A Synthetic Small RNA Homologous to the D-Loop Transcript of mtDNA Enhances Mitochondrial Bioenergetics

Abstract

Mitochondrial malfunction is a hallmark of many diseases, including neurodegenerative disorders, cardiovascular and lung diseases, and cancers. We previously found that alveolar progenitor cells, which are more resistant to cigarette smoke-induced injury than the other cells of the lung parenchyma, upregulate the mtDNA-encoded small non-coding RNA mito-ncR-805 after exposure to smoke. The mito-ncR-805 acts as a retrograde signal between the mitochondria and the nucleus. Here, we identified a region of mito-ncR-805 that is conserved in the mammalian mitochondrial genomes and generated shorter versions of mouse and human transcripts (mmu-CR805 and hsa-LDL1, respectively), which differ in a few nucleotides and which we refer to as the “functional bit”. Overexpression of mouse and human functional bits in either the mouse or the human lung epithelial cells led to an increase in the activity of the Krebs cycle and oxidative phosphorylation, stabilized the mitochondrial potential, conferred faster cell division, and lowered the levels of proapoptotic pseudokinase, TRIB3. Both oligos, mmu-CR805 and hsa-LDL1 conferred cross-species beneficial effects. Our data indicate a high degree of evolutionary conservation of retrograde signaling via a functional bit of the D-loop transcript, mito-ncR-805, in the mammals. This emphasizes the importance of the pathway and suggests a potential to develop this functional bit into a therapeutic agent that enhances mitochondrial bioenergetics.

Mitochondrial ncRNA LDL-805 declines in alveolar epithelial type 2 cells of chronic obstructive pulmonary disease patients

“…This suggests that the full-length transcripts likely form similar secondary structures and/or interact with similar regulatory proteins in both species and likely others, potentially revealing a novel evolutionarily conserved mitochondria-to-nucleus signaling pathway. Notably, we demonstrated via two approaches that nuclear localization of mito-ncR-LDL805 triggers upregulation of NeMitos and increased mitochondrial respiration, which validates and extends our previous findings (22).…”

“…The human mito-ncR-LDL805 transcript ( hsa - mito - ncR-LDL805 ) was identified from the small ncRNA library prepared from human primary AEC2s according to evolutionary conservation of its 20-nucleotide functional bit (22) (colored red in the murine sequence and yellow in the human sequence in Figure 1A ). No nuclear mitochondrial transcript sequences corresponding to hsa - mito - ncR-LDL805 were identified by bioinformatics analysis ( Figure 1B ) hsa - mito - ncR-LDL805 is located at coordinates 16,472–16,546 of the D-loop of the light strand of mtDNA ( Figure 1C ).…”

“…Evolutionary conservation of mitochondrial ncRNAs is rare. Although the mmu-mito-ncR-LDL805 sequence is mouse specific (according to sequence homology of the full-length transcript), a 20-nucleotide-long “functional bit” is conserved in most mammalian mitochondrial genomes (22). This conservation enabled us to identify the human homologue, which is comparable to the mouse transcript in length and location in mitochondrial genome.…”

“…Post nuclear fractions were obtained and treated with RNase (to eliminate all but mitochondrial RNAs) as previously described (22).…”

“…This transcript, along with three other mtDNA-encoded ncRNAs, is tightly regulated depending on the energy demand, the ability to supply mitochondrial ribosomes, and whether mtDNA replication-transcription are balanced (20,21). Although mmu-mito-ncR-LDL805 is a mouse-specific transcript, it contains a 20-nucleotide “functional bit” that is highly conserved among mammals (22). Therefore, a homologous transcript is likely expressed by human cells that may be relevant to the development and progression of smoke-related lung diseases such as COPD.…”