Human RCC1L is involved in the maintenance of mitochondrial nucleoids and mtDNA

Matsumoto, Emi; Sasaki, Taeko; Higashiyama, Tetsuya; Sasaki, Narie

doi:10.1038/s41598-025-98397-y

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Published: 21 April 2025

Human RCC1L is involved in the maintenance of mitochondrial nucleoids and mtDNA

Emi Matsumoto¹,
Taeko Sasaki^2,3,
Tetsuya Higashiyama¹ &
…
Narie Sasaki ORCID: orcid.org/0000-0002-9784-3674^1,4

Scientific Reports volume 15, Article number: 13811 (2025) Cite this article

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Abstract

Mitochondrial DNA (mtDNA) is organized with proteins into mitochondrial nucleoid (mt-nucleoid). The mt-nucleoid is a unit for the maintenance and function of mtDNA. The regulator of chromosome condensation 1-like protein (RCC1L) performs various functions in mitochondria, including translation, but its involvement in regulating mt-nucleoid maintenance is unknown. Herein, we found that human RCC1L was required to maintain mt-nucleoids and mtDNA. Human RCC1L has three splicing isoforms: RCC1L^V1, RCC1L^V2, and RCC1L^V3. Knockout (KO) cells lacking all RCC1L isoforms, which were lethal without pyruvate and uridine, exhibited a decrease in mt-nucleoids and mtDNA, along with swollen and fragmented mitochondria. Among the three RCC1L isoforms, only RCC1L^V1 recovered all phenotypes observed in RCC1L KO cells. As the treatment of wild-type cells with chloramphenicol, a mitochondrial translation inhibitor, did not lead to the decrease in mt-nucleoids accompanied by mtDNA depletion, the decrease in mt-nucleoids and mtDNA in RCC1L KO cells was not solely attributed to impaired mitochondrial translation. Using conditional RCC1L KO cells, we observed a rapid decrease in mt-nucleoids and mtDNA during a specific period following RCC1L loss. Our findings indicate that RCC1L regulates the maintenance of mt-nucleoids and mtDNA besides its role in mitochondrial translational regulation.

Introduction

Mitochondria produce energy through oxidative phosphorylation (OXPHOS) and possess their own DNA (mtDNA). Human mtDNA is a 16.6 kbp circular DNA that encodes 13 essential subunits of the OXPHOS system, 2 rRNA, and 22 tRNA¹. Defects in mtDNA maintenance result in mitochondrial dysfunction, linked to human disease and aging^2,3,4. In mitochondria, mtDNA is packaged with proteins into the mitochondrial nucleoid (mt-nucleoid), a highly organized structure, which is the functional unit for mtDNA replication, distribution, and transcription^5,6. In humans, thousands of mtDNA copies per cell have been observed as hundreds of punctate mt-nucleoids^5,6,7. We have previously developed a method for specific labeling of mt-nucleoids using SYBR Green I in HeLa cells and demonstrated that mt-nucleoids double from G₁ to G₂ phases and are distributed approximately equally among daughter cells, suggesting that the maintenance of mt-nucleoids is regulated during the cell cycle⁸. However, the mechanisms underlying the regulation of mt-nucleoid maintenance remain poorly understood.

In this study, we found that the regulator of chromosome condensation 1-like (RCC1L) is involved in the maintenance of mt-nucleoids and mtDNA. Human RCC1L, also known as Williams–Beuren Syndrome Chromosome Region 16 (WBSCR16), is encoded in the deletion region of Williams–Beuren syndrome (WBS), a neurodevelopmental disorder characterized by cardiovascular disease, mental retardation, and dysmorphic facial features⁹. For human RCC1L, three splicing isoforms, namely RCC1L^V1, RCC1L^V2, and RCC1L^V3, have been reported in the NCBI Consensus CDS (CCDS) database, all of which share the same N-terminus. Previously, we have reported that RCC1L^V1 adopts the seven-bladed β-propeller fold similar to RCC1^10,11. RCC1, a guanine nucleotide exchange factor (GEF) in the nucleus, is involved in the regulation of various functions, such as nuclear protein transport, spindle formation, and nuclear membrane assembly¹². Various functions of RCC1L have also been reported. RCC1L is involved in mitochondrial ribosome assembly, and mitochondrial 16S rRNA processing^13,14. Loss of RCC1L leads to defects in mitochondrial translation^13,14,15. GTPases involved in mitochondrial ribosome assembly, including GTPBP10, ERAL1, and C4orf14, are putative targets of RCC1L¹³. RCC1L also has GEF activity for OPA1, a GTPase important for mitochondrial inner membrane fusion¹⁶. Knockout (KO) of RCC1L in HeLa cells results in mitochondrial fragmentation, suggesting the importance of RCC1L in mitochondrial fusion¹⁶. Moreover, RCC1L contributes to pyrimidine nucleotide metabolism in mitochondria by interacting with the nucleoside diphosphate kinase NME6^17,18.

