nrdc  (Santa Cruz Biotechnology)

Journal: Journal of Sport and Health Science

Article Title: Exercise training-induced extracellular miR-136-3p modulates glucose uptake and myogenesis through targeting of NRDC in human skeletal muscle

doi: 10.1016/j.jshs.2025.101091

Figure Lengend Snippet: NRDC is a direct target of miR-136-3p in human myotubes. Skeletal muscle NRDC mRNA is responsive to training and inactivity. (A) Tissue mRNA expression of NRDC from the Human Protein Atlas database showing enriched expression of NRDC in human skeletal muscle. (B) The miR-136-3p target site in the NRDC gene is highly conserved in mammals. (C) Luciferase activity in HEK293 cells co-transfected the NRDC 3’UTR and miR-136-3p with or without anti-miR136-3p inhibitors. miR-136-3p transfection downregulates NRDC (D) mRNA and (E) representative image of protein abundance in human myotubes. (F) Publicly available data ( GSE14413 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 6 weeks of endurance training ( n = 8). (G) Publicly available data ( GSE120862 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 2 months of aerobic training ( n = 10). (H) Publicly available data ( GSE14901 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 14 days of immobilization ( n = 24). * p < 0.05, ** p < 0.005. GSE = gene set enrichment; HEK293 = human embryonic kidney; miR = microRNA; NC = negative control; NRDC = nardilysin convertase; nTPM = normalized transcripts per million; si NRDC = small interfering RNA of NRDC ; UTR = untranslated region.

Article Snippet: Membranes were incubated with primary antibodies directed to NRDC (sc-137199; Santa Cruz Biotechnology, Dallas, TX, USA), AMP-activated protein kinase α (AMPKα) (#2532; Cell Signaling Technology, Danvers, MA, USA), Akt substrate of 160 kDa (AS160) (#2670; Cell Signaling Technology), phospho-AS160 (#8730; Cell Signaling Technology), protein kinase B (AKT) (#9272; Cell Signaling Technology), phospho-AKT (Ser473) (#9271; Cell Signaling Technology), or puromycin (MABE343; Merck Burlington, MA, USA).

Techniques: Expressing, Luciferase, Activity Assay, Transfection, Quantitative Proteomics, Negative Control, Small Interfering RNA

Journal: Journal of Sport and Health Science

Article Title: Exercise training-induced extracellular miR-136-3p modulates glucose uptake and myogenesis through targeting of NRDC in human skeletal muscle

doi: 10.1016/j.jshs.2025.101091

Figure Lengend Snippet: NRDC is a direct target of miR-136-3p in human myotubes. Skeletal muscle NRDC mRNA is responsive to training and inactivity. (A) Tissue mRNA expression of NRDC from the Human Protein Atlas database showing enriched expression of NRDC in human skeletal muscle. (B) The miR-136-3p target site in the NRDC gene is highly conserved in mammals. (C) Luciferase activity in HEK293 cells co-transfected the NRDC 3’UTR and miR-136-3p with or without anti-miR136-3p inhibitors. miR-136-3p transfection downregulates NRDC (D) mRNA and (E) representative image of protein abundance in human myotubes. (F) Publicly available data ( GSE14413 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 6 weeks of endurance training ( n = 8). (G) Publicly available data ( GSE120862 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 2 months of aerobic training ( n = 10). (H) Publicly available data ( GSE14901 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 14 days of immobilization ( n = 24). * p < 0.05, ** p < 0.005. GSE = gene set enrichment; HEK293 = human embryonic kidney; miR = microRNA; NC = negative control; NRDC = nardilysin convertase; nTPM = normalized transcripts per million; si NRDC = small interfering RNA of NRDC ; UTR = untranslated region.

Article Snippet: Skeletal muscle NRDC mRNA is responsive to training and inactivity. (A) Tissue mRNA expression of NRDC from the Human Protein Atlas database showing enriched expression of NRDC in human skeletal muscle. (B) The miR-136-3p target site in the NRDC gene is highly conserved in mammals. (C) Luciferase activity in HEK293 cells co-transfected the NRDC 3’UTR and miR-136-3p with or without anti-miR136-3p inhibitors. miR-136-3p transfection downregulates NRDC (D) mRNA and (E) representative image of protein abundance in human myotubes. (F) Publicly available data ( GSE14413 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 6 weeks of endurance training ( n = 8). (G) Publicly available data ( GSE120862 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 2 months of aerobic training ( n = 10). (H) Publicly available data ( GSE14901 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 14 days of immobilization ( n = 24).

