DOI: 10.7274/25596288.v1
title: Multi-Omics Analyses to Uncover Gene Expression Alterations and the Contribution of Human Methyltransferase-Like Protein 16 in Miller-Dieker Syndrome

description: Miller-Dieker Syndrome (MDS) is a rare neurogenetic disorder resulting from a heterozygous deletion of 26 genes in the MDS locus on human chromosome 17. MDS patients often die in utero and only 10% of those who are born reach ten years of age. Current treatments for MDS are based on the patient’s symptoms, which mostly prevent complications and control seizures. A detailed understanding of the pathogenesis of MDS through gene expression studies would be useful in developing precise medical approaches toward MDS. To better understand MDS at the molecular level, we performed RNA sequencing on RNA and mass spectrometry on total protein isolated from BJ (non-MDS) cells and GM06097 (MDS) cells, which were derived from a healthy individual and an MDS patient, respectively. Differentially expressed genes (DEGs) at the RNA and protein levels involved genes associated with phenotypic features reported in MDS patients (CACNG4, ADD2, SPTAN1, SHANK2), signaling pathways (WNT, GABBR2, CAMK2B, TRAM-1), and nervous system development (CAMK2B, BEX1, ARSA). Phenotypic assays validated enhanced calcium signaling, cell migration defects and downregulated protein translation in MDS. Although DNA- and RNA-modifying enzymes were among the DEGs and the intracellular S-adenosylmethionine (SAM) levels were 2-fold lower in MDS cells, the global nucleoside modifications remained unchanged. 
Interestingly, methyltransferase-like protein 16 (METTL16), a protein encoded in the MDS locus, displayed a significant role in restoring the protein translation in MDS cells. Several studies have shown SAM regulation in the mammalian target of rapamycin (mTOR) pathway in controlling protein translation. Our RT-qPCR validation of alternative splicing analysis showed relatively higher expression levels of intron retained isoforms of MAT2A (methionine adenosyltransferase 2A which is a SAM synthase) in MDS cells. Moreover, SAM levels were restored in the presence of full length METTL16, while R82A/N184A catalytic mutant exhibited reduced SAM levels suggestive of a tight regulation of SAM levels via METTL16 expression. Besides, the inhibition of mTOR pathway showed reduced SAM production justifying the mTOR pathway dependency on SAM regulation. Additionally, our validation of the phosphor states of the mTOR pathway downstream regulators: p-mTOR, p-P70 S6K, p-4e-BP1 showed increased phosphorylation in the presence of METTL16, supporting our SUnSET (SUrface SEnsing of Translation) studies on METTL16 control in protein translation. Furthermore, our co-immunoprecipitation study with METTL16 followed by mass spectrometry analysis (co-IP-MS) identified some eukaryotic initiation factors (eIF4A1 (unwinds RNA secondary structure), eIF4A2 (facilitates RNA binding), and eIF4A3 (part of exon-junction complex)), eEF2 and several splicing-related proteins as METTL16 interacting partners in protein translation initiation. Western blot validation of co-immunoprecipitation results underscores the importance of the methyltransferase (MTase) domain, particularly in the amino acid residues of 1-249, in mediating the interaction between eIF4A2, eIF4A3, and SRSF1 with METTL16. Simultaneously, this corroborates our SUnSET assay findings, indicating a potential contribution of the MTase domain in facilitating translation. 
Thus, this dissertation work will pave the way for the understanding of genes or proteins that are altered in MDS condition and provide insights into the aberrant protein translation and cell migration. Hence, our multi-omics work sheds light on therapeutic approaches aimed at inhibiting the STAT3 pathway (such as JAK inhibitors), normalizing the SAM/SAH methylation ratio (such as betaine therapy, SAMe for the SAM level restoration), and boosting protein translation (for instance, with NGF and EGF treatments) as treatment options for MDS. This will provide a very strong starting point in leading to the identification of promising candidates in designing therapeutics for MDS.