This study explores the role of RCC1L using RCC1L KO cells, with an emphasis on mt-nucleoids. RCC1L KO cells exhibited a decrease in the number of mt-nucleoids and the amount of mtDNA, accompanied by growth defects and mitochondrial fragmentation, all of which were recovered by the expression of RCC1L^V1. The decrease in the number of mt-nucleoids and the amount of mtDNA in RCC1L KO cells cannot be explained solely by impaired mitochondrial translation. Moreover, we observed a rapid decrease in mt-nucleoids and mtDNA during a specific period following the loss of RCC1L. This study proposes a novel function of RCC1L in maintaining mt-nucleoids and mtDNA besides its role in mitochondrial translation.

Results

RCC1L is required for the maintenance of mt-nucleoids and mtDNA

We first investigated the expression of RCC1L isoforms in HeLa cells by quantitative PCR (qPCR). RCC1L^V1 was expressed at levels 150 times and 2,900 times higher than RCC1L^V2 and RCC1L^V3, respectively (Supplementary Fig. S1). To analyze the effect of RCC1L loss on mt-nucleoids, we established stable RCC1L KO HeLa cell lines using the CRISPR/Cas9 technology. Exon1-8 are common in all humans RCC1L splicing isoforms, RCC1L^V1, RCC1L^V2, and RCC1L^V3 (Supplementary Fig. S2). We used sgRNA targeting a sequence in exon5 to knockout all isoforms. Although no homozygous RCC1L KO cells were obtained in DMEM, we established two types of RCC1L KO cell lines in DMEM supplemented with pyruvate and uridine (Supplementary Fig. S3). These KO cells had a single nucleotide deletion and/or insertion, resulting in a frameshift that introduced a stop codon immediately following mutation.

To confirm the loss of RCC1L in the two KO cell lines, we performed western blot analysis using two anti-RCC1L antibodies raised against the polypeptides encoded in exon2-3 or exon2-4 (Fig. 1A). The mitochondria-targeting peptide was predicted to be the first 38 amino acids at the N-terminus of RCC1L by the TargetP 2.0¹⁹, and so the molecular sizes of mature RCC1L^V1, RCC1L^V2, and RCC1L^V3 were expected to be 46, 45, and 34 kDa, respectively. In the wild-type, two bands at approximately 44 and 32 kDa were observed for both anti-RCC1L antibodies (Fig. 1A). The band at 32 kDa did not disappear in either KO cell line, suggesting that this band was nonspecific. In contrast, the band at 44 kDa disappeared in both RCC1L KO cell lines (Fig. 1A). These results suggest that the 44 kDa band represents RCC1L^V1, and RCC1L^V2 and RCC1L^V3 are undetectable due to their low expression.

In wild-type cells, the growth rate was unaffected by the presence or absence of pyruvate and uridine in the culture medium. RCC1L KO cells did not grow without pyruvate and uridine but did at a reduced rate in the presence of pyruvate and uridine (Fig. 1B).

Cells were co-stained with MitoTracker and SYBR Green I to clarify the effect of RCC1L loss on mitochondria and mt-nucleoids. In both RCC1L KO cell lines, the tubular mitochondrial network observed in wild-type cells was disrupted, resulting in swollen and fragmented mitochondria (Fig. 1C). Furthermore, the number of mt-nucleoids significantly decreased in KO cells (Fig. 1C, D). Similar phenotypes were observed by immunostaining with anti-Tom20 and anti-Cytochrome c antibodies for mitochondria, and anti-DNA and TFAM antibodies for mt-nucleoids (Supplementary Fig. S4). In addition, a decrease in the amount of mtDNA in KO cells was confirmed using qPCR analysis (Fig. 1E). These results suggest that RCC1L is involved in the maintenance of mt-nucleoids and mtDNA. To evaluate mtDNA replication ability, we analyzed the relative amounts of mtDNA and nuclear DNA per 1 mL of culture at 0, 48, 96, and 120 h using qPCR (Fig. 1F). RCC1L KO cells exhibit a lower mtDNA replication rate than wild-type cells. Moreover, the replication rate of mtDNA was similar to that of nuclear DNA in both wild-type and RCC1L KO cells (Fig. 1F), indicating that RCC1L KO cells could replicate mtDNA in proportion to cell growth.

RCC1L^V1 is required for the maintenance of mt-nucleoids and mtDNA

To determine whether each RCC1L isoform could recover the phenotypes observed in RCC1L KO cells, we transfected each FLAG-tagged RCC1L isoform into RCC1L KO cells, and the transfected cells were selected with G418 in a culture medium containing pyruvate and uridine. When the expression of the FLAG-tagged isoforms in RCC1L KO cells was confirmed by western blot analysis, all three isoforms were detected using anti-FLAG antibodies (Fig. 2A). In contrast, FLAG-tagged RCC1L^V2 and RCC1L^V3 were not detected with anti-RCC1L antibodies, likely due to their low expression levels and the low sensitivity of the antibody.

