Identification and analysis of short open reading frames (sORFs) in the initially annotated noncoding RNA LINC00493 from human cells

Abstract

Whole transcriptome analyses have revealed that mammalian genomes are massively transcribed, resulting in the production of huge numbers of transcripts with unknown functions (TUFs). Previous research has categorized most TUFs as noncoding RNAs (ncRNAs) because most previously studied TUFs do not encode open reading frames (ORFs) with biologically significant lengths (>100 amino acids). Recent studies, however, have reported that several transcripts harboring small ORFs (sORFs) that encode peptides shorter than 100 amino acids are translated and play important biological functions. Here, we examined the translational capacity of transcripts annotated as ncRNAs in human cells, and identified several hundreds of ribosome-associated transcripts previously annotated as ncRNAs. Ribosome footprinting and polysome profiling analyses revealed that 61 of them are potentially translatable. Among them, 45 were non-nonsense-mediated mRNA decay targets, suggesting that they are productive mRNAs. We confirmed the translation of one ncRNA, LINC00493, by luciferase reporter assaying and western blotting of a FLAG-tagged LINC00493 peptide. While proteomic analysis revealed that the LINC00493 peptide interacts with many mitochondrial proteins, immunofluorescence assays showed that its peptide is mitochondrially localized. Our findings indicate that some transcripts annotated as ncRNAs encode peptides and that unannotated peptides may perform important roles in cells.

LINC00493‐encoded microprotein SMIM26 exerts anti‐metastatic activity in renal cell carcinoma

“…Our earlier works dedicated to the global discovery of new smORFs by using full‐length translating mRNA sequencing and ribosome profiling (Wang et al , 2013), and reported a total of 314 lncRNA‐encoded new microproteins (Lu et al , 2019). As one of these microproteins, we first provided evidence of translation and MS to support the existence of SMIM26 as smORF in LINC00493 (Lu et al , 2019), which was further confirmed by another group (Yeasmin et al , 2021). However, the detailed molecular function of SMIM26 remains unclear.…”

“…SMIM26, one of the microproteins identified in our previous study, was reported to be involved in the regulation of cell viability (Konina et al , 2021; Yeasmin et al , 2021). In this study, we validated that microprotein SMIM26 was derived from lncRNA LINC00493 and resided in mitochondria.…”

Investigation of LINC00493/SMIM26 Gene Suggests Its Dual Functioning at mRNA and Protein Level

“…A recent study of Fouzia Yeasmin et al [38] confirmed the translation of LINC00493 by using luciferase reporter assays and Western blotting analysis. It was shown that the SMIM26 protein interacts with many mitochondrial proteins.…”

A 66 amino acid micro-peptide encoded by long non-coding RNA RP11-119F7.5 was identified in hepatocellular carcinoma

“…Recent studies have found that there are millions of smORF sequences in eukaryotic genomes that have the ability to encode and translate proteins (<100 codons). [40,41] These ncRNAs encode functional peptides that are important in regulating many physiological and pathological processes. [42] Some reports have indicated that the functional peptides encoded by ncRNAs may play an important role in the development of tumors.…”