Translation and protein expression of lncRNAs: Impact for liver disease and hepatocellular carcinoma
Authors: Mirjam B. Zeisel, Thomas F. Baumert
Journal: Hepatology (2016)

Although the genome is pervasively transcribed, extensive annotation efforts over the past years revealed that only a minority of the human genome is composed of genes encoding messenger RNAs (mRNAs) that are translated into proteins. Indeed, the majority of DNA appears to give rise to RNAs that are thought to not code for protein (noncoding RNAs; ncRNAs) given their lack of long open reading frame (ORF). Among these are microRNAs and long noncoding RNAs (lncRNAs; Fig. 1). Interestingly, recent evidence from computational and experimental studies has suggested that some lncRNAs may not be noncoding after all and can have biological functions through encoded peptides. However, the extent of this process remains largely elusive. To address this issue, Ji et al. developed a new computational method (RibORF) recently reported to systematically analyze ribosome profiling data in order to assess to which extent ncRNAs are translated into small proteins or peptides.1 Ribosome profiling uses high-throughput sequencing to provide a global measurement of RNAs that are associated with ribosomes within cells to monitor RNA translation. However, this technique does not necessarily distinguish between the RNAs that are truly translated and those that are just bound to ribosomes. Furthermore, translation does not equal generation of a functional protein or peptide. RibORF is a Support Vector Machine classifier that has been developed by Ji et al. to identify bona-fide translated ORFs from ribosome profiling data in an unbiased way. Using this classifier, the researchers show that many more than previously recognized noncanonical peptides are translated from lncRNAs: Indeed, ∼40% of lncRNAs and pseudogene RNAs are translated into peptides longer than 10 amino acids in human cells. In line with the cytoplasmic localization of the translation machinery, they noticed a differential localization between translated and nontranslated ncRNAs: Whereas nontranslated RNAs are mainly found in the nucleus, translated ncRNAs are generally found in the cytoplasm. Noteworthy, most of the peptides translated from lncRNAs are likely to be highly unstable and nonfunctional. However, a small fraction (∼9%) of them appeared to be evolutionarily conserved between mice and humans and under stabilizing selection (Fig. 1). This suggests that these peptides may have yet unrecognized biological functions. Taken together, this study adds another piece to the puzzle of the elusive biological functions of lncRNAs and indicates that some of them may regulate biological processes through noncanonical peptides—and may thus not be truly ncRNAs. However, to which extent, how, and why such peptides may participate in regulatory networks remains to be determined. Future experiments will provide evidence whether and under which circumstances defined lncRNAs could function as RNA molecules, thereby interacting with RNAs, DNA, and proteins and/or through peptides to fine-tune gene expression. Furthermore, it will be interesting to uncover whether these concepts apply to any cell type or may be restricted to certain cell types or be regulated in a pathophysiological manner. The role of lncRNAs in disease biology is only about to emerge. They may likely play a role in a variety of diseases, including liver disease and cancer. Hepatocellular carcinoma (HCC) is the second-leading cause of cancer death worldwide. Although the major risk factors of HCC are well known, lack of early detection and limited preventive and therapeutic options hamper the clinical management of the patient with disease. This is partly attributed to the fact that despite recent progress, the molecular mechanisms underlying HCC development still remain poorly understood. Within the past decade, scientists have started to explore the role of lncRNAs in HCC and discovered several lncRNAs that may play a role in liver carcinogenesis (reviewed in Huang et al.2 and George and Patel3). Profiling studies revealed that numerous lncRNAs are deregulated in HCC tumors. Whether or not lncRNA dysregulation is a cause or a consequence of cancer development remains to be determined. Given their tissue- and cell-specific expression and because lncRNAs can be detected in body fluids, circulating lncRNAs have been suggested as noninvasive HCC biomarkers (reviewed in Huang et al.2 and George and Patel3). Indeed, several studies have suggested that defined lncRNAs may be useful for detection and diagnosis of HCC (e.g., higher frequency of highly up-regulated in liver cancer expression in patients with HCC than healthy individuals) and/or prediction of HCC recurrence after liver transplantation (e.g., overexpression of Hox transcript antisense intergenic RNA in patients with tumor recurrence in the liver graft; reviewed in Huang et al.2 and George and Patel3). Furthermore, studies aiming at the functional characterization of the role of lncRNAs showed that defined lncRNAs are linked to apoptosis/proliferation/invasiveness of liver-derived cells, but to date only a few molecular function(s) of these lncRNAs related to HCC have been reported.2-4 It is worth noting that recent studies have also linked viral-derived lncRNAs to the pathogenesis of hepatitis B (HBV) and C virus (HCV) infection, two major causes of HCC: Whereas HCV has been suggested to encode a subgenomic lncRNA, that is a stable decay intermediate generated from the viral genome (reviewed in Charley and Wilusz5), the integration of the HBV genome leads to a chimeric viral-human lncRNA-like transcript, which has been reported to have cancer-promoting properties.6 Collectively, lncRNAs may contribute to virus-induced pathogenesis of liver disease and HCC development.2, 3, 5-7 Besides pathogenesis, lncRNAs may also play a role in the treatment of liver disease since defined lncRNAs have been linked to drug resistance in HCC (reviewed in Huang et al.2). Furthermore, given their multiple functions in HCC, lncRNAs may themselves represent interesting therapeutic targets to prevent and/or treat liver cancer. It has been suggested that lncRNAs acting as transcriptional enhancers could be used to increase gene expression, and their potential as therapeutic strategies is currently being explored. In vivo proof of concept of the use of "antagoNATs" (oligonucleotides or small interfering RNA designed to inhibit natural antisense transcripts [NATs], i.e., RNA transcripts that originate from the opposite strand of a sense RNA transcript) for therapeutic intervention has recently been reported (reviewed in Wahlestedt8). Taken together, the previously unrecognized extent of lncRNA translation uncovered by the Regev and Struhl groups will likely lead to a rethinking in the ncRNA field and stimulate further experiments to better characterize the three classes of ncRNAs they have suggested: (1) nontranslated ncRNAs; (2) RNAs translated into nonfunctional peptides; and (3) RNAs translated into noncanonical peptides with biological function.1 Given the emerging evidence for a functional role of lncRNAs as drivers and biomarkers in liver disease and HCC, the study of Ji et al.1 opens novel perspectives to better understand the role of lncRNAs for pathogenesis of liver disease biology and cancer. Whether defined lncRNAs may contribute to HCC development through their translation into peptides remains to be explored. The study of the role of translated lncRNAs in liver disease biology may ultimately contribute to the identification of urgently needed novel biomarkers and therapeutic targets for liver disease. Furthermore, applying these concepts to other types of cancer will broaden our understanding of conserved mechanisms of carcinogenesis and develop novel anticancer strategies. Mirjam B. Zeisel, Pharm.D., Ph.D.1,2 Thomas F. Baumert, M.D.1-3 1Inserm U1110 Institut de Recherche sur les Maladies Virales et Hépatiques Strasbourg, France 2Université de Strasbourg Strasbourg, France 3Institut Hospitalo-Universitaire Pôle hépato-digestif Nouvel Hôpital Civil Strasbourg, France