Abstract 3502: Simultaneous characterization of mrtl and MycHex1 along with c-Myc p64 and p67 from c-myc locus
Authors: Jun Hyun Jin , Ji Mi Hong , Blume Scott W. , Choi Hyoung Soo 
Published: May 2015

Abstract
Background
In addition to the canonical c-Myc p64 and p67 proteins, the human c-myc locus also encodes two distinct proteins designated “mrtl” (ORF114) and “MycHex1” (ORF188) which are entirely devoid of homology with each other or with c-Myc. The coding sequences for mrtl and MycHex1 are located upstream of the c-Myc coding sequence, within the 5’-“untranslated” region of the c-myc P0 mRNA. The 15 kDa mrtl protein (myc-related translation/localization regulatory factor) has recently been characterized and appears to function in the regulation of c-Myc localization to the nucleus. The 32 kDa MycHex1 (Myc Human Exon 1) protein was first reported over 20 years ago. Yet to this date, no studies of MycHex1 function have ever been undertaken. The aim of this study is to examine simultaneously, for the first time, mrtl, MycHex1, c-Myc p64, and p67 in human tumor cell lines.
Materials and methods
T47D (breast carcinoma), IMR-32 (neuroblastoma), U343 (glioblastoma), D341 and DAOY (medulloblastoma), Ramos and Daudi (Burkitt's lymphoma) were obtained from ATCC. The mrtl and MycHex1 coding sequences were PCR amplified and analyzed. In addition to monoclonal mouse anti-mrtl previously developed using a synthetic 16-mer peptide (CQTVLLRRSSRERERV, aa 99-114), rabbit polyclonal anti-MycHex1 antisera was developed using a synthetic 15-mer peptide (LTRCSNSSERQRERA, aa 62-76). Western analysis was done with whole cell lysates and immunofluorescence (IF) staining and confocal imaging performed following methanol fixation.
Results
Sequence analyses confirmed a known polymorphism at base 1965 (T&gt;G) of mrtl sequence and revealed a new mutation in mrtl codon 83 in IMR32 cells, both of which represent non-synonymous amino acid substitutions. MycHex1 sequence revealed no variation from published sequence.
Western analyses clearly visualized mrtl, MycHex1, c-Myc (N262) p64 and p67 simultaneously. Both mrtl (15 kDa) and MycHex1 (22 kDa and 42 kDa forms) were readily detected in all tumor cell lines. The relative intensities of mrtl and MycHex1 were positively correlated.
IF staining showed mrtl dominantly localized to the nuclear envelope, along with prominent reticular pattern in the cytoplasm consistent with endoplasmic reticulum (ER). MycHex1 was observed as a series of bright foci located exclusively within the nucleus. In many cases, especially the T47D cells, only one MycHex1 focus per cell was seen, colocalizing with mrtl in the nucleoplasmic reticulum. Upon cycloheximide treatment, MycHex1 appeared to be highly stable retaining its typical distribution pattern, while mrtl disappeared somewhat more rapidly.
Conclusion
Evidence is presented for expression and stable accumulation of all 4 proteins (mrtl, MycHex1, c-Myc p64, and p67) encoded by three non-overlapping reading frames from within the human c-myc locus. Further work is warranted to elucidate the functional or regulatory relationships between these molecules.
Citation Format: Hyun Jin Jun, Mi Hong Ji, Scott W. Blume, Hyoung Soo Choi. Simultaneous characterization of mrtl and MycHex1 along with c-Myc p64 and p67 from c-myc locus. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3502. doi:10.1158/1538-7445.AM2014-3502