Despite the crucial roles of mitochondrial RNA transport in maintaining mitochondrial functionality, how mitochondrial RNAs are transported across mitochondrial membranes remains largely unknown. In a recent work published in Cell Research, Wang et al. identified adenine nucleotide translocase-2 as the first mammalian mitochondrial RNA translocon using a series of innovative biochemical systems and revealed its regulatory role in innate immunity triggered by mitochondrial double-stranded RNA efflux.

Mitochondria, often described as the cell’s powerhouses, are central to ATP production and play pivotal roles in regulation of cellular metabolism. Despite possessing their genome, mitochondria import nuclear-encoded non-coding RNAs, which is essential for maintaining mitochondrial function and regulating gene expression.1 Moreover, mammalian mitochondria export double-stranded RNA (dsRNA) that contributes to cellular signaling, especially in activating innate immune responses during antiviral defenses.2,3 Although mitochondrial RNA transport is crucial for mitochondrial RNA dynamics and overall mitochondrial functionality, the exact mechanisms involved are yet to be fully elucidated.

Wang et al.4 identified adenine nucleotide translocase-2 (ANT2) as a potential RNA translocon in mitochondrial membranes using an in vitro RNA cross-membrane transport system (Fig. 1a). They developed this system with ribonuclease-free liposomes derived from mitochondrial membranes, and utilized UV cross-linking and mass spectrometry to analyze potential RNA translocons with biotin-labeled mitochondrial dsRNA (mt-dsRNA) and telomerase RNA component (TERC) as RNA substrates. Notably, ANT2 emerged as a significant hit in mitochondrial RNA transporter screening and demonstrated the ability to bind to various previously reported mitochondrial RNAs, including dsRNA, TERC, 5S rRNA, and growth arrest-specific transcript 5 (GAS5). The ANT family, primarily recognized as ADP/ATP transporter, facilitates the exchange of ATP from the mitochondrial matrix with ADP from the cytoplasm, which is essential for maintaining cellular energy homeostasis. Additionally, ANT proteins contribute to the formation of the mitochondrial permeability transition pore, which is implicated in regulated necrotic cell death. This study is the first to suggest ANT2’s potential role as a translocon for mitochondrial cross-membrane RNA translocation.

Fig. 1: ANT2 functions as an RNA translocon for cross-membrane translocation in mitochondria.
figure 1

a ANT2 acts as a bidirectional RNA translocon in the inner mitochondrial membrane of mammals. b Electrostatic surface potential analysis reveals an overall positive charge in the region of the nucleotide-binding channel (left). The side view of the structure highlights Arg-237 of the nucleotide carrier signature motif and Thr-247 in magenta (right). c ANT2 regulates immune and inflammation responses by mediating the efflux of mt-dsRNA.

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To further verify ANT2’s involvement in RNA transport, the authors utilized purified mitochondria and mitochondrial membrane-derived liposomes for the transport assay. The results showed that ANT2 overexpression significantly enhanced the mitochondrial transport of dsRNA (~5-fold), TERC and 5S rRNA (~3-fold), and GAS5 (~2-fold), suggesting that ANT2 is somehow involved in RNA transport. Subsequently, the authors established a system inherently incapable of RNA transport using bacterial cell membrane-derived liposomes or pure liposome uptake system. The introduction of ANT2 into this system enabled RNA permeability, directly demonstrating that ANT2 functions as an inner membrane transport channel for mitochondrial RNA. Notably, in contrast to liposome RNA transport, ATP is required for RNA transport into purified mitochondria to counteract the unfavorable mitochondrial inner membrane potential against negatively charged RNA. ANT2’s role in mitochondrial RNA import (TERC and 5S rRNA) and export (TERC-53) was confirmed via an in organelle system, underscoring bidirectional transport capability and dual functionality of ANT2.

The RNA translocating activity is unique to ANT2 among the human ANT isoforms, including ANT1, ANT2, ANT3, and ANT4. Although ANT1 and ANT3 share high homology and similar ADP/ATP carrier activity with ANT2, they lack the RNA translocating activity in bacterial or pure liposome system. Interestingly, T247A mutation of ANT2 significantly diminished its RNA translocating activity without affecting ADP/ATP carrier activity. Additionally, inhibitors of ADP/ATP carrier activity did not influence RNA translocation, underscoring the distinct and independent nature of ANT2-mediated RNA transport. Mechanistically, ANT2 forms a channel with positively charged residues for binding and transporting RNA, as revealed by structural analysis; here T247 plays a crucial role in stabilizing the interaction with RNA through hydrogen bonding (Fig. 1b). These findings highlight the specialized function of ANT2 in RNA transport, independent of its traditional role as a mitochondrial solute carrier.

mt-dsRNA is formed from overlap** transcripts generated by bidirectional transcription of mitochondrial DNA.5,6 Recently, Dhir et al.3 discovered that mt-dsRNA accumulates extensively and escapes into the cytoplasm in a PNPase-dependent manner, initiating a melanoma differentiation-associated gene 5 (MDA5)-driven type I interferon response and subsequent immune activation. In the present research, ANT2 overexpression increased cytosolic mt-dsRNA levels and subsequently upregulated immune response markers MDA5, retinoic acid-inducible gene 1 (RIG-I), and interferon-stimulated gene 15 (ISG15). Conversely, ANT2 knockdown produced the opposite effect. Furthermore, the ANT2-T247A mutant had much weaker effects on cytosolic mt-dsRNA levels and downstream immune responses, highlighting the critical role of ANT2 in mt-dsRNA translocation. Importantly, following the degradation of mt-dsRNA by Dicer, the immune responses in both ANT2-overexpressing and control cells were reduced, further confirming that ANT2-mediated efflux of mt-dsRNA is the primary trigger of immune responses. In addition, previous studies highlight the clinical significance of mt-dsRNA efflux in autoimmune diseases such as osteoarthritis, Sjӧgren’s syndrome, and lupus.7,8,9 In this study, ANT2 knockdown in immunized mice reduced mt-dsRNA levels and associated inflammation markers (IL6 and ISG15), effectively mitigating immune responses in an ovalbumin-induced inflammation model. Targeting ANT2 to inhibit mt-dsRNA efflux presents a promising therapeutic strategy for managing excessive inflammation in immune-related disorders, thereby offering the potential for new treatments to control inflammation and improve outcomes of patients with autoimmune conditions (Fig. 1c).

The discovery of ANT2 as an RNA translocon for mitochondrial cross-membrane translocation opens a new avenue for understanding mitochondrial RNA dynamics and metabolism. Future research should aim to delineate the molecular mechanisms underlying ANT2-mediated RNA translocation and investigate how ANT2 transports various RNA types to enhance our understanding of mitochondrial biology. Additionally, exploring ANT2-mediated RNA transport in various physiological and pathological scenarios, including mitochondrial diseases, cancer, and metabolic disorders, is crucial. Utilizing ANT2 as a target to adjust mitochondrial RNA levels and immune responses offers a viable therapeutic approach for conditions marked by excessive inflammation and immune dysregulation.