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1.
Cell Chem Biol ; 30(10): 1248-1260.e4, 2023 10 19.
Article in English | MEDLINE | ID: mdl-37442135

ABSTRACT

Speckle-type pox virus and zinc finger (POZ) protein (SPOP), a substrate recognition adaptor of cullin-3 (CUL3)/RING-type E3 ligase complex, is investigated for its role in cardiac fibrosis in our study. Cardiac fibroblasts (CFs) activation was achieved with TGF-ß1 (20 ng/mL) and MI mouse model was established by ligation of the left anterior descending coronary, and lentivirus was employed to mediate interference of SPOP expression. SPOP was increased both in fibrotic post-MI mouse hearts and TGF-ß1-treated CFs. The gain-of-function of SPOP promoted myofibroblast transformation in CFs, and exacerbated cardiac fibrosis and cardiac dysfunction in MI mice, while the loss-of-function of SPOP exhibited the opposite effects. Mechanistically, SPOP bound to the receptor of activated protein C kinase 1 (RACK1) and induced its ubiquitination and degradation by recognizing Ser/Thr-rich motifs on RACK1, leading to Smad3-mediated activation of CFs. Forced RACK1 expression canceled the effects of SPOP on cardiac fibrosis. The study reveals therapeutic targets for fibrosis-related cardiac diseases.


Subject(s)
Myocardial Infarction , Transforming Growth Factor beta1 , Animals , Mice , Fibrosis , Myocardial Infarction/complications , Myocardial Infarction/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Receptors for Activated C Kinase , Transforming Growth Factor beta1/metabolism , Transforming Growth Factor beta1/pharmacology
2.
FASEB J ; 37(3): e22797, 2023 03.
Article in English | MEDLINE | ID: mdl-36753405

ABSTRACT

Cardiac fibrosis is a common pathological manifestation in multiple cardiovascular diseases and often results in myocardial stiffness and cardiac dysfunctions. LncRNA (long noncoding RNA) participates in a number of pathophysiological processes. However, its role in cardiac fibrosis remains unclear. The purpose of this study was to investigate the role and molecular mechanism of MetBil in regulating cardiac fibrosis. Our data showed that METTL3 binding lncRNA (MetBil) was significantly increased both in fibrotic tissue following myocardial infarction (MI) in mice and in cardiac fibroblasts (CFs) exposed to TGF-ß1 (20 ng/mL) or 20% FBS. Overexpression of MetBil augmented collagen deposition, CF proliferation and activation while silencing MetBil exhibited the opposite effects. Importantly, heterozygous knockout of MetBil alleviated cardiac fibrosis and improved cardiac function after MI. RNA pull-down and RNA-binding protein immunoprecipitation assay showed that METTL3 is a direct downstream target of MetBil; consistently, MetBil and METTL3 were co-localized in both the nucleus and cytoplasm of CFs. Interestingly, MetBil regulated METTL3 expression at protein level, but not mRNA level, in ubiquitin-proteasome pathway. Enforced expression of METTL3 canceled the antifibrotic effects of silencing MetBil reflected by increased collagen production, CF proliferation and activation. Most notably, the m6A-modified fibrosis-regulated genes mediated by METTL3 are profoundly involved in the regulation of MetBil in the cardiac fibrosis following MI. Our study reveals that MetBil as a novel regulator of fibrosis promotes cardiac fibrosis via interacting with METTL3 and regulating the expression of the methylated fibrosis-associated genes, providing a new intervening target for fibrosis-associated cardiac diseases.


Subject(s)
Heart Diseases , Myocardial Infarction , RNA, Long Noncoding , Mice , Animals , RNA, Long Noncoding/genetics , Myocardial Infarction/metabolism , Fibrosis , Methyltransferases/genetics , Methyltransferases/metabolism , Collagen/genetics , Collagen/metabolism
3.
Genetics ; 205(4): 1489-1501, 2017 04.
Article in English | MEDLINE | ID: mdl-28213476

ABSTRACT

Respiration, the core of mitochondrial metabolism, depends on the function of five respiratory complexes. Many respiratory chain-related proteins are encoded by the mitochondrial genome and their RNAs undergo post-transcriptional modifications by nuclear genome-expressed factors, including pentatricopeptide repeat (PPR) proteins. Maize defective kernel 10 (dek10) is a classic mutant with small kernels and delayed development. Through positional cloning, we found that Dek10 encodes an E-subgroup PPR protein localized in mitochondria. Sequencing analysis indicated that Dek10 is responsible for the C-to-U editing at nad3-61, nad3-62, and cox2-550 sites, which are specific editing sites in monocots. The defects of these editing sites result in significant reduction of Nad3 and the loss of Cox2. Interestingly, the assembly of complex I was not reduced, but its NADH dehydrogenase activity was greatly decreased. The assembly of complex IV was significantly reduced. Transcriptome and transmission electron microscopy (TEM) analysis revealed that proper editing of nad3 and cox2 is critical for mitochondrial functions, biogenesis, and morphology. These results indicate that the E-subgroup PPR protein Dek10 is responsible for multiple editing sites in nad3 and cox2, which are essential for mitochondrial functions and plant development in maize.


Subject(s)
Mitochondria/genetics , RNA Editing , Zea mays/genetics , Electron Transport Complex IV/genetics , Electron Transport Complex IV/metabolism , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Plant Proteins/genetics , Plant Proteins/metabolism , Seeds/genetics , Seeds/metabolism , Zea mays/growth & development , Zea mays/metabolism
4.
BMC Biotechnol ; 16(1): 58, 2016 08 11.
Article in English | MEDLINE | ID: mdl-27515683

ABSTRACT

BACKGROUND: CRISPR/Cas9 genome editing strategy has been applied to a variety of species and the tRNA-processing system has been used to compact multiple gRNAs into one synthetic gene for manipulating multiple genes in rice. RESULTS: We optimized and introduced the multiplex gene editing strategy based on the tRNA-processing system into maize. Maize glycine-tRNA was selected to design multiple tRNA-gRNA units for the simultaneous production of numerous gRNAs under the control of one maize U6 promoter. We designed three gRNAs for simplex editing and three multiple tRNA-gRNA units for multiplex editing. The results indicate that this system not only increased the number of targeted sites but also enhanced mutagenesis efficiency in maize. Additionally, we propose an advanced sequence selection of gRNA spacers for relatively more efficient and accurate chromosomal fragment deletion, which is important for complete abolishment of gene function especially long non-coding RNAs (lncRNAs). Our results also indicated that up to four tRNA-gRNA units in one expression cassette design can still work in maize. CONCLUSIONS: The examples reported here demonstrate the utility of the tRNA-processing system-based strategy as an efficient multiplex genome editing tool to enhance maize genetic research and breeding.


Subject(s)
Gene Editing/methods , Genes, Plant/genetics , Glycine/genetics , Plant Proteins/genetics , RNA, Transfer/genetics , Zea mays/genetics , CRISPR-Associated Proteins , Clustered Regularly Interspaced Short Palindromic Repeats , Plants, Genetically Modified/genetics
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