employ this cell line in an RNA-seq analysis to initially investigate the effect of DNPS and ADSL deficiency on the transcriptome as a first step in establishing a cellular model of ADSL deficiency. We report transcriptome changes in genes relevant to development, vascular development, muscle, and cancer biology, which provide interesting avenues for future research.

In de novo purine biosynthesis (DNPS), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (EC 2.1.2.3)/inosine monophosphate cyclohydrolase (EC 3.5.4.10) (ATIC) catalyzes the last two reactions of the pathway: conversion of 5-aminoimidazole-4-carboxamide ribonucleotide [aka Z -nucleotide monophosphate (ZMP)] to 5-formamido-4-imidazolecarboxamide ribonucleotide (FAICAR) then to inosine monophosphate (IMP). Mutations in ATIC cause an untreatable and devastating inborn error of metabolism in humans. ZMP is an adenosine monophosphate (AMP) mimetic and a known activator of AMP-activated protein kinase (AMPK). Recently, a HeLa cell line null mutant for ATIC was constructed via CRISPR-Cas9 mutagenesis. This mutant, crATIC, accumulates ZMP during purine starvation. Given that the mutant can accumulate ZMP in the absence of treatment with exogenous compounds, crATIC is likely an important cellular model of DNPS inactivation and ZMP accumulation. In the current study, we characterize the crATIC transcriptome versus the HeLa transcriptome in purine-supplemented and purine-depleted growth conditions. We report and discuss transcriptome changes with particular relevance to Alzheimer's disease and in genes relevant to lipid and fatty acid synthesis, neurodevelopment, embryogenesis, cell cycle maintenance and progression, extracellular matrix, immune function, TGFβ and other cellular processes.

Purines are molecules essential for many cell processes, including RNA and DNA synthesis, regulation of enzyme activity, protein synthesis and function, energy metabolism and transfer, essential coenzyme function, and cell signaling. Purines are produced via the de novo purine biosynthesis pathway. Mutations in purine biosynthetic genes, for example phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS, E.C. 6.3.2.6/E.C. 4.1.1.21), can lead to developmental anomalies in lower vertebrates. Alterations in PAICS expression in humans have been associated with various types of cancer. Mutations in adenylosuccinate lyase (ADSL, E.C. 4.3.2.2) or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC, E.C. 2.1.2.3/E.C. 3.5.4.10) lead to inborn errors of metabolism with a range of clinical symptoms, including developmental delay, severe neurological symptoms, renal stones, combined immunodeficiency, and autistic features. The pathogenetic mechanism is unknown for any of these conditions, and no effective treatments exist. The study of cells carrying mutations in the various de novo purine biosynthesis pathway genes provides one approach to analysis of purine disorders. Here we report the characterization of AdeD Chinese hamster ovary (CHO) cells, which carry genetic mutations encoding p.E177K and p.W363* variants of PAICS. Both mutations impact PAICS structure and completely abolish its biosynthesis. Additionally, we describe a sensitive and rapid analytical method for detection of purine de novo biosynthesis intermediates based on high performance liquid chromatography with electrochemical detection. Using this technique we detected accumulation of AIR in AdeD cells. In AdeI cells, mutant for the ADSL gene, we detected accumulation of SAICAR and SAMP and, somewhat unexpectedly, accumulation of AIR. This method has great potential for metabolite profiling of de novo purine biosynthesis pathway mutants, identification of novel genetic defects of purine metabolism in humans, and elucidating the regulation of this critical metabolic pathway.