Identification of gene expression in cells that may express micropeptide from RN7SL1

RN7SL1 is a 299-base single-stranded RNA that binds to five SRP proteins and contributes to the generation of transmembrane and secretory proteins as a signal recognition particle. It theoretically possesses two small open reading frames (smORFs) and encodes a micropeptide of 7 and 16 amino acids from the 5' side. However, it is unknown if those smORFs are translated. Therefore, we constructed a plasmid vector expressing RN7SL1-GFP, in which the former sequence was replaced by AcGFP1, and examined whether GFP is expressed. In 293T cells electroporated or lipofected with the RN7SL1-GFP-expressing plasmid, GFP fluorescence was detected under the microscope in both cases, although there were a few GFP-positive cells. GFP-positive and GFP-negative cells were separated by FACS and gene expression of each sample was confirmed by RNA-seq. As a result, GFP-positive cells showed activation of cancer-related pathways compared to GFP-negative cells. These results suggest that RN7SL1 smORFs may be translated under special conditions. 293T cells were electroporated or lipofected with RN7SL1-GFP expression plasmid and GFP positive and GFP negative cells were separated by FACS. Total RNA was extracted from 293T cells under 4 conditions (electroporation GFP negative, electroporation GFP positive, lipofection GFP negative, lipofection GFP positive) and RNA-seq was performed.