In the culture medium without pyruvate and uridine, RCC1L KO cells transfected with RCC1L^V1 recovered their reduced growth (Fig. 2B and Supplementary Fig. S5). In contrast, the growth of RCC1L KO cells transfected with RCC1L^V2 or RCC1L^V3 did not recover (Fig. 2B and Supplementary Fig. S5).

To observe mitochondria in RCC1L KO cells transfected with RCC1L^V1, RCC1L^V2, and RCC1L^V3, cells were co-immunostained with anti-FLAG and anti-Tom20 antibodies. Immunostaining with anti-FLAG antibodies was used to select and observe cells expressing RCC1L^V1, RCC1L^V2, and RCC1L^V3 at comparable levels. FLAG-positive cells expressing RCC1L^V1 exhibited a tubular mitochondrial network, whereas those expressing RCC1L^V2 or RCC1L^V3 did not (Fig. 2C). Furthermore, co-immunostaining with anti-FLAG and anti-DNA antibodies showed that the number of mt-nucleoids increased only in FLAG-positive cells expressing RCC1L^V1 (Fig. 2D). Similar recovery of mitochondrial morphology and the number of mt-nucleoids was also observed via co-staining with MitoTracker and SYBR Green I (Supplementary Fig. S5). The number of mt-nucleoids and amount of mtDNA in RCC1L^V1-expression cells was comparable to that in the wild-type cells (Fig. 2E, F). These results suggest that RCC1L^V1 is also involved in mitochondrial morphology and the maintenance of mt-nucleoids and mtDNA.

A decrease in mt-nucleoids accompanied by mtDNA depletion is not solely induced by impaired mitochondrial translation

Loss of RCC1L impairs mitochondrial ribosome assembly, thereby inducing mitochondrial translation dysfunction^13,14,15. To determine whether the phenotypes observed in RCC1L KO cells were due to mitochondrial translation dysfunction, we examined the effects of the mitochondrial translation inhibitor, chloramphenicol (CAP), on wild-type cells and compared them with the phenotypes of RCC1L KO cells. To examine the long-term effect of mitochondrial translation dysfunction, wild-type cells were treated with 100 µg/mL CAP in the presence of pyruvate and uridine for 1 month. CAP-treated cells (CAP cells) showed swollen and fragmented mitochondria, closely resembling the phenotype of RCC1L KO cells (Fig. 3A and Supplementary Fig. S6). Electron microscopy revealed a reduction in the number of cristae and alterations in their orientation in RCC1L KO and CAP cells (Fig. 3B). The length of the cristae per mitochondrial area was similar in both cell types (Fig. 3C). Accompanying morphological changes in the mitochondria and cristae, the mitochondrial membrane potential was reduced in both cell types (Supplementary Fig. S7). We also showed that the number of mt-nucleoids in CAP cells decreased (Fig. 3A, D, and Supplementary Fig. S6). However, unlike RCC1L KO cells, mt-nucleoids were larger than those of wild-type cells, and the amount of mtDNA remained unchanged in CAP cells (Fig. 3A, E, and Supplementary Fig. S6). These results suggest that the phenotypes of mt-nucleoids and mtDNA observed in RCC1L KO cells are not directly caused by mitochondrial translation dysfunction.

MALSU1, TFAM, and NME6 are specifically reduced in RCC1L KO cells but not in CAP cells

We compared the changes in mitochondrial proteins between RCC1L KO and CAP cells using western blot analysis (Fig. 4). In both RCC1L KO and CAP cells, the expression of MTCO2, which is encoded by mtDNA, was not detected due to mitochondrial translation dysfunction. MALSU1, a protein involved in the maturation of the large subunit of mitochondrial ribosome, is unaffected by CAP treatment and undetectable in human cells without mtDNA (ρ0 cells)^20,21. As expected, MALSU1 expression was reduced in RCC1L KO cells but not in CAP cells (Fig. 4). Among the proteins involved in mtDNA replication, including POLG, an mtDNA polymerase; SSBP1, a mitochondrial single-stranded DNA-binding protein; TWINKLE, a mitochondrial helicase; TFAM, an mtDNA packaging protein, POLG and TFAM were reduced in RCC1L KO cells. Reduced POLG levels were also observed in CAP cells, suggesting that POLG is not involved in RCC1L-mediated mtDNA maintenance. In contrast, no reduction in TFAM was observed in CAP cells. We also examined several RCC1L-interacting proteins potentially associated with mtDNA maintenance, including NME6, C4orf14, and OPA1. NME6 and C4orf14 expression was reduced in RCC1L KO cells. Between these two proteins, a reduction in NME6 expression was not observed in CAP cells. Eight OPA1 isoforms are expressed as combinations of the long (L-OPA1) and short (S-OPA1) forms in humans²². Five bands (a–e) were detected using anti-OPA1 antibodies in the wild-type (Fig. 4). In both RCC1L KO and CAP cells, the L-OPA1 (a) form, was reduced, and the S-OPA1 forms (c, e), were increased. We also confirmed that the levels of all proteins were recovered by RCC1L^V1 expression in the RCC1L KO cells. These results suggest that the reduction in MALSU1, TFAM, and NME6 expression in RCC1L KO cells is not attributed to mitochondrial translation dysfunction.

