CRISPR-Cas9 Screening Reveals Microproteins Regulating Adipocyte Proliferation and Lipid Metabolism [RNA-seq]
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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:Adipocyte lipid metabolism is coordinated by circadian rhythms, diet, and environmental temperature. Yet how these diverse signals are molecularly integrated remains unknown. We show that these cues converge on the orphan mitochondrial transporter SLC25A34 to orchestrate lipid cycling. During the sleep phase, the adipocyte clock suppresses Slc25a34 expression through the REV-ERB transcriptional repressors. Entering the active phase, consuming lipid-rich diets, or exposure to cold abolishes REV-ERB repression, and lipolytic signals stimulate Slc25a34 transcription through the peroxisome proliferator-activated receptors. SLC25A34 is proposed to import oxaloacetate into mitochondria, dually supporting the tricarboxylic acid cycle and cytosolic acetyl-CoA production. Elevated cytosolic acetyl-CoA then fuels the synthesis of lipids and promotes transcription of genes enhancing mitochondrial oxidation. Thus, SLC25A34 confers circadian, dietary, and temperature control of adipocyte lipid metabolism.
2026-09-23 | GSE348259 | GEO
Project description:Objective: In vivo studies in humans and mice have implicated the pseudokinase Tribbles 3 (TRIB3) in various aspects of energy metabolism. Whilst cell-based studies indicate a role for TRIB3 in adipocyte differentiation and function, it is unclear if and how these cellular functions may contribute to overall metabolic health. Methods: We investigated the metabolic phenotype of whole-body Trib3 knockout (Trib3KO) mice, focusing on adipocyte and adipose tissue functions. In addition, we combined lipidomics, transcriptomics, interactomics and phosphoproteomics analyses to elucidate cell-intrinsic functions of TRIB3 in pre- and mature adipocytes. Results: Trib3KO mice display increased adiposity, but their insulin sensitivity remains unaltered. Trib3KO adipocytes are smaller and display higher Proliferating Cell Nuclear Antigen (PCNA) levels, indicating potential alterations in either i) proliferation-differentiation balance, ii) impaired expansion after cell division, or iii) an altered balance between lipid storage and release, or a combination thereof. Lipidome analyses suggest TRIB3 involvement in the latter two processes, as triglyceride storage is reduced and membrane composition, which can restrain cellular expansion, is altered. Integrated interactome, phosphoproteome and transcriptome analyses support a role for TRIB3 in all three cellular processes through multiple cellular pathways, including Mitogen Activated Protein Kinase- (MAPK/ERK), Protein Kinase A (PKA)-mediated signaling and Transcription Factor 7 like 2 (TCF7L2) and Beta Catenin-mediated gene expression. Conclusions: Our !ndings support TRIB3 playing multiple distinct regulatory roles in the cytoplasm, nucleus and mitochondria, ultimately controlling adipose tissue homeostasis, rather than affecting a single cellular pathway.
2026-01-26 | PXD046703 | Pride
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:The dark proteome comprises a rapidly expanding catalog of microproteins with largely unknown functions that have been historically ignored in genome annotations. Here, we exploit an in vivo single-cell CRISPR screening strategy in the mouse epidermis to systematically investigate the tissue-wide function of microproteins. We document the global and cell-type-specific roles of microproteins during epidermal development and homeostasis at single-cell transcriptomic resolution and identify a cohort of functional ribosomal protein-like microproteins. Focusing on select candidates, we characterize a novel microprotein on Gm10076, identical to the ribosomal intersubunit bridge protein RPL41, whose perturbation strongly impairs proliferation and protein synthesis. Single-particle cryo-electron microscopy reveals that, unlike canonical RPL41, Gm10076-encoded RPL41 is not incorporated into the ribosomal intersubunit bridge. Instead, Gm10076-encoded RPL41 localizes exclusively to the nucleolus. By contrasting the translational landscapes of Gm10076 and Rpl41 knockouts, we document that Gm10076 loss profoundly remodels translational programs, altering the translational efficiency of ribosome biogenesis factors and disrupting the balance between 40S and 60S maturation programs. Together, our study comprehensively charts the tissue-wide functional landscape of the dark proteome, implicates transcript context-dependent diversification of identical microproteins in shaping tissue-wide translational landscapes and establishes a basis for exploring the impact of microproteins on disease pathogenesis.
2026-09-16 | PXD078928 | Pride
Project description:DCHP targets lipid associated macrophages to disrupt lipid metabolism via crosstalk with adipocyte precursor cells
| PRJNA1033638 | ENA