Techniques: Expressing, Luciferase, Activity Assay, Transfection, Quantitative Proteomics, Negative Control, Small Interfering RNA

Journal: Journal of Sport and Health Science

Article Title: Exercise training-induced extracellular miR-136-3p modulates glucose uptake and myogenesis through targeting of NRDC in human skeletal muscle

doi: 10.1016/j.jshs.2025.101091

Figure Lengend Snippet: An mRNA analysis of miR-136-3p-transfected or NRDC-silenced cultured myotubes. (A) Volcano plot of gene expression in primary myotubes transfected with miR-136-3p (left) and/or siRNA against NRDC (right) as compared to respective controls. (B) Venn diagram of DEGs. (C) Bubble plots of the GSEA. CKM and MYOD gene expression in (D) miR-136-3p-transfected myoblasts and in (E) NRDC -silenced myoblasts. Gene expression showing transition of (F) CKM and (G) MYOD during differentiation of myoblasts to myotubes. * p < 0.05, ** p < 0.005; # p < 0.0005. CKM = creatine kinase M-type; DEGs = differentially expressed genes; FDR = false discovery rate; GSEA = gene set enrichment analysis; MYOD = myoblast determination protein 1; miR = microRNA; NES = normalized enrichment score; NC = negative control; NRDC = nardilysin convertase; ns = no significance; scr = negative control for small interfering RNA; si NRDC = small interfering RNA of NRDC .

Article Snippet: Skeletal muscle NRDC mRNA is responsive to training and inactivity. (A) Tissue mRNA expression of NRDC from the Human Protein Atlas database showing enriched expression of NRDC in human skeletal muscle. (B) The miR-136-3p target site in the NRDC gene is highly conserved in mammals. (C) Luciferase activity in HEK293 cells co-transfected the NRDC 3’UTR and miR-136-3p with or without anti-miR136-3p inhibitors. miR-136-3p transfection downregulates NRDC (D) mRNA and (E) representative image of protein abundance in human myotubes. (F) Publicly available data ( GSE14413 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 6 weeks of endurance training ( n = 8). (G) Publicly available data ( GSE120862 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 2 months of aerobic training ( n = 10). (H) Publicly available data ( GSE14901 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 14 days of immobilization ( n = 24).

Techniques: Transfection, Cell Culture, Gene Expression, Negative Control, Small Interfering RNA

Journal: bioRxiv

Article Title: Mutational scanning by multiplexed genome editing of the essential transcription termination factor Nrd1

doi: 10.1101/2025.09.09.675026

Figure Lengend Snippet: A. Schematic representation of the functional domains along the Nrd1 protein. (CID: CTD-interacting domain; NAID: Nab3 interaction domain; RS: R/S rich region; SD: Split domain; RRM: RNA recognition motif; PrD: Prion-like domain) B. Overview of the platform to scan essential genes in yeast. First, a complementation system is introduced at a safe-harbor locus on Chr. XVI by recoding the sequence of the target gene. This second wild-type copy is expressed under the control of a degron system. During editing, CRISPR-Cas9 is guided only to the endogenous target gene, leaving the recoded wild-type copy to complement potential deleterious mutations. Finally, the phenotype associated with the introduced edit can be unveiled by auxin (IAA) mediated depletion of the recoded wild-type copy. C. Dispersion plot of the fitness scores of the edits computed over a 20-generation time course in the presence of IAA (phenotype exposed), and in YPD (phenotype complemented). D. List of the deleterious edits identified by filtering for the top 5% of effects, IAA-conditional effects and depletion by the end of the experiment. The number of barcodes (Bc) associated to each edit is indicated, as well as their agreement of the phenotype.

Article Snippet: The complementation system was introduced into the MAGESTIC base strain yKR965 by assembling the following fragments via transformation-associated recombination upon CRISPR-Cas9 cleavage at YPRCΔ15 on Chromosome XVI (referred to as “site 20” in ) :(i) a cassette encoding OsTIR1::3xMyc under the control of a constitutive ADH1 promoter, amplified by PCR from pOsTIR1w/oGFP ( ); (ii) a constitutively active ribosomal promoter (p RPLB8 ), amplified from wild type S. cerevisiae genome; (iii) a recoded NRD1 gene, which sequence follows the logic described below and synthesized by Twist Bioscience; an AID::6xFLAG tag, amplified by PCR from pHyg-AID*-6FLAG ( ).

Techniques: Functional Assay, Sequencing, Control, CRISPR, Dispersion

Journal: bioRxiv

Article Title: Mutational scanning by multiplexed genome editing of the essential transcription termination factor Nrd1

doi: 10.1101/2025.09.09.675026

Figure Lengend Snippet: A. Western blotting analysis of the Nrd1rec expression level in presence and absence of IAA treatment (1 hour). The detected levels of Tir1 are also shown. B. Quantification of A and other 2 independent replicates. C. Growth test of plates of the indicated strains in presence and absence of IAA and at the indicated temperature. Plates were incubated for 2 days before being photographed. D. Pie plot representation of the percentages and the overlap of the edits present in the Nrd1 alanine scanning library compared to the total amount of residues in Nrd1. E. Pies of the percentages of amino acid positions in Nrd1 associated to the indicated numbers of barcodes. F. Distribution of the edits along the Nrd1 gene. Mutation categories (missense, synonymous and premature termination codons, PTCs) are shown with different colors as indicated in the legend. G. Schematic representation of the 20-generation time course experiment and the downstream fitness score quantification. Cells are grown for 20 generations in an automatized platform, with samples collected each four generations (see more in Materials and Methods). After gDNA extraction and PCR amplification of the barcode locus , the products are sequenced and the normalized count of barcodes quantified along the time course to fit a linear regression, the slope of which is used as a fitness index. H. Dispersion plots of the of the barcode counts for the indicated replicates. Correlation scores are shown for each replicate pair. J. Edit distribution along the NRD1 gene from barcode counts during library prep (pre-editing stage).

