Mitochondrial non-coding RNA: An atlas of organelle non-coding RNA

Mitochondrial non-coding RNA: An atlas of organelle non-coding RNA. Mitochondrial non-coding RNA. The Lin Aifu team of Zhejiang University established a non-coding RNA map of organelles and analyzed its functions.

The organelle obtained by the organelle immunoaffinity purification system can greatly avoid cross-contamination between organelles and crosstalk of membraneless subcellular structures.

Mitochondria, lysosomes, endoplasmic reticulum and other organelles are composed of relatively independent structures and closely linked intracellular membrane systems that efficiently support the body’s normal physiological activities. The imbalance of the cell inner membrane system leads to the imbalance of cell metabolism and the abnormal regulation of intracellular signal network, which causes uncontrollable malignant regulation of cells and promotes the occurrence of cancer and other malignant diseases.

Different organelles have their own unique compositions of nucleic acids, proteins, and metabolites, which promote the efficient operation of various biochemical reactions and support their own unique functions such as energy metabolism. Analysis of organelles, proteomics and metabolomics, has effectively revealed the unique functions and dynamic relationships of each organelle in important cell activities such as energy metabolism balance.

In contrast, nucleic acid, which is one of the three main biological molecular components of cells, has little research on its distribution as organelle components and its participation in cell metabolism. Therefore, detailed research on the unique nucleic acid components of organelles and their related proteins and metabolites will be an important breakthrough in the analysis of the dynamic behavior of organelles regulating energy metabolism.

Long non-coding RNA (LncRNA), as a type of non-coding RNA with a length greater than 200 nt, plays an important regulatory function in cell metabolism and tumorigenesis and development.

A series of preliminary studies by Lin Aifu and other research groups of Zhejiang University found that the binding of LINK-A to cell membrane lipids promoted the plasma membrane PIP3-AKT signal transduction, cytoplasmic CamK-A mediated the interaction of Ca2+ signals with NF-κB signals, and nuclear BCAR4 Mediated Hedgehoge-Hippo signal transcription coordination, showing the close connection between the biological function of lncRNA and its subcellular location.

Therefore, it is of great significance to explore the cellular spatial hierarchical distribution of LncRNAs and how to dynamically regulate cell metabolism and tumor pathogenesis under physiological and pathological conditions.

On January 4, 2021, Lin Aifu’s research group from the School of Life Sciences, Zhejiang University published a research paper entitled: Mitochondrial Long Non-coding RNA GAS5 Tunes TCA Metabolism in Response to Nutrient Stress in Nature Metabolism.

The study established the organelle immune affinity purification system, mapped the lncRNA map of the organelle, and revealed the important regulatory role of mitochondrial lncRNA GAS5 in helping cells cope with energy-deficient conditions and mediating the dissociation of the metabolic compartment of the TCA cycle, in order to study lncRNA The relationship between the dynamic regulation of subcellular space and the occurrence of human diseases provides a new perspective.

In order to accurately analyze the distribution of organelle RNA components, researchers have established a set of organelle immunoaffinity purification system through exploration and optimization. Traditional organelle purification methods mainly rely on the density gradient centrifugation of sucrose, percoll and other media, which are separated according to the different sedimentation coefficients of different organelles. However, due to the overlap of sedimentation coefficient ranges between some organelles (such as mitochondria, lysosomes), it is difficult to achieve Accurate RNA detection resolution.

In addition, recently revealed a large number of granular membraneless subcellular structures, especially RNA granule particles that are resistant to RNase digestion, often lead to contamination in various organelle components due to their more discrete sedimentation coefficients.

Therefore, the author developed a purification scheme for organelles based on immunoaffinity purification, using a simplified cell homogenization-centrifugation method to obtain a light mitochondrial fraction suspension (LMF), and then pass the mitochondrial surface marker protein Tom20 or lysosomal surface marker protein Lamp1 The antibodies captured the mitochondria and lysosome components in the LMF suspension, and finally enriched by Protein A/G magnetic beads and magnetically separated to obtain the immunoaffinity purified complete organelles.

The organelles obtained by this organelle immunoaffinity purification system can greatly avoid cross-contamination between organelles and crosstalk of membraneless subcellular structures, and because there is no need for exogenous transfection or chemical treatment of cells in advance, the integrity of the biochemical characterization of organelles can be better preserved. It is suitable for the dynamic analysis of various organelle components under different stress conditions and the extraction and analysis of organelles in tissues.