A rapid decrease in mt-nucleoids and mtDNA occurs during a specific period following the loss of RCC1L

To monitor the progression of a decrease in mt-nucleoids and mtDNA following RCC1L loss, we generated conditional RCC1L KO cells using the Cre-loxP system. RCC1L KO cells were transfected with pCMV-loxP-RCC1L^V1FLAG-loxP-DsRed (referred to as RCC1L^V1-loxP) and selected using G418 in a culture medium containing pyruvate and uridine. In RCC1L^V1-loxP-transfected RCC1L KO cells, cleavage at the loxP position by Cre resulted in the deletion of the RCC1L^V1 sequence and expression of DsRed, which labels mitochondria (Fig. 5A).

Before Cre addition, we confirmed that RCC1L^V1-loxP-transfected RCC1L KO cells showed recovered cell growth and mtDNA levels (Supplementary Fig. S8). To label mitochondria with DsRed without Cre, we transfected a vector encoding DsRed fused to a mitochondria-targeting sequence into RCC1L^V1-loxP-transfected RCC1L KO cells. In these cells, the mitochondrial morphology and the number of mt-nucleoids were recovered (Fig. 5B).

Following the addition of Cre, we observed conditional RCC1L KO cells expressing DsRed every three days (Fig. 5B). Immunostaining with anti-FLAG antibodies revealed that RCC1L was rapidly depleted within 3–6 days following Cre addition, and at day 9, its expression was almost completely undetectable (Supplementary Fig. S9). Twelve days following Cre addition, some conditional RCC1L KO cells exhibited swollen and fragmented mitochondria. At 15 and 18 days following Cre addition, most conditional RCC1L KO cells displayed these mitochondrial abnormalities. A decrease in mt-nucleoids was observed 12 days following Cre addition (Fig. 5B). Interestingly, 12–15 days following Cre addition, some conditional RCC1L KO cells exhibited uneven localization of mt-nucleoids stained with SYBR Green I, whereas swollen mitochondria lacking mt-nucleoids clustered on one side of the cell (Fig. 5C). This phenotype was observed in up to 20% of conditional RCC1L KO cells, peaking 12 days following Cre addition (Supplementary Fig. S10). The average mitochondrial length decreased 12 days following Cre addition, and a significant difference was observed at 15 days (Fig. 5D). The amount of mtDNA, measured by the total SYBR Green I fluorescence intensity per cell, excluding the nuclei, showed a significant decrease from 12 days following Cre addition (Fig. 5E). A rapid decrease in mtDNA was observed 12–15 days following Cre addition, which stabilized 18 days following Cre addition. These results suggest that the loss of RCC1L caused mitochondrial fragmentation and decreased mtDNA almost simultaneously during a specific period.

Discussion

In this study, we identified a novel function of RCC1L in the maintenance of mt-nucleoids and mtDNA. It has been reported that RCC1L knockdown in human cells and adipocyte-specific RCC1L knockout mice (RCC1L^flox/flox × Adiponectin-Cre), neither of which result in complete RCC1L depletion, lead to the loss of mitochondrial translation function but do not decrease mtDNA^13,14. We established RCC1L KO cell lines with complete loss of RCC1L, and these cells exhibited a significant decrease in mtDNA levels (Fig. 1E). This difference from previous reports suggests that mtDNA depletion may occur only when the level of RCC1L falls below a certain threshold. Additionally, the number of mt-nucleoids was decreased in RCC1L KO cells (Fig. 1D). Although mitochondrial translation-inhibited CAP cells also showed a decrease in mt-nucleoids, their mtDNA levels remained unchanged, and the mt-nucleoids were enlarged (Fig. 3A, E, and Supplementary Fig. S6), indicating that each enlarged mt-nucleoid contains more copies of mtDNA than those in wild-type cells. Consistently, most proteins associated with mtDNA within mt-nucleoids such as SSBP1, TWINKLE, and TFAM did not decrease in CAP cells (Fig. 4). These results suggest that RCC1L regulates the maintenance of mt-nucleoids and mtDNA through a mechanism involving other factors besides mitochondrial translation.

To establish RCC1L KO cell lines, we needed to add pyruvate and uridine to the culture medium. Supplementation with pyruvate and uridine compensates for mitochondrial dysfunction. Pyruvate supplementation restores the redox balance disrupted by mitochondrial OXPHOS dysfunction by replenishing NAD⁺²⁴, whereas uridine supplementation supports de novo pyrimidine synthesis, a mitochondria-dependent process²⁵. As expected, RCC1L KO cells could not proliferate in a culture medium without pyruvate and uridine (Fig. 1B), indicating that RCC1L is essential for OXPHOS-dependent cell growth. This result is consistent with previous findings showing RCC1L KO mice exhibit embryonic lethality¹⁶.