Article Snippet: The complementation system was introduced into the MAGESTIC base strain yKR965 by assembling the following fragments via transformation-associated recombination upon CRISPR-Cas9 cleavage at YPRCΔ15 on Chromosome XVI (referred to as “site 20” in ) :(i) a cassette encoding OsTIR1::3xMyc under the control of a constitutive ADH1 promoter, amplified by PCR from pOsTIR1w/oGFP ( ); (ii) a constitutively active ribosomal promoter (p RPLB8 ), amplified from wild type S. cerevisiae genome; (iii) a recoded NRD1 gene, which sequence follows the logic described below and synthesized by Twist Bioscience; an AID::6xFLAG tag, amplified by PCR from pHyg-AID*-6FLAG ( ).

Techniques: Western Blot, Expressing, Incubation, Mutagenesis, Extraction, Amplification, Dispersion

Journal: bioRxiv

Article Title: Mutational scanning by multiplexed genome editing of the essential transcription termination factor Nrd1

doi: 10.1101/2025.09.09.675026

Figure Lengend Snippet: A. Heatmaps of the fitness score for the edits at the indicated position along Nrd1. Both missense and synonymous edits are shown, in presence and absence of IAA. The positions of the functional domains of Nrd1 are indicated on the top. B. Rendering of the structures of the CID domain bound to an Rpb1 (RNAPII) CTD repeat (left) (PDB: 2LO6), the Nrd1-interaction motif (NIM) of Sen1 (center) (PDB: 6GC3) and the NIM of Trf4 (right) (PDB: 2MOW). Lethal residues of Nrd1 identified in this study are highlighted in red. C. As in B, but for the RRM motif bound to an RNA (PDB: 5O20). D. Heatmap of the predicted (Pred.) instability of the CID (top) and the RRM (bottom) of Nrd1 using the FoldX AlaScan suite. The experimental fitness scores (Exp.) of these regions are also displayed. E. Growth defects for the indicated strains in the presence of IAA. The defect was computed as a ratio (IAA/YPD) of the area under the curve (AUC) of the normalized OD density over 6 generations of actively growing yeast cells.

Article Snippet: The complementation system was introduced into the MAGESTIC base strain yKR965 by assembling the following fragments via transformation-associated recombination upon CRISPR-Cas9 cleavage at YPRCΔ15 on Chromosome XVI (referred to as “site 20” in ) :(i) a cassette encoding OsTIR1::3xMyc under the control of a constitutive ADH1 promoter, amplified by PCR from pOsTIR1w/oGFP ( ); (ii) a constitutively active ribosomal promoter (p RPLB8 ), amplified from wild type S. cerevisiae genome; (iii) a recoded NRD1 gene, which sequence follows the logic described below and synthesized by Twist Bioscience; an AID::6xFLAG tag, amplified by PCR from pHyg-AID*-6FLAG ( ).

Techniques: Functional Assay

Journal: bioRxiv

Article Title: Mutational scanning by multiplexed genome editing of the essential transcription termination factor Nrd1

doi: 10.1101/2025.09.09.675026

Figure Lengend Snippet: A. Western blotting analysis of GFP-tagged Nrd1 analogs. B. (Top) Summary table of the different phenotypes displayed by each strain; (bottom) representative microscopy fields and schematic representation of the different categories of Nrd1 localization identified. C. RNA quantification by RT-qPCR of the extended SNR13 transcript in the indicated strains upon IAA treatment. D. Fitness score and alignment of the protein sequences of S. cerevisiae Nrd1, S. pombe Seb1, H. sapiens SCAF4 and SCAF8, S. cerevisiae Pcf11 and H. sapiens PCF11, in the indicated regions. Stars indicate positions in SCAF4 the mutation of which has been linked to neurodevelopmental disorders.

Article Snippet: The complementation system was introduced into the MAGESTIC base strain yKR965 by assembling the following fragments via transformation-associated recombination upon CRISPR-Cas9 cleavage at YPRCΔ15 on Chromosome XVI (referred to as “site 20” in ) :(i) a cassette encoding OsTIR1::3xMyc under the control of a constitutive ADH1 promoter, amplified by PCR from pOsTIR1w/oGFP ( ); (ii) a constitutively active ribosomal promoter (p RPLB8 ), amplified from wild type S. cerevisiae genome; (iii) a recoded NRD1 gene, which sequence follows the logic described below and synthesized by Twist Bioscience; an AID::6xFLAG tag, amplified by PCR from pHyg-AID*-6FLAG ( ).

Techniques: Western Blot, Microscopy, Quantitative RT-PCR, Mutagenesis