CRISPR-Cas9 Screening Reveals Microproteins Regulating Adipocyte Proliferation and Lipid Metabolism [RNA-seq]
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ABSTRACT:
PROVIDER: PRJNA1263618 | ENA |
REPOSITORIES: ENA
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Project description:Small open reading frames (smORFs) encode microproteins that play crucial roles in various biological processes, yet their functions in adipocyte biology remain largely unexplored. In a previous study, we identified thousands of smORFs in white and brown adipocytes derived from the stromal vascular fraction (SVF) of mice using ribosome profiling (Ribo-Seq). Here, we expand on this work by identifying additional smORFs related to adipocytes using the in vitro 3T3-L1 preadipocyte model. To systematically investigate the functional relevance of these smORFs, we designed a custom CRISPR/Cas9 guide RNA (sgRNA) library and screened for smORFs influencing adipocyte proliferation and differentiation. Through a dropout screen and fluorescence-assisted cell sorting (FACS) of lipid droplets, we identified dozens of smORFs that regulate either cell proliferation or lipid accumulation. Among these, we validated a novel microprotein as a key regulator of adipocyte differentiation. These findings highlight the potential of CRISPR/Cas9-based screening to uncover functional smORFs and provide a framework for further exploration of microproteins in adipocyte biology and metabolic regulation.
2025-08-27 | GSE297349 | GEO
Project description:Small open reading frames (smORFs) encode microproteins that play crucial roles in various biological processes, yet their functions in adipocyte biology remain largely unexplored. In a previous study, we identified thousands of smORFs in white and brown adipocytes derived from the stromal vascular fraction (SVF) of mice using ribosome profiling (Ribo-Seq). Here, we expand on this work by identifying additional smORFs related to adipocytes using the in vitro 3T3-L1 preadipocyte model. To systematically investigate the functional relevance of these smORFs, we designed a custom CRISPR/Cas9 guide RNA (sgRNA) library and screened for smORFs influencing adipocyte proliferation and differentiation. Through a dropout screen and fluorescence-assisted cell sorting (FACS) of lipid droplets, we identified dozens of smORFs that regulate either cell proliferation or lipid accumulation. Among these, we validated a novel microprotein as a key regulator of adipocyte differentiation. These findings highlight the potential of CRISPR/Cas9-based screening to uncover functional smORFs and provide a framework for further exploration of microproteins in adipocyte biology and metabolic regulation.
2025-08-27 | GSE297348 | GEO
Project description:CRISPR-Cas9 Screening Reveals Microproteins Regulating Adipocyte Proliferation and Lipid Metabolism [Ribo-seq]
| PRJNA1263619 | ENA
Project description:MicroProteins are short, single domain proteins that act by sequestering larger, multidomain proteins into non-functional complexes. MicroProteins have been identified in plants and animals, where they are mostly involved in the regulation of developmental processes. Here we show that two Arabidopsis thaliana microProteins, miP1a and miP1b, physically interact with CONSTANS (CO) a potent regulator of flowering time. The miP1a/b-type microProteins evolved in dicotyledonous plants and have an additional carboxy-terminal PF(V/L)FL motif. This motif enables miP1a/b microProteins to interact with TOPLESS/TOPLESS-RELATED (TPL/TPR) proteins. Interaction of CO with miP1a/b/TPL causes late flowering due to a failure in the induction of FLOWERING LOCUS T (FT) expression under inductive long day conditions. Both miP1a and miP1b are expressed in vascular tissue, where CO and FT are active. Genetically, miP1a/b act upstream of CO thus our findings unravel a novel layer of flowering time regulation via microProtein-inhibition. RNA-Seq transcriptome analysis of four biological samples were analysed with two technical replicates. Columbia wildtype plants Col-0, constans mutant plants co-SAIL, and two transgenic lines overexpressing a microProtein (miP1a and miP1b) were sequenced.