Huang et al. (2017) reported that RCC1L KO caused mitochondrial fragmentation, as RCC1L acts as a GEF for the mitochondrial fusion factor OPA1¹⁶. In this study, western blot analysis showed that a reduction in L-OPA1 and an increase in S-OPA1 in RCC1L KO cells (Fig. 4). It is possible that these changes in the OPA1 processing may also contribute to mitochondrial fragmentation, as L-OPA1 is required for mitochondrial fusion, whereas S-OPA1 facilitates mitochondrial fission²⁶. The reduction in L-OPA1 is caused by its cleavage of L-OPA1 into S-OPA1 via the inner membrane protease OMA1 in dysfunctional mitochondria with low membrane potential^27,28,29. Consistent with this, we observed a reduction in mitochondrial membrane potential in RCC1L KO cells (Supplementary Fig. S7). Since similar changes in the OPA1 processing and membrane potential were observed in CAP cells, it is likely that impaired mitochondrial translation leads to the cleavage of L-OPA1 into S-OPA1.

Most mammalian species, except primates, possess only RCC1L^V1, while isoforms RCC1L^V2 and RCC1L^V3 are thought to have appeared during primate evolution¹³. Consistent with our results, the Genotype-Tissue Expression (GTEx) project has reported that the expression level of endogenous RCC1L^V1 is significantly higher than that of RCC1L^V2 and RCC1L^V3 in humans³⁰. RCC1L^V1 and RCC1L^V3 are essential for proper mitochondrial ribosome assembly and play important roles in mitochondrial translation, influencing cell growth and mitochondrial morphology¹³. Meanwhile, RCC1L^V1 and RCC1L^V2 possess DNA-binding ability, with RCC1L^V2 exhibiting a stronger affinity for DNA¹³. In this study, the expression of RCC1L^V1 recovered all phenotypes observed in RCC1L KO cells, whereas that of RCC1L^V2 and RCC1L^V3 did not (Fig. 2B–F). This indicates that RCC1L^V1 is necessary for OXPHOS-dependent cell growth, mitochondrial morphology, and the maintenance of mt-nucleoids and mtDNA.

MALSU1, TFAM, and NME6 were specifically reduced in RCC1L KO cells by western blot analysis (Fig. 4). Mitochondrial membrane potential is necessary for transport of nuclear encoded proteins into the mitochondria³¹. These three proteins did not decrease in CAP cells, suggesting that the reduction in membrane potential observed in both RCC1L KO and CAP cells may not affect their mitochondrial import. MALSU1 plays a role in the maturation of the large subunit of mitochondrial ribosome^20,21. MALSU1 stability does not depend on mitochondria-encoded proteins but rather on the mtDNA itself or on mitochondria-encoded rRNAs or tRNAs²¹. Therefore, the reduction in MALSU1 expression in RCC1L KO cells may result from decreased mtDNA. TFAM, the most abundant mt-nucleoid protein, binds directly to mtDNA and compacts it into a packaged structure^32,33. The TFAM band that decreased in RCC1L KO cells is likely the mature form because its band size was the same as the 24 kDa mature form observed in wild-type cells. Reduced TFAM levels decrease in the mtDNA copy number³⁴, while decreased mtDNA levels reduce TFAM³⁵. Therefore, it is important to analyze the relationship between mtDNA and TFAM in RCC1L KO cells. NME6 is involved in the mitochondrial pyrimidine nucleotide metabolism^17,18. Proteomic analysis of immunoprecipitates demonstrated an interaction between NME6 and RCC1L^17,18. NME6 is required to maintain mtDNA when access to cytosolic pyrimidine deoxyribonucleotides is limited due to the loss of mitochondrial pyrimidine nucleotide transporters¹⁷. These transporters likely depend on the mitochondrial membrane potential to facilitate substrate transport³⁶, indicating that their transport function could be impaired in RCC1L KO cells. Therefore, the decrease in mtDNA observed in RCC1L KO cells may result from the simultaneous occurrence of both NME6 reduction and impaired mitochondrial pyrimidine nucleotide transport. Since dysfunction of mitochondrial translation is known to induce a reduction in mitochondrial membrane potential, mitochondrial translation may be partially involved in the RCC1L-dependent regulation of mtDNA. In contrast, C4orf14 and OPA1 exhibited similar changes in expression levels in both RCC1L KO and CAP cells. C4orf14 is involved in the assembly of the small subunit of mitochondrial ribosome³⁷ and co-immunoprecipitates with RCC1L^V313. The role of C4orf14 in mtDNA maintenance remains controversial; knockdown of C4orf14 in humans has been shown to decrease mtDNA levels³⁷, whereas C4orf14 knockout in mice does not³⁸. In this study, the reduction in C4orf14 did not affect mtDNA levels in CAP cells (Fig. 3E), suggesting that C4orf14 may not be involved in mtDNA maintenance. OPA1 knockout decreases the number of mt-nucleoids and the amount of mtDNA^39,40,41, and all OPA1 isoforms can recover these phenotypes⁴¹. In both RCC1L KO and CAP cells, most OPA1 isoforms, except for L-OPA1, were retained, suggesting that OPA1 is not involved in RCC1L-mediated mtDNA maintenance. Considering these findings, TFAM and NME6 are potential candidates involved in the maintenance of mtDNA in association with RCC1L^V1, and further studies remain warranted to elucidate the underlying mechanism.

