Secondary Structure of Human De Novo Evolved Gene Product NCYM Analyzed by Vacuum-Ultraviolet Circular Dichroism

NCYM, a cis-antisense gene of MYCN, encodes a Homininae-specific protein that promotes the aggressiveness of human tumors. Newly evolved genes from non-genic regions are known as de novo genes, and NCYM was the first de novo gene whose oncogenic functions were validated in vivo. Targeting NCYM using drugs is a potential strategy for cancer therapy; however, the NCYM structure must be determined before drug design. In this study, we employed vacuum-ultraviolet circular dichroism to evaluate the secondary structure of NCYM. The SUMO-tagged NCYM and the isolated SUMO tag in both hydrogenated and perdeuterated forms were synthesized and purified in a cell-free in vitro system, and vacuum-ultraviolet circular dichroism spectra were measured. Significant differences between the tagged NCYM and the isolated tag were evident in the wavelength range of 190-240 nm. The circular dichroism spectral data combined with a neural network system enabled to predict the secondary structure of NCYM at the amino acid level. The 129-residue tag consists of alpha-helices (approximately 14%) and beta-strands (approximately 29%), which corresponded to the values calculated from the atomic structure of the tag. The 238-residue tagged NCYM contained approximately 17% alpha-helices and 27% beta-strands. The location of the secondary structure predicted using the neural network revealed that these secondary structures were enriched in the Homininae-specific region of NCYM. Deuteration of NCYM altered the secondary structure at D90 from an alpha-helix to another structure other than alpha-helix and beta-strand although this change was within the experimental error range. All four nonsynonymous single-nucleotide polymorphisms (SNPs) in human populations were in this region, and the amino acid alteration in SNP N52S enhanced Myc-nick production. The D90N mutation in NCYM promoted NCYM-mediated MYCN stabilization. Our results reveal the secondary structure of NCYM and demonstrated that the Homininae-specific domain of NCYM is responsible for MYCN stabilization.

Automated summary

NCYM, a newly evolved gene from non-genic regions specific to Homininae, has been shown to promote human tumor aggressiveness. The secondary structure of NCYM was determined using vacuum-ultraviolet circular dichroism, revealing that the Homininae-specific domain is responsible for MYCN stabilization. This study provides insights into the oncogenic functions of NCYM and potential strategies for cancer therapy.