Identification And Function Of Noncoding RNA-encoded Micropeptides On Lipid Droplets

Objective: Over the past decades,great progress in sequencing technologies and computational biology suggests that the noncoding majority in mammalian genome is rich in functional elements to produce proteins.Many RNA molecules,mis-annotated as noncoding,actually harbor small open reading frames(sORFs)that are predicted to code proteins called micropeptides.Some micropeptides have been discovered and verified to play critical roles in multiple biological processes.Lipid droplet(LD)is a unique cellular organelle,with a core of neutral lipids bound by phospholipid monolayer membrane and associated proteins.LD is conserved from bacteria to humans and closely associated with cellular lipid metabolism and metabolic disorders.However,no micropeptide has been identified on LDs.Here we for the first time explore this possibility and its biological funcitons.Methods: To accomplish the above research objectives,lipid droplets were isolated first and the small molecular weight proteins were enriched.The possible micropeptides on lipid droplets were identified by mass spectrometry coupled with micropeptide database.The intracellular localization of these micropeptides was further screened.The effects of micropeptides localized to lipid droplets on lipid storage and insulin signaling were studied.1.Construction of the database for identification of micropeptides.Non-coding transcripts from Gencode database were extracted using bioinformatics method and those sequences harboring small open reading frames were translated for construction the database for proteomic mass spectrometry data searching.2.The lipid droplets were isolated from C2C12 skeletal muscle cells using the well-establised method in our laboratory.Then the quality of isolated lipid droplets was verified by silver staining and Western blot.3.Enrichment and identification of small molecular weight proteins on lipid droplets.The purified high-quality lipid droplets were separated by 12% bis-tris gel,and the proteins below 17 kDa were cut for mass spectrometry study.The micropeptides on the lipid droplets were identified by mass spectrometry coupled with the home-made micropeptide database.4.Study on the intracellular localizations of identified micropeptides.GFP was fused to C-termimus of the identified micropeptides and the plasmids were transfected into Huh7 and C2C12 cells,and then confocal laser scanning microscopy was used to observe whether they located on lipid droplets.At the same time,the fusion protein of the truncation and GFP was also constructedand the localization of the truncation was observed by laser scanning confocal microscope to find outlipid droplet targeting sequence.5.Study on the regulation of micropeptide stablitiy.Proteasome inhibitor and molecular biology methods,to mutate lysine sites that might be involved in ubiquitination,were used to study the possible degradation way.6.Confirmation of the localization to lipid droplets.The micropeptides that could locate on lipid droplets were studied.The intracellular localization was further analyzed by cell fractionation,immunofluorescence staining and immunoelectron microscopy.7.Verificaiton of endogenous expression of micropeptides.The CRISPR-CAS9 gene editing technology was used to construct stable cell lines with 3×FLAG-HA tags in situ before the termination codon of the target micropeptide,and the endogenous expression of micropeptide was detected by immunoprecipitation and Western blot experiments.8.Study on the function of micropeptides.The effects of target micropeptides on triglyceride storage in skeletal muscle cells and insulin sensitivity were investigated using triglyceride detection kits and an insulin resistance model stimulated by saturated fatty acids.In brief,oleic acid was used to treat skeletal muscle cells overexpressing micropeptide and control cells to compare their triacylglycerol levels.After 12 hours of treatment with oleic acid and/or palmitic acid,micropeptide overexpressed cells and control cells were stimulated with insulin for 10 minutes.The changes of pAKT protein in the two types of cells were detected by Western blot experiment to analyze the effect of micropeptide on insulin signaling.Results: 1.We found 44 micropeptides on lipid droplets.These micropeptides are mainly encoded by transcripts annotated as processed_transcript and retained_intron.2.Two micropeptides localized to lipid droplets,revealed by fusion protein with GFP,were screened and named LDANP1 and LDANP2?3.After treatment with proteasome inhibitor MG132,the expression level of LDANP1 increased significantly,indicating that the stability of LDANP1 is regulated by proteasome.Lysine at sites 7,49 and 61 may play an important role in the ubiquitination of LDANP1 and inhence its degradation by proteasome.4.Cell fractionation results showed that LDANP1 was mainly enriched in lipid droplet fraction.Immunofluorescence staining results showed that LDANP1 surrounded the lipid droplets.The immunocolloidal gold labeling results showed that the gold particles mainly surrounded lipid droplets.All these results confirmed that LDANP1 was localized to lipid droplets.5.Through CRISPR-CAS9 gene editing technology,LDANP1 cell line with 3× FLAG-HA tag was constructed,and endogenous expression of LDANP1 was confirmed.6.LDANP1 was overexpressed and triacylglycerol content in skeletal muscle cells was significantly reduced after oleic acid treatment.At the same time,the treatment of skeletal muscle cells by saturated fatty acids resulted in a significant decrease in AKT phosphorylation under insulin stimulation.Oleic acid rescued the phosphorylation of AKT reduced by palmitic acid,but this recovery was significantly weakened after the overexpression of LDANP1.These results indicate that LDANP1 plays an important role in the storage of triacylglycerol in skeletal muscle cells and the regulation of insulin signaling sensitivity.7.The truncation experiment of LDANP2 showed that the 35-56 truncation and 36-56 truncation of LDANP2,differing by only one amino acid residue,showed two localizations,lipid droplets and mitochondria respectively.Conclusion: In this study,44 micropeptides were found on lipid droplets of skeletal muscle cells by means of mass spectrometry coupled with micropeptide database,and the endogenous expression of LDANP1 was verified.Besides,LDANP1 was verified to localize on LDs a variety of methods and was found to function in regulation of triacylglycerol storage and insulin signaling in skeletal muscle cells.In addition,interestingly,we found that different truncations of LDANP2 located on different organelles.This is the first time to systematically study the existence and function of micropeptides on lipid droplets.This study suggests a new way to explore new LD-associated proteins as well as factors modulating LD dynamics and affecting the insulin sensitivity in skeletal muscle cells.