We monitored the progression of a decrease in mt-nucleoids and mtDNA following RCC1L loss using conditional RCC1L KO cells. Our results suggest that the decrease in mtDNA mainly occurred during a specific period after the loss of RCC1L. We also observed an uneven mt-nucleoid distribution during only the period of rapid mtDNA reduction (Fig. 5C). In general, within the mitochondrial network, mt-nucleoids are semi-regularly distributed^42,43,44. This distribution is thought to facilitate the accurate transmission of mt-nucleoids to daughter cells during cell division⁴³. Therefore, the uneven distribution in conditional RCC1L KO cells may contribute to the rapid decrease in mt-nucleoids due to the asymmetric distribution of mt-nucleoids during cell division. Understanding the rapid decrease in mt-nucleoids and mtDNA following the loss of RCC1L will provide valuable insights into the mechanisms by which RCC1L regulates the maintenance of mt-nucleoids and mtDNA.

Methods

Cell culture

HeLa cells (RCB0007; RIKEN Cell Bank, Tsukuba, Japan) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, 044-29765; Fujifilm Wako Pure Chemicals, Osaka, Japan) containing 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 50 µg/mL uridine, and 100 U/mL penicillin/streptomycin under 5% CO₂ at 37℃.

Establishment of RCC1L knockout cell lines and plasmid transfection

To establish stable human RCC1L KO cell lines in HeLa cells, we used the CRISPR/Cas9 KO Plasmid (Santa Cruz Biotechnology, Dallas, TX, USA). sgRNA for the CRISPR system was designed to target the sequence shown in Supplemental Fig. S2. HeLa cells, grown in a 10 cm dish at 80% confluency, were transfected with 10 µg of CRISPR/Cas9 KO Plasmid using jetPRIME kit (Polyplus, France). After 36 h of incubation, the GFP-positive cells were collected using Cell Soter SH800 (SONY, Tokyo, Japan). After repeating the transfection and sorting steps twice, the cells were collected and plated in 96 well plates by serial dilution of single cells. After colony growth, the clones were tested using sequencing and immunoblotting to confirm the RCC1L KO cell lines.

To establish stable RCC1L KO cell lines expressing FLAG-tagged each of the three isoforms of RCC1L, RCC1L^V1, RCC1L^V2, and RCC1L^V3, cloned into p3xFLAG-CMV-14 expression vector (Sigma-Aldrich, MO, USA), 5–7.5 µg of each construct was transfected into RCC1L KO cells using jetPRIME kit. Upon growth, the cells were treated with 300 µg/mL G418.

Confocal microscopy settings and image acquisition

All fluorescent images were obtained using a spinning disk confocal system (CellVoyager CV1000; Yokogawa Electric, Tokyo, Japan) equipped with 488-nm and 561-nm diode lasers. Confocal images were acquired with a 100× oil immersion objective lens (UPLSAPO 100XO, WD = 0.13 mm, NA = 1.40; Olympus, Tokyo, Japan). The exposure time was 100 ms, and fluorescence was acquired using band-pass filters: BP525/50 for SYBR Green I, Alexa488, or ATTO488, and BP617/73 for MitoTracker Red, Alexa594, or DsRed. Images were acquired using 35 z-plane sectioning with 0.3-µm intervals and processed with CV1000 software (Yokogawa Electric) to create maximum-intensity projection (MIP) images.

Quantification of mitochondrial morphology was performed with the Fiji software using the Mitochondria Analyzer plugin²³.

Live cell staining with SYBR green I and mitotracker

Cells were plated onto an eight-well cover glass chamber (5232-008; IWAKI, Japan; hereinafter 8 well chamber) and cultured at 37℃ for 24 to 36 h. SYBR Green I (Thermo Fisher Scientific, MA, USA) was diluted with DMEM 1:300,000 times, and MitoTracker™ Red (Thermo Fisher Scientific) was used at 100 nM. Cells were incubated with the staining solution at 37℃ for 5 min in a CO₂ incubator and washed three times with fresh growth medium. The number of mt-nucleoids was manually counted following SYBR Green I staining, and the fluorescence intensity of mt-nucleoids per cell, excluding nuclei, was measured on MIP images using the Fiji software.