2016-03-08 | E-GEOD-56811 | biostudies-arrayexpress
Project description:Proteogenomics methods have identified many non-annotated protein-coding genes in the human genome. Many of the newly discovered protein-coding genes encode peptides and small proteins, referred to collectively as microproteins. Microproteins are produced through ribosome translation of small open reading frames (smORFs). The discovery of many smORFs reveals a blind spot in traditional gene-finding algorithms for these genes. Biological studies have found roles for microproteins in cell biology and physiology, and the potential that there exists additional bioactive microproteins drives the interest in detection and discovery of these molecules. A key step in any proteogenomics workflow is the assembly of RNA-Seq data into likely mRNA transcrips that are then used to create a searchable protein databases. Here we demonstrate that specific features of the assembled transcriptome impact microprotein detection by shotgun proteomics. By tailoring transcript assembly for downstream mass spectrometry searching, we show that we can detect more than double the number of high-quality microprotein candidates and introduce a novel open-source mRNA assembler for proteogenomics (MAPS) that incorporates all of these features. By integrating our specialized assembler, MAPS, and a popular generalized assembler into our proteogenomics pipeline, we detect 45 novel human microproteins from a high quality proteogenomics dataset of a human cell line. We then characterize the features of the novel microproteins, identifying two classes of microproteins. Our work highlights the importance of specialized transcriptome assembly upstream of proteomics validation when searching for short and potentially rare and poorly conserved proteins.
2017-12-20 | GSE92659 | GEO
Project description:MicroProteins are short, single domain proteins that act by sequestering larger, multidomain proteins into non-functional complexes. MicroProteins have been identified in plants and animals, where they are mostly involved in the regulation of developmental processes. Here we show that two Arabidopsis thaliana microProteins, miP1a and miP1b, physically interact with CONSTANS (CO) a potent regulator of flowering time. The miP1a/b-type microProteins evolved in dicotyledonous plants and have an additional carboxy-terminal PF(V/L)FL motif. This motif enables miP1a/b microProteins to interact with TOPLESS/TOPLESS-RELATED (TPL/TPR) proteins. Interaction of CO with miP1a/b/TPL causes late flowering due to a failure in the induction of FLOWERING LOCUS T (FT) expression under inductive long day conditions. Both miP1a and miP1b are expressed in vascular tissue, where CO and FT are active. Genetically, miP1a/b act upstream of CO thus our findings unravel a novel layer of flowering time regulation via microProtein-inhibition.
2016-03-08 | GSE56811 | GEO
Project description:DCHP targets lipid associated macrophages to disrupt lipid metabolism via crosstalk with adipocyte precursor cells
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Project description:Microproteins, as hidden players in the proteomic club, are encoded by small open reading frames (sORFs) and have garnered increasing attention in recent years. T cells play a critical role in the immune system's response to cancer, however, the exploration of novel microproteins in T cells remains an unexplored area. In this study, we employed an integrated approach combined nascent protein profiling and quantitative global proteomics to identify novel microproteins in human T cells.
2024-09-24 | GSE277522 | GEO
Project description:LUM1_969842: Brown adipocyte lipid droplet proteomics (Figure 4d):
126, 127N, 127C, 128N: WT
128C, 129N, 129C, 130N: KO (clstn3b-/-)
LUM1_997681: White adipocyte lipid droplet proteomics (Figure 4e):
126, 127C, 127N, 128C, 128N: WT
129C, 129N, 130C, 130N, 131N: KO (clstn3b-/-)
| MSV000095763 | MassIVE
Project description:Perotein corona is formed surrouding interface of nanopsrticles upon administered in biological fluids. The quality of protein corona is recognized as the crucial factor for in vivo fate of the administered nanoparticles. In this project, Cas9 ribnonucleoprotein (RNP)-loaded lipid nanoparticles (LNPs) capable of robust gene knockout of hepatic genes of interest after single intravenous injection were developed. Compared with short interfering RNA (siRNA)-loaded LNPs, RNP-loaded LNPs showed different morphology, biodistribution, and mechanism of hepatic acccumulation. Therefore, proteome analysis of the corona proteins on the RNP and siRNA-loaded LNPs were performed to elucidate the impact of payloads on nano-bio interactions.
2024-09-05 | PXD055572 | JPOST Repository