Additional file 1 of A novel protein encoded by circRsrc1 regulates mitochondrial ribosome assembly and translation during spermatogenesis
Creators: name: Zhang, Shu
name: Wang, Chang
name: Wang, Yue
name: Zhang, Hao
name: Xu, Chen
name: Cheng, Yiwei
name: Yuan, Yan
name: Sha, Jiahao
name: Guo, Xuejiang
name: Cui, Yiqiang
Publisher: name: figshare
Year: 2023
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Developmental Biology
subject: Cancer
subject: Computational Biology

description: Additional file 1: Fig. S1. Peptides translated by detected circRNAs are present in mouse testes. a. The specific peptides translated by circRNAs were detected by mass spectrometry and the corresponding spectra are shown. Fig. S2. The detected circRNAs are present in mouse testes. a. Linear splicing forms of the gene of interest were detected specifically in mouse testes by convergent primers. Both divergent primers detected circular forms in cDNA but not in gDNA of mouse testes. Sanger sequencing confirmed the presence of circular RNAs. Fig. S3. Different knockout mice were constructed using the CRISPR/Cas9 system. a. Relative expression levels of Rsrc1 in mouse testes at different ages in weeks. b. Relative expression levels of Rsrc1 in different germ cells and somatic cells of mouse testes. c. Subcellular localization of circRsrc1 in mouse testes was determined by RT-qPCR. 18S and U6 were used as internal controls. d. Schematic of the targeted exon 4 of mouse and sequence from wild-type and Rsrc1−/−/circRsrc1+/+ mouse. Sanger sequencing after PCR confirmed that the knockout was successful. e. Identification of Rsrc1−/−/circRsrc1+/+ mice by PCR. f. RT- PCR analysis of circRsrc1 and Rsrc1 mRNA levels in wild-type and Rsrc1−/−/circRsrc1+/+ mice. g. Western blotting of Rsrc1 protein in wild-type and Rsrc1−/−/circRsrc1+/+ mice. h. Schematic of the targeted intron of mouse and sequence from wild-type and Rsrc1+/+/circRsrc1−/− mouse. Sanger sequencing after PCR confirmed that the knockout was successful. i. Identification of Rsrc1+/+/circRsrc1−/− mice by PCR. j. RT-PCR analysis of circRsrc1 mRNA levels in wild-type and Rsrc1+/+/circRsrc1−/− mice. Sanger sequencing was performed after PCR. k. Schematic of the targeted intron and exon 3 of sequences from wild-type and Rsrc1−/−/circRsrc1−/− mouse. Sanger sequencing after PCR confirmed that the knockout was successful. l. RT-PCR analysis of circRsrc1 and Rsrc1 mRNA levels in wild-type and Rsrc1−/−/circRsrc1−/− mice. m. RT-qPCR analysis of circRsrc1 and Rsrc1 mRNA levels in wild-type and Rsrc1−/−/circRsrc1−/− mice. n. Western blotting of Rsrc1 protein in wild-type, Rsrc1+/+/circRsrc1−/− and Rsrc1−/−/circRsrc1−/− mice. Fig. S4. Phenotypic analysis of different knockout mice. a. The testicular/body weight ratio of different knockout and wild-type mice are shown. b. Percentage of TUNEL positive tubules in mouse testes. A TUNEL positive tubule contained at least one TUNEL positive cell. c. The average number of TUNEL positive cells in each tubule of mouse testes. d. H&amp;E staining of the testis, caupt epididymidis, cauda epididymidis and sperm from adult wild-type and different knockout mice. Bar, 50 μm. All data are means ± SEM, tested using one-way ANOVA with Tukey’s post hoc analysis *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001. Fig. S5. Construction of knockout cell line. a. RT-qPCR was performed using divergent primers to detect the expression of circRsrc1 in cDNA from GC-1 spg cells and GC-2spdcells. All data are means ± SEM, tested using a two-tailed unpaired t-test; ***P &lt; 0.001. b. Detection of the circular form of circRsrc1 in cDNA of GC-1 spg cells by using divergent primers. Sanger sequencing was performed to confirm the junction site of circRsrc1. c. Mass spectrometry detection of peptides translated by circRsrc1 from wild-type and Rsrc1+/+/circRsrc1−/− cells. d. Establishment of Rsrc1+/+/circRsrc1−/− cell line using CRISPR/Cas9 technology. e. Validation of Rsrc1+/+/circRsrc1−/− cells by PCR. f. FISH with junction-specific and linear-specific probes for circRsrc1 and linear Rsrc1, respectively, in vitro. Bar, 10 μm. g. RT-PCR analysis of circRsrc1 mRNA levels in wild-type and Rsrc1+/+/circRsrc1−/− cells. Sanger sequencing was performed following PCR. h. Rsrc1-161aa effect on cell apoptosis by TUNEL staining. All data are means ± SEM, tested using one-way ANOVA with Tukey’s post hoc analysis. Fig. S6. Rsrc1-161aa affects the levels of reactive oxygen species. a. Representative images showing DHE staining of wild-type, Rsrc1+/+/circRsrc1−/− and rescue cells. Bar, 20 μm. b. ROS levels were quantified by measuring the average gray value of the DHE signal in cells by the ImageJ software. All data are means ± SEM, tested using one-way ANOVA with Tukey’s post hoc analysis; **P &lt; 0.01. c. Western blotting showing protein levels in wild-type, Rsrc1+/+/circRsrc1−/− and rescue cells. Fig. S7. Rsrc1-161aa does not affect C1qbp interaction with the mitochondrial ribosome. a. RT-qPCR quantification of mtDNA amounts based on the ratio of mtDNA/nuclearDNA. All data are means ± SEM, tested using one-way ANOVA with Tukey’s post hoc analysis. b. RT-qPCR detection of certain gene transcripts of the mitochondrial respiratory chain in wild-type and Rsrc1+/+/circRsrc1−/− cells. RNA data were normalized to 18S levels. All data are means ± SEM, tested using a two-tailed unpaired t-test. c. circRsrc1 regulating the association of C1qbp to mitoribosomal proteins remained unchanged in Rsrc1+/+/circRsrc1−/− cells. d. Western blotting analysis of mitochondrial ribosomal proteins in wild-type, Rsrc1+/+/circRsrc1−/− and rescue cells. β-tubulin was the loading control. e. Schematic model. The loss of Rsrc1-161aa results in weakened binding ability between C1qbp and mitochondrial mRNA, as well as affecting the assembly of mitochondrial ribosomes, leading to decreased translation of mitochondrial proteins and ultimately causing damage to oxidative phosphorylation.