Journal: bioRxiv

Article Title: The non-coding facet of Pink1 mRNA regulates mitochondria homeostasis

doi: 10.64898/2026.01.05.697839

Figure Lengend Snippet: A. The location of Pink1 mRNA in MuSCs assessed by FISH assay. Mitochondria were stained by mito-dsRed. White arrows indicate Pink1 mRNAs colocalized with mitochondria. Scale bar, 2μm. B. Quantification of the percentage of Pink1 mRNAs localized in mitochondria in MuSCs based on FISH analysis. C. Schematic of the experimental workflow of identifying Pink1 -associated proteins. D. Silver staining of proteins pulled down by the Pink1 mRNA or Pink1-ATGmut RNA. Negative controls included blank beads and two unrelated RNA sequences. Red arrows indicated proteins specifically bound by Pink1 mRNA or Pink1-ATGmut RNA. E. Ven diagram showing proteins specifically bound by Pink1-ATGmut RNA, categorized based on 3 distinct negative controls. Numbers indicated proteins enriched by Pink1-ATGmut relative to each control. Overlapping areas highlighted proteins that were enriched by Pink1-ATGmut RNA across controls. F. Representative proteins specifically enriched by Pink1-ATGmut RNA shown in panel E, along with their unique peptides and log2 fold enrichment relative to 3 negative controls. Proteins located in mitochondria were colored in purple. G. RNA pulldown followed by Immunoblotting analysis showing the interaction between YME1L1 and Pink1 mRNA. beta-ACTIN serves as the negative control. YME1L1, p: precursor form of YME1L1. YME1L1, m: mature form of YME1L1. Non-specific bands were marked by asterisk. H. Representative images of the mitochondria stained by MitoTracker (red) and DAPI (blue) in scramble (siNC) or Yme1l1 RNAi MuSCs and myotubes, respectively. Scale bars, 4μm and 10μm, respectively. I. Quantification of the mitochondria length in MuSCs and myotubes treated by scramble or Yme1l1 siRNA, respectively. J. Immunoblotting analysis showing OPA1 isoforms in scramble (siNC) and Yme1l1 RNAi (siYme1L1) MuSCs, respectively. Beta-ACTIN served as an internal control. The schematic diagram on the left illustrated the pattern of intermediate products during proteolytic processing of OPA1. S2 indicated the sites cleaved by YME1L1. L-OPA1: long OPA1 isoforms; S-OPA1: short OPA1 isoforms. Yme1l1 knockdown led to insufficient YME1L1 level, which impairs OPA1 processing, leading to accumulation of L-OPA1 and the intermediate proteolytic protein product and decreased S-OPA1 d form. K. Immunoblotting analysis showing OPA1 isoforms in scramble (siNC) and Pink1 RNAi (si Pink1 ) MuSCs, respectively. Beta-ACTIN served as an internal control. L. Immunoblotting analysis showing OPA1 isoforms in WT, Tet2 KO, Tet2 KO with Pink1 overexpressed, and Tet2 KO with Pink1-ATGmut overexpressed MuSCs, respectively. Beta-ACTIN served as an internal control. M. A working model of coding independent function of Pink1 mRNA maintaining mitochondria homeostasis in skeletal muscle cells.

Article Snippet: Pink1 cDNA fused with a HA tag or a Flag tag at the C terminal, mito-dsRed sequence (Addgene, 44386, 4777bp – 5755bp, US) were cloned into the pLenti-CMV-puro (Addgene, 17448, US) lentiviral expression vector using ClonExpress II One Step Cloning kit (Vazyme, #C112, China), respectively.

Techniques: Staining, Silver Staining, Control, Western Blot, Negative Control, Knockdown

Journal: bioRxiv

Article Title: The non-coding facet of Pink1 mRNA regulates mitochondria homeostasis

doi: 10.64898/2026.01.05.697839

Figure Lengend Snippet: A. The location of Pink1 mRNA in myotubes assessed by FISH assay. Mitochondria were marked by mito-dsRed. White arrows indicate Pink1 mRNAs colocalized with mitochondria. Scale bar, 4μm. B. Quantification of the percentage of Pink1 mRNAs localized in mitochondria in myotubes based on FISH assay. C. RT-qPCR quantification of the mRNA level after scramble (negative control, NC), and Yme1l1 siRNA knockdown (n=3). D. Immunoblotting analysis showing precursor (p) or mature (m) form of YME1L1 in scramble (siNC) and Yme1l1 RNAi (si Yme1l1 ) MuSCs, respectively. GAPDH served as an internal control. E. Immunoblotting analysis showing precursor (p) or mature (m) form of YME1L1 in scramble (siNC) and Pink1 RNAi (si Pink1 ) MuSCs, respectively. Beta-ACTIN served as an internal control. F. Immunoblotting analysis showing precursor (p) or mature (m) form of YME1L1 in WT and Tet2 KO MuSCs, respectively. Beta-ACTIN served as an internal control. G. Dot plot illustrating mRNA abundance rank (the mRNA with the highest abundance ranks 1, the mRNA with the lowest abundance ranks 16,590; mRNAs with same RPM values have the same rank) and protein abundance rank (the protein with the highest abundance ranks 1, the protein with the lowest abundance ranks 8,300, proteins with same abundance have the same rank) for 17,135 protein-coding genes detected either by RNA-seq or spectrometry in WT MuSCs. Black-colored dots indicated genes with discrepant mRNA level and protein level. Red-colored dots indicated genes with high mRNA level but undetectable protein level. Pink1 is highlighted with a green dot.

Article Snippet: Pink1 cDNA fused with a HA tag or a Flag tag at the C terminal, mito-dsRed sequence (Addgene, 44386, 4777bp – 5755bp, US) were cloned into the pLenti-CMV-puro (Addgene, 17448, US) lentiviral expression vector using ClonExpress II One Step Cloning kit (Vazyme, #C112, China), respectively.

Techniques: Quantitative RT-PCR, Negative Control, Knockdown, Western Blot, Control, Quantitative Proteomics, RNA Sequencing