SDS-PAGE and Western blot analysis

As sample preparation, cells grown in a plate at 80% confluency were collected and mixed with the sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 1.43 mM 2-mercaptoethanol, Bromophenol Blue). After sonication using an ultrasonic liquid processor XL2000 SERIES (Misonix, Farmingdale, NY, USA), samples were incubated at 96℃ for 5 min. The protein samples were separated by SDS-PAGE conducted at 100 V with 7.5, 10, or 15% polyacrylamide gels.

After SDS-PAGE, proteins were transferred to polyvinylidene fluoride membranes (Immobilon^®-P Transfer Membrane, 0.45 μm pore size; Merck Millipore, MA, USA) for 60 min at 100 V in transfer buffer (20% methanol, 2.5 mM Tris, 19.2 mM glycine), with or without 0.02% SDS. Membranes were blocked with 5% skimmed milk powder in buffer A (5 mM Tris, 13.8 mM NaCl, 0.27 mM KCl, 0.05% Tween20, pH 7.6) overnight at 4℃. After blocking, membranes were incubated with a primary antibody diluted in 5% skimmed milk powder in buffer A, either shaking for 2 h at room temperature or overnight at 4℃. After three washes with 5% skimmed milk powder in buffer A at room temperature for 10 min, the membranes were incubated with a secondary antibody (anti-Rabbit IgG HRP or anti-mouse IgG HRP) diluted in 5% skimmed milk powder in buffer A for 2 h at room temperature or overnight at 4℃. The washing procedure was repeated three times with buffer A at room temperature for 10 min. After washing with buffer B (5 mM Tris, 13.8 mM NaCl, 0.27 mM KCl, pH 7.6), the membranes were exposed to immobilon western chemiluminescent HRP substrate (Merck Millipore) for 5 min at room temperature and visualized using an iBright 1500 (Thermo Fisher Scientific).

Immunostaining

Cells were plated onto 8 well chamber and fixed with 8% paraformaldehyde (PFA) in PBS (166-23555, Fujifilm Wako Pure Chemicals) at 37℃ for 5 min and permeabilized with 0.5% TritonX-100 in PBS. After blocking with 5% skimmed milk powder in PBS, cells were incubated with the primary antibody in 1% skimmed milk powder in PBS and left overnight at 4℃, followed by the secondary antibody. Washing was repeated three times with PBS for 10 min at room temperature.

Antibodies

We raised anti-TFAM antibodies against the purified recombinant protein. TFAM (aa43-246) was expressed in Escherichia coli (XL1-Blue) as a 6× histagged protein using the vector pQE-30 (Qiagen, Hilden, Germany). The protein, purified using Ni-NTA agarose, was used to generate polyclonal rabbit antisera. Anti-TFAM antibodies were purified from serum using Protein G affinity chromatography (MABTrap Kit; GE Healthcare). Other primary antibodies used in this study were rabbit anti-RCC1L (ab247142, Abcam), rabbit anti-RCC1L (LS-C170613, LSBio, CA, USA), mouse anti-β-Tubulin (66240-1-Ig, Proteintech), rabbit anti-FLAG (F7425, Sigma-Aldrich), mouse anti-FLAG (F1804, Sigma-Aldrich), mouse anti-MTCO2 (ab110258, Abcam), rabbit anti-MALSU1 (HPA020487, Sigma-Aldrich), rabbit anti-NME6 (HPA017909, Sigma-Aldrich), mouse anti-OPA1 (612606, BD Biosciences, NJ, USA), rabbit anti-C4orf14 (ab251841, Abcam), rabbit anti-POLG (PA521314, Thermo Fisher Scientific), rabbit anti-SSBP1 (HPA002866, Sigma-Aldrich), rabbit anti-TWINKLE (18793-1-AP, Proteintech), rabbit anti-Tom20 (sc-11415, SANTA CRUZ), mouse anti-DNA (MAB030, Merck Millipore). Secondary antibodies used for western blotting were goat anti-rabbit IgG HRP (074-1506, KPL, MD, USA) and goat anti-mouse IgG HRP (214–1806, KPL). Goat Alexa 488-conjugated anti-rabbit (A-11008; Thermo Fisher Scientific), goat ATTO 488-conjugated anti-mouse (610-152-121; Rockland, PA, USA), goat Alexa 594-conjugated anti-rabbit (A-11012; Thermo Fisher Scientific), and goat Alexa 594-conjugated anti-mouse (A-11032; Thermo Fisher Scientific) antibodies were used for immunostaining.

qPCR of mitochondrial DNA

The relative copy number of mtDNA per cell was determined using qPCR with TB Green^® Premix ExTaq™ II (Takara Bio, Shiga, Japan) following DNA isolation with the Blood/Tissue DNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocols.

Primer sequences used were tRNA-Leu (5′-CACCCAAGAACAGGGTTTGT-3′ and 5′-TGGCCATGGGTATGTTGTTA-3′) for mtDNA and β2 microglobulin B2M (5′-TGCTGTCTCCATGTTTGATGTATCT-3′ and 5′-TCTCTGCTCCCCACCTCTAAGT-3′) for nuclear DNA.

