Given the crucial role of communication between the nucleus and mitochondria in maintaining cellular homeostasis, it is important to identify biological molecules that facilitate inter-compartmental crosstalk. LncRNAs can be categorically classified based on their origin into nulncRNAs and mtlncRNAs. In the field of RNA biology, extensive research has focused on lncRNAs, particularly nuclear lncRNAs (nulncRNAs), due to their involvement in a wide range of biological processes. Furthermore, specific nulncRNAs have been implicated in the regulation of mitochondrial function28–32. Nevertheless, whether and how mtlncRNAs operate in the regulation of nuclear events remains largely unknown. In this study, we have identified several mtlncRNAs involved in retrograde regulation from the mitochondria to the nucleus. Facilitated by the associated HuR and PNPT1 proteins, the mtlncRNAs MDL1AS, lncND5, and lncCyt b are involved in mitochondria-to-nucleus shuttling. Additionally, the nuclear-translocated lncCyt b may cooperate with the splicing factor hnRNPA2B1 to modulate cellular metabolism, potentially exerting a splicing regulatory effect on relevant nuclear genes.
Nuclear genes influence mitochondrial activities through a mechanism known as anterograde regulation. Conversely, mitochondrial signaling can modulate nuclear events through a mechanism called retrograde regulation. Several nulncRNAs, such as CARL, Cerox1, and Tug1, function as anterograde messengers and directly or indirectly regulate mitochondrial events including apoptosis, energy metabolism, and oxidative stress33–35. In the case of mtlncRNAs involved in retrograde regulation, limited information is currently available36. However, our related study revealed that the mtlncRNA MDL1 collaborates with Tid1 to inhibit the nuclear translocation of p53, a key transcription factor, thereby regulating a network of nuclear genes involved in cell cycle control37.
It is widely recognized that the subcellular localization of lncRNAs is closely linked to their functionality. It has long been known that nuclear-encoded housekeeping tRNAs and 5S rRNA localize to mitochondria to regulate mitochondrial function. Additionally, specific nulncRNAs like SAMMSON, SRA, and GAS5 have been suggested to have roles in particular cellular contexts following their import into mitochondria28–31. Furthermore, the nulncRNA hTERC undergoes bidirectional transportation between the nucleus and mitochondria38. Subsequently, it is processed into a shorter transcript that reflects alterations in mitochondrial function38. Nonetheless, the role of mtlncRNAs as retrograde messengers through nuclear translocation remains an unresolved question. To address this knowledge gap, we analyzed the subcellular localization of a panel of mtlncRNAs. The results of cellular fractionation and FISH assays revealed a distinct nuclear distribution of the mtlncRNAs MDL1AS, lncND5, and lncCyt b, indicating their involvement in mitochondria-to-nucleus shuttling. Furthermore, we detected the nuclear distribution of MDL1AS, lncND5, and lncCyt b in all cell types tested, indicating that their mitochondria-to-nucleus shuttling is a widespread phenomenon across various cellular contexts.
RNA binding proteins (RBPs) play a crucial role in binding to RNA molecules and influencing their fate and function39. In our study, we identified that both HuR and PNPT1 interact with the mtlncRNAs MDL1AS, lncND5, and lncCyt b, and they play a role in facilitating their nuclear transportation. HuR, a multifunctional RBP, is known for its involvement in regulating the stability and translation of target mRNAs. It binds to RNA molecules, including both coding and noncoding transcripts, within the nucleus and aids in their nuclear export23,40. On the other hand, PNPT1 controls adenylation of mitochondrial RNA tails and facilitates the import of cytoplasmic RNAs into the mitochondria24. Interestingly, our results indicate that HuR and PNPT1 may function in a reverse manner, contributing the nuclear localization of MDL1AS, lncND5, and lncCyt b. mtlncRNAs undergo translocation from the mitochondria to the cytoplasm and subsequently enter the nucleus. Knockdown of either HuR or PNPT1 results in a decrease in the nuclear distribution of mtlncRNAs, suggesting their involvement in the transportation of mtlncRNAs. HuR, which shuttles between the nucleus and cytoplasm, interacts with the F7 segment of lncCyt b. Overexpression of this segment inhibits the interaction between endogenous mtlncRNAs and HuR, resulting in a decrease in the nuclear localization of the endogenous mtlncRNAs. Additionally, PNPT1 is localized in both the mitochondria and cytoplasm27, and its binding to the F3 segment of lncCyt b is also crucial for the nuclear transportation of mtlncRNAs. These findings suggest that PNPT1 may contribute to the export of mtlncRNAs from the mitochondria.
We further investigated the functional consequences of the nuclear translocation of mtlncRNAs. RNA-seq analysis revealed that knockdown of the nuclear-localized mtlncRNAs using ASOs affected a network of NGs. Interestingly, it has been known that some RBPs can be modulated in an inverse manner by the RNA molecules they bind to, rather than directly regulating their RNA targets41. Consistent with this concept, numerous lncRNAs have been shown to regulate gene expression by modulating the activity of their protein partners involved in various processes such as epigenetic modification, RNA transcription, pre-mRNA processing, and protein translation10. In our study, we identified the splicing factor hnRNPA2B1 as a binding partner of lncCyt b. Furthermore, the nuclear-translocated lncCyt b was found to cooperate with hnRNPA2B1 to influence multiple aspects of cell metabolism, including glycolysis. This cooperation potentially relies on their synergistic effect on pre-mRNA splicing and mRNA maturation of relevant NGs.
In summary, our study demonstrates that the mtlncRNAs MDL1AS, lncND5, and lncCyt b function as messenger molecules in mediating a retrograde regulation from mitochondria to the nucleus, thereby contributing to our understanding of mitochondrial biology and inter-compartmental crosstalk within cells. Furthermore, we propose that a comprehensive exploration of the functional mechanisms underlying mtlncRNA-mediated communication between mitochondria and the nucleus can provide valuable insights into various physiological and pathological processes in human.