The New Protein Cytb-187aa Regulates Mammalian Early Development

Academic Background

Mitochondria are multifunctional organelles that provide energy for cells. In addition to energy supply, they are involved in the regulation of apoptosis, cell signaling, and various biosynthetic pathways. Among these functions, substances within mitochondria can be released into the cytoplasm or nucleus, acting as signaling molecules. For example, mitochondrial reactive oxygen species (ROS) and calcium ions (Ca2+) can be released into the cytoplasm to participate in apoptosis and cell fate determination. The release of mitochondrial RNA and peptides also plays a crucial role in various metabolic stress responses and biological processes.

Previous studies have shown that the mitochondrial genome contains 37 genes, of which 13 encode proteins used for oxidative phosphorylation, all translated within the mitochondria using a variant genetic code. Until now, no studies have explored whether the mRNA encoded by these mitochondrial genes can also be translated in the cytoplasm using cytoplasmic ribosomes with the standard genetic code.

Research Source

This study was jointly conducted by Zhijuan Hu, Liang Yang, Maolei Zhang, and other researchers from the Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences Institute of Biophysics, Guangzhou Medical University, and Hong Kong Institute of Science & Innovation. The research results were published on July 2, 2024, in the journal “Cell Metabolism” under the title “A novel protein cytb-187aa encoded by the mitochondrial gene cytb modulates mammalian early development.”

Research Process

Experimental Process

1. Discovery and Validation of the New Protein: Using open reading frame (ORF) finder tools, researchers discovered a new protein, cytb-187aa, encoded within the mitochondrial gene cytb, which consists of 187 amino acids. This was confirmed through liquid chromatography-tandem mass spectrometry (LC-MS/MS) and antibody detection.

2. Cytoplasmic Ribosomal Translation: Utilizing the Moontag system, researchers confirmed the translation of cytb-187aa mRNA on cytoplasmic ribosomes, indicating the use of the standard genetic code.

3. Subcellular Localization and Functional Investigation: Microscopic examination revealed that cytb-187aa primarily localizes to the mitochondrial matrix. By constructing a series of deletion mutants and microscopic imaging, the mitochondrial targeting signal peptide was identified as located at the N-terminal region encompassing the first 1-37 amino acids.

4. Functional Verification: Through overexpression and knockdown experiments in induced pluripotent stem cells (iPSCs), the study showed that the protein influences the transition of iPSCs to a pluripotent state. Utilizing anti-Flag co-immunoprecipitation, its interaction with the mitochondrial phosphate carrier Slc25a3 was identified, and ATP level testing confirmed its role in ATP-dependent pluripotency transition.

5. Mouse Model Validation: Using the CRISPR-Cas9 gene-editing technique, a cytb-187aa knockdown mouse model was created. It was found that knocking down cytb-187aa led to a reduced number of ovarian follicles and decreased fertility in female mice.

Data Analysis Methods and Tools

Throughout the study, the ORF finder tool was used for open reading frame searches, and LC-MS/MS was applied for protein identification and validation. Custom antibodies were used for specific detection of the new protein. For cytoplasmic translation verification, the Moontag system was primarily employed. Co-immunoprecipitation experiments used anti-Flag antibodies, and data analysis was performed using parallel reaction monitoring mass spectrometry (PRM-MS) and real-time monitoring with microscopic instruments.

Experimental Results and Conclusions

Main Experimental Results

1. Discovery and Validation of the New Protein: A new protein, cytb-187aa, consisting of 187 amino acids, was found within the cytb gene and verified through mass spectrometry and specific antibodies. LC-MS/MS data and PRM-MS analysis showed high expression of this protein in various cell types.

2. Cytoplasmic Ribosomal Translation: The