LINC00116 codes for a mitochondrial peptide linking respiration and lipid metabolism Anastasia Chugunovaa,b, Elizaveta Losevaa, Pavel Mazinc,d,e, Aleksandra Mitinac, Tsimafei Navalayeua, Dmitry Bilanf,g, Polina Vishnyakovah, Maria Mareyh, Anna Golovinai, Marina Serebryakovab,i, Philipp Pletneva,b, Maria Rubtsovaa,b,i, Waltraud Mairc, Anna Vanyushkinac, Philipp Khaitovichc, Vsevolod Belousovf,g,j, Mikhail Vysokikhh,i,1, Petr Sergieva,b,i,k,1, and Olga Dontsovaa,b,f,i aDepartment of Chemistry, Lomonosov Moscow State University, 119992 Moscow, Russia; bCenter of Life Sciences, Skolkovo Institute of Science and Technology, Moscow, 143028, Russia; cSkoltech Center for Neurobiology and Brain Restoration, Skolkovo Institute of Science and Technology, Moscow, 143028, Russia; dInstitute for Information Transmission Problems (Kharkevich Institute) RAS, 127051 Moscow, Russia; eFaculty of Computer Science, National Research University Higher School of Economics, 119991 Moscow, Russia; fShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia; gPirogov Russian National Research Medical University, 117997 Moscow, Russia; hResearch Center for Obstetrics, Gynecology and Perinatology, 117198 Moscow, Russia; iBelozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia; jInstitute for Cardiovascular Physiology, Georg August University Göttingen, 37073 Göttingen, Germany; and kInstitute of Functional Genomics, Lomonosov Moscow State University, 119992 Moscow, Russia Edited by Igor Ulitsky, Weizmann Institute of Science, and accepted by Editorial Board Member David J. Mangelsdorf January 28, 2019 (received for review June 7, 2018) Genes coding for small peptides have been frequently misanno- proteome is composed of over 1,100 proteins (16), 5% of that tated as long noncoding RNA (lncRNA) genes. Here we have dem- number being small proteins of less than 100 amino acids in onstrated that one such transcript is translated into a 56-amino- length (17). Both mitochondrial (18, 19) and nuclear genomes acid-long peptide conserved in chordates, corroborating the work code for the short mitochondrial peptides being integral com- published while this manuscript was under review. The Mtln pep- ponents of oxidative phosphorylation (OXPHOS) complexes tide could be detected in mitochondria of mouse cell lines and (17), or their assembly factors (20), and play significant roles in tissues. In line with its mitochondrial localization, lack of the Mtln longevity (21), insulin resistance (19), and modulation of apo- decreases the activity of mitochondrial respiratory chain complex I. ptosis (18). Mutations in genes coding for small peptides residing BIOCHEMISTRY Unlike the integral components and assembly factors of NADH: in mitochondria might have pathological outcomes, such as mi- ubiquinone oxidoreductase, Mtln does not alter its enzymatic activ- tochondrial encephalomyopathies (22) and Leigh disease (23). ity directly. Interaction of Mtln with NADH-dependent cytochrome Here we describe a murine peptide encoded in a gene b5 reductase stimulates complex I functioning most likely by pro- misannotated as 1500011k16Rik lncRNA, corresponding to human viding a favorable lipid composition of the membrane. Study of LINC00116. The peptide resides in mitochondria and is important Mtln illuminates the importance of small peptides, whose genes for the activity of respiratory chain complex I. After our first report might frequently be misannotated as lncRNAs, for the control of on the function of this peptide on the conference (24) and while this vitally important cellular processes. manuscript was under review two groups published similar conclu- sions on the functional role of this peptide (25, 26), named sORF | peptide | respiration | mitochondria | lipid metabolism Mitoregulin, Mtln. ’ xamination of eukaryotic cells transcriptome revealed a di- Significance Eversity of long transcripts exceeding an annotated set of protein coding genes by a factor of five (1). A number of those Short peptides are encoded in genomes of all organisms and have transcripts, designated long noncoding RNAs (lncRNAs), were important functions. Due to the small size of such open reading extensively studied and ascribed important functions in chro- frames, they are frequently overlooked by automatic genome matin remodeling, transcriptional control, miRNA sponging, annotation. We investigated the gene that was misannotated as transcription and splicing factor sequestration, and other func- long noncoding RNA LINC00116 and demonstrated that this gene – tions described in recent reviews (2 5). However, with the de- codes for a 56-amino-acid-long peptide, Mtln, which is localized in velopment of a ribosome profiling method (6), a significant mitochondria. Inactivation of the Mtln coding gene leads to re- proportion of lncRNAs was found to be associated with ribo- duction of oxygen consumption attributed to respiratory complex somes and presumably translated (7). Subsequently, it became I activity and perturbs lipid composition of the cell. This influence apparent that short peptides whose genes might be overlooked is mediated by Mtln interaction with NADH-dependent cyto- by computational annotation pipelines due to their minimalistic chrome b5 reductase. Disruption of the mitochondrial localization size play a significant role in cells (8–10). For example, a peptide of the latter phenocopies Mtln inactivation. encoded by the tarsal-less (tal) lncRNA appeared to be important for epidermal differentiation in Drosophila (11). The Pri peptide, Author contributions: A.C., P.K., M.V., P.S., and O.D. designed research; A.C., E.L., A.M., interacting with Ubr3 ubiquitin ligase, activates Svb processing by T.N., D.B., P.V., M.M., A.G., M.S., P.P., M.R., A.V., V.B., and M.V. performed research; D.B., P.V., M.M., M.S., P.P., M.R., A.V., and V.B. contributed new reagents/analytic tools; A.C., the proteasome (12). The pgc RNA encoded peptide (13) turned P.M., A.M., D.B., P.V., M.M., P.P., M.R., W.M., A.V., V.B., and M.V. analyzed data; and A.C., out to be crucial for RNAPII C-terminal domain Ser2 phos- P.K., M.V., P.S., and O.D. wrote the paper. phorylation. The peptide interacts with P-TEFb, a key regulator of The authors declare no conflict of interest. RNAPII-dependent transcription of most cellular genes (14), and This article is a PNAS Direct Submission. I.U. is a guest editor invited by the Editorial Board. prevents its recruitment to transcription sites. This open access article is distributed under Creative Commons Attribution-NonCommercial- Many small peptides are localized in mitochondria, an or- NoDerivatives License 4.0 (CC BY-NC-ND). ganelle whose main functions are respiration coupled to ATP 1To whom correspondence may be addressed. Email: [email protected] or 2+ production, Ca homeostasis, cellular redox-potential mainte- [email protected]. nance, synthesis of steroids, heme, FeS clusters, induction of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. apoptosis, and many others, finally controlling cell fate and tis- 1073/pnas.1809105116/-/DCSupplemental. sues’ functional integrity (15). The mammalian mitochondrial www.pnas.org/cgi/doi/10.1073/pnas.1809105116 PNAS Latest Articles | 1of6 Downloaded by guest on October 2, 2021 Results expressed at natural expression level from an endogenous Mtln Analysis of 1500011k16Rik Coding Potential. Many lncRNAs contain promoter, could be detected in the cells by immunoblotting (SI putative ORFs occurring by chance and not translated into a Appendix, Fig. S3A) and immunocytochemistry (SI Appendix, Fig. functional peptide entity. The murine 1500011k16Rik transcript S3B). To further determine exact peptide localization, we contains a 56-amino acid ORF with a predicted single pass ectopically expressed Mtln-mCherry fusion and observed its transmembrane segment (27), but not any detectable domain colocalization with mitochondria using an organelle-specific dye, relative. Several sequence characteristics are indicative of coding MitoTracker Green FM (Fig. 2A). Cell fractionation revealed potential. Analysis of nucleotide conservation of the genes ho- that the Mtln peptide is always copurified with mitochondria mologous to 1500011k16Rik (Fig. 1A), among 60 vertebrate similarly to the mitochondrial Tom20 protein (Fig. 2B, Left and species (28), revealed that a region of putative ORF is the most, SI Appendix, Fig. S3C). Our results perfectly match the conclu- and almost only, conserved region of the gene. Analysis of the sions of two independent groups, which showed that Mtln lo- aggregated ribosome profiling data (29) revealed substantial ri- calizes in the inner membrane of mitochondria (25, 26). To bosome coverage of the putative ORF (SI Appendix, Fig. S1A). exclude the possibility of any influence of the mCherry tag on the Alignment of the putative products of ORF translation revealed localization of Mtln, we raised polyclonal rabbit antibodies to Mtln and detected an endogenous peptide presence in mito- a high degree of conservation at the amino acid level (Fig. 1B) chondria of the NIH 3T3 cell lines, but not in the generated cell with a high ratio of synonymous over nonsynonymous codon lines with an inactivated Mtln gene (Fig. 2B, Left). To check that substitutions and absence of premature in-frame stop codons (SI the Mtln peptide is present not only in the NIH 3T3 cell line but Appendix, Fig. S1 B and C). As a result, we concluded that