Electron microscopy analysis

Cells were fixed with 2% PFA and 2% glutaraldehyde (GA) in 0.1 M phosphate buffer (PB) pH 7.4 at 37℃. The cells were then incubated at 4℃ for 30 min and fixed with 2% GA in 0.1 M PB at 4℃ overnight, after which samples were analyzed by Tokai Em, Inc. On the following day, the samples were washed three times with 0.1 M PB for 30 min each and postfixed with 2% osmium tetroxide (OsO₄) in 0.1 M PB at 4℃ for 1 h. Samples were dehydrated in graded ethanol solutions (50%, 70%, and 90% anhydrous). The schedule was as follows: 50% and 70% for 5 min each at 4℃, 90% for 5 min at room temperature, and three changes of anhydrous ethanol for 5 min each at room temperature. For embedding, the samples were transferred to a resin (Quetol-812; Nissin EM Co., Tokyo, Japan) and polymerized at 60℃ for 48 h. The polymerized resins were ultra-thin-sectioned at 70 nm with a diamond knife using an ultramicrotome (Ultracut UCT; Leica, Vienna, Austria). The sections were mounted on copper grids, stained with 2% uranyl acetate at room temperature for 15 min, washed with distilled water, and secondarily stained with lead stain solution (Sigma-Aldrich) at room temperature for 3 min. The grids were observed under a transmission electron microscope (JEM-1400Plus; JEOL Ltd., Tokyo, Japan) at an acceleration voltage of 100 kV. Digital images (3296 × 2472 pixels) were captured using a CCD camera (EM-14830RUBY2; JEOL Ltd., Tokyo, Japan).

Establishment of conditional RCC1L knockout cells

To obtain the pCMV-loxP-RCC1L^V1FLAG-loxP-DsRed (RCC1L^V1-loxP) vector, the FLAG-tagged RCC1L^V1 sequence, including the poly-A signal, was amplified from the FLAG-tagged RCC1L^V1 expression vector used in the complementation experiment. A loxP sequence was then added to the front of this sequence by overlap extension using PCR to create a loxP-RCC1L^V1FLAG-polyA fragment. pDsRed2-Mito (632421, Takara Bio) was digested with the restriction enzyme NheI, and the loxP-RCC1L^V1FLAG-poly A fragment and the back loxP fragment were inserted between the promoter and DsRed sequences of the vector using Gibson assembly.

To establish conditional RCC1L KO cells, the RCC1L^V1-loxP vector was transfected into RCC1L KO cells. After cell growth, selection was performed using 300 µg/mL G418 for more than 1 month. On the previous day of Cre addition, 2.0 × 10⁴ cells per well were cultured in an 8-well chamber in antibiotic-free DMEM. Following incubation for 24 h, the medium was replaced with polybrene media, which is antibiotic-free DMEM with 6 µg/mL polybrene (Hexadimethrine bromide, H9268, Sigma-Aldrich). Then, 2 µL of Cre Recombinase Gesicles (631449, Takara Bio) per well was added, and the cells were incubated at 37℃ for 24–48 h. Following incubation, the medium was replaced with fresh DMEM without polybrenes, or the cells were seeded onto new plates.

Data availability

The data supporting the findings of this study are included in the manuscript and Supplementary file. All the other data are available from the corresponding author upon request.

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Acknowledgements

We thank Dr. Yoshikatsu Sato for the helpful comments and discussions and Ms. Yoriko Tanaka for technical assistance. We are grateful to Dr. Haruko Kuroiwa and Dr. Tsuneyoshi Kuroiwa for their technical advice. We would like to thank Editage (www.editage.jp) for English language editing.

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Authors and Affiliations

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan
Emi Matsumoto, Tetsuya Higashiyama & Narie Sasaki
Laboratory of Molecular Membrane Biology, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma, 371-8512, Japan
Taeko Sasaki
Laboratory of Molecular Traffic, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma, 371-8512, Japan
Taeko Sasaki
Center for Diversity, Equity & Inclusion, Tohoku University, 2-1-1 Katahira Aoba-ku, Sendai, Miyagi, 980-8577, Japan
Narie Sasaki

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Contributions

E.M., T.S., T.H., and N.S. conceived and designed the study. E.M. and T.S. performed all the experiments. T.H. and N.S. supervised the study. All authors contributed to the writing of the manuscript.

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Correspondence to Narie Sasaki.

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Matsumoto, E., Sasaki, T., Higashiyama, T. et al. Human RCC1L is involved in the maintenance of mitochondrial nucleoids and mtDNA. Sci Rep 15, 13811 (2025). https://doi.org/10.1038/s41598-025-98397-y

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Received: 12 December 2024
Accepted: 11 April 2025
Published: 21 April 2025
Version of record: 21 April 2025
DOI: https://doi.org/10.1038/s41598-025-98397-y