With the help of the above-mentioned organelle immunoaffinity purification system combined with RNA-seq high-throughput sequencing analysis, the author established the organelle lncRNA map. This map shows that the immunoaffinity purification method has better RNA enrichment efficiency and resolution effect than the traditional centrifugal method, revealing that the organelle lncRNA accounts for about 5.2% of the total lncRNA. The author further revealed that the organelle lncRNA is potentially involved in the regulation of related functions of the organelle through biometric analysis, qPCR verification, and siRNA screening.

Figure 1 Schematic diagram of organelle immunoaffinity purification system and selection of organelle lncRNA candidate molecules

In the established organelle lncRNA map, the author screened high abundance of organelle lncRNA through organelle extraction-qPCR, and then used mitochondrial/lysosome localization glucose sensitivity and mitochondrial ATP production/lysosome surface AMPK activation as indicators. Screened organelle lncRNAs with potential organelle-related metabolic regulation functions, including lncRNA GAS5 (Growth-Arrest-Specific 5) closely related to mitochondrial energy metabolism. The researchers further confirmed the sugar-sensitive mitochondrial localization of GAS5 and its effect on the production of mitochondrial ATP and NADH through experiments such as FISH, mitochondrial separation-qPCR, ATP detection, NADH/NAD+ detection. GAS5 has been reported to inhibit cell growth and proliferation through competitive inhibition of GR (glucocorticoid receptor) transcription activity, microRNA buffer sponge and other ways, and it is a broad tumor suppressor molecule.

However, these studies cannot fully explain the sugar-sensitive subcellular spatial dynamic behavior of GAS5 and its regulatory function on mitochondrial metabolism. The author identified the mitochondrial protein MDH2 (Malate dehydrogenase, mitochondrial) directly targeted by GAS5 through biotin-labeled RNA-pulldown combined with protein profiling analysis.

MDH2 is the node molecule that maintains the steady-state cycle of energy metabolism in the mitochondrial TCA cycle. The researchers further found that MDH2 can be coupled with its upstream and downstream FH and CS to form a metabolic compartment, and help MDH2 complete the counter-standard Gibb in the cell through the series of reaction substrates Free energy (ΔG0′) response.

Knockdown of GAS5 will enhance the stability of the metabolic compartment of MDH2 and weaken the sensitivity of the metabolic compartment to sugar; while overexpression of GAS5 can mimic the sugar-deficient signal, inhibit the formation of the FH-MDH2-CS metabolic compartment, and cause citric acid , Malic acid level is abnormal, mitochondrial energy metabolism is imbalanced, and then inhibit cell growth and proliferation. Mitochondria often have a high concentration of acetyl-CoA, and many proteins related to mitochondrial metabolism are regulated by acetylation modification.

Through the 4KR mutation of the four acetylation sites of MDH2-K185/301/307/314, the authors found that the acetylation modification of MDH2 has a central effect on the stability of the metabolic compartment of FH-MDH2-CS. Through dynamic mitochondrial translocation, GAS5 can respond to and transmit glucose deprivation signals, promote MDH2 to recruit SIRT3 and down-regulate MDH2 acetylation modification, inhibit MDH2 and FH/CS interaction to form compartments, thereby down-regulating the TCA cycle metabolic flow and inhibiting mitochondrial metabolism.

Metabolic rearrangement is an important driving factor for tumor progression, and mitochondrial metabolism provides a large amount of biosynthetic material raw materials, which is essential for tumor occurrence and development. Researchers found that GAS5 is low in breast cancer and is positively correlated with a good prognosis, while MDH2, CS, and FH are highly expressed in tumors and are related to poor prognosis. Overexpression of GAS5 significantly inhibits the formation of FH-MDH2-CS metabolic compartments in tumors, TCA cycle and tumor proliferation, thus confirming the important supporting role of mitochondrial TCA cycle on tumor occurrence and development, while low expression of GAS5 in tumors helps tumor cells avoid energy substances The inhibitory effect of mitochondrial metabolism in the absence of conditions promotes tumor progression.

Figure 2 Schematic diagram of LncRNA subcellular distribution and mitochondrial GAS5 mechanism

In summary, this study established the organelle immunoaffinity purification system, mapped the lncRNA map of the organelle, and revealed the important regulatory function of mitochondrial lncRNA GAS5 on the metabolic compartment of the TCA cycle MDH2, which provides support for mitochondrial metabolism to support tumorigenesis and development. The new evidence provides a new potential molecular target GAS5 for tumor clinical diagnosis.

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