The Novel Gene CRNDE Encodes a Nuclear Peptide (CRNDEP) Which Is Overexpressed in Highly Proliferating Tissues

CRNDE, recently described as the lncRNA-coding gene, is overexpressed at RNA level in human malignancies. Its role in gametogenesis, cellular differentiation and pluripotency has been suggested as well. Herein, we aimed to verify our hypothesis that the CRNDE gene may encode a protein product, CRNDEP. By using bioinformatics methods, we identified the 84-amino acid ORF encoded by one of two CRNDE transcripts, previously described by our research team. This ORF was cloned into two expression vectors, subsequently utilized in localization studies in HeLa cells. We also developed a polyclonal antibody against CRNDEP. Its specificity was confirmed in immunohistochemical, cellular localization, Western blot and immunoprecipitation experiments, as well as by showing a statistically significant decrease of endogenous CRNDEP expression in the cells with transient shRNA-mediated knockdown of CRNDE. Endogenous CRNDEP localizes predominantly to the nucleus and its expression seems to be elevated in highly proliferating tissues, like the parabasal layer of the squamous epithelium, intestinal crypts or spermatocytes. After its artificial overexpression in HeLa cells, in a fusion with either the EGFP or DsRed Monomer fluorescent tag, CRNDEP seems to stimulate the formation of stress granules and localize to them. Although the exact role of CRNDEP is unknown, our preliminary results suggest that it may be involved in the regulation of the cell proliferation. Possibly, CRNDEP also participates in oxygen metabolism, considering our in silico results, and the correlation between its enforced overexpression and the formation of stress granules. This is the first report showing the existence of a peptide encoded by the CRNDE gene.CRNDE是近年来被鉴定为编码长链非编码RNA（long non-coding RNA, lncRNA）的基因，在人类恶性肿瘤中呈现RNA水平的过表达。其在配子发生、细胞分化及细胞多能性中的作用亦有相关报道。本研究旨在验证我们提出的假说：CRNDE基因可编码一种蛋白质产物CRNDEP。本研究通过生物信息学方法，鉴定出本团队此前报道的两条CRNDE转录本之一所编码的84个氨基酸开放阅读框（open reading frame, ORF）。将该开放阅读框克隆至两个表达载体中，随后将载体用于海拉（HeLa）细胞的亚细胞定位研究。我们还制备了针对CRNDEP的多克隆抗体，并通过免疫组化实验、细胞定位实验、蛋白质印迹（Western blot）实验及免疫沉淀实验，以及在经瞬时短发夹RNA（short hairpin RNA, shRNA）介导敲低CRNDE的细胞中，内源性CRNDEP的表达出现具有统计学意义的显著下调这一结果，验证了该抗体的特异性。内源性CRNDEP主要定位于细胞核，且在高增殖活性组织中表达水平升高，例如鳞状上皮的基底旁层、肠隐窝及精母细胞。将CRNDEP与增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）或单体红色荧光蛋白（DsRed Monomer）融合后在海拉细胞中进行人工过表达，结果显示CRNDEP可诱导应激颗粒的形成并定位于该颗粒中。尽管CRNDEP的确切功能尚未明确，但本研究的初步结果提示其可能参与细胞增殖的调控过程。结合我们的in silico分析结果，以及其强制过表达与应激颗粒形成之间的相关性，CRNDEP可能也参与氧代谢过程。本研究首次报道了CRNDE基因所编码的肽类产物的存在。