A putative long noncoding RNA-encoded micropeptide maintains cellular homeostasis in pancreatic β-cells

Abstract

Micropeptides(microproteins) encoded by transcripts previously annotated as long noncoding RNA (IncRNAs) are emerging as important mediators of fundamental biological processes in health and disease.Here we applied two computational tools to identify putative micropeptides encoded by lncRNAs that are expressed in human pancreas. We experimentally verified one such micropeptide encoded by the β-celland neural cell-enriched lncRNA TUNAR (TUNA / HI-LNC78 / LINC00617). We named this highly conserved 48-amino-acid micropeptide Beta cell-and Neural cell-regulin (BNLN). BNLN contains a singlepass transmembrane domain. In pancreatic β-cells, BNLN mainly localized at the endoplasmic reticulum.Overexpression of BNLN lowered ER calcium levels, increased cytosolic calcium levels, and maintained ER homeostasis in response to high glucose challenge. To determine the physiological and pathological roles of BNLN, we assessed the BNLN expression in islets from mice fed with a high-fat diet and a regular diet, and found that BNLN is suppressed by diet-induced obesity (DIO). Conversely, overexpression of BNLN elevated glucose-stimulated insulin secretion in INS-1 cells. Lastly, BNLN overexpression enhanced insulin secretion in islets from lean and obese mice as well as from humans. Taken together, our study provides the first evidence that lncRNA-encoded micropeptides play a critical role in pancreatic β-cell function and provides a foundation on which to perform future comprehensive analyses of micropeptide function and pathophysiological impact on diabetes. Abbreviations:ATF6: activating transcription factor-6; BNLN: beta cell and neuron regulin; Cre: Cre recombinase; DIO: diet-induced obesity; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; Glc: glucose; HFD: high-fat diet; IncRNA: long noncoding RNA; RD: regular diet; sORF: small open reading frame; T2D: type 2 diabetes; TUNAR: TCL1 Upstream Neural Differentiation-Associated RNA. INTRODUCTION:Large-scale projects such as ENCODE and FANTOM have found thousands of long noncoding RNAs (lncRNAs) in the human genome 1-4 . lncRNAs play an essential role in regulating islet function in health and diseases including diabetes 5 . It has been demonstrated that lncRNAs are key components of the islet regulatome, with more than 1000 lncRNAs identified in human islets and mouse β-cells 6,7 . These lncRNAs play critical roles in islet and β-cell survival, cell development, and physiological function through regulation of the islet transcriptome 5-7 . Of note, a genome-wide association study that examined T2D susceptibility loci demonstrated that dysregulation of IncRNAs is involved in pancreatic pathologies in humans 6 . In addition, dysregulation of lncRNAs has been implicated in diabetes complications, such as diabetic nephropathy and retinopathy 5,8 . Recently, several circulating lncRNAs have been identified as biomarkers of diabetes and diabetic complications 9,10 . However, although much knowledge has accumulated correlating d...

Noncoding translation mitigation

“…Our curation of the literature further showed that the majority of functional proteins translated from noncoding sequences localize to cellular membranes 11,[115][116][117][118][119][120][121][122][123][124][125][126][127][128][129][130]132,133,138 . One question that remains to be answered is what underlies this preferential membrane targeting of noncoding proteins.…”

“…We searched the available literature for examples of functional peptides derived from the translation of 5' UTRs or previously annotated lncRNAs in mammalian cells for which localization of the peptide had been experimentally determined. Indeed, a large majority (47 of 64 total peptides) of the identified peptides demonstrated localization to various cellular membranes 11,[115][116][117][118][119][120][121][122][123][124][125][126][127][128][129][130]132,133,138 (Figure 4.3A).…”

“…These proto-genes also tend to contain putative transmembrane domains 113,114 . We manually curated 64 translated functional noncoding peptides in humans from the available literature to determine if there was a preference for localization to cellular membranes 11,[115][116][117][118][119][120][121][122][123][124][125][126][127][128][129][130][131][132][133] . Indeed, 47 of the 64 translated functional noncoding peptides we curated appear to localize to cellular membranes.…”

A unified model for the surveillance of translation in diverse noncoding sequences

“…While most peptides synthesized from noncanonical ORFs are likely nonfunctional, on the evolutionary timescale noncanonical ORF translation is necessary to expose the noncoding genome to natural selection, and ultimately, the origination of new protein-coding genes. There have been numerous recent discoveries of functional peptides translated from 5’ UTRs and previously annotated lncRNAs in mammalian cells (Anderson et al, 2015; Chen et al, 2020; Li et al, 2021; Nelson et al, 2016; Polycarpou-Schwarz et al, 2018; Senís et al, 2021; Wang et al, 2020). Intriguingly, of the 64 functional peptides whose cellular localization had been determined experimentally, about three quarters (47) localize to membranes, including the plasma membrane and membranes of ER, mitochondria, and lysosome ( Table S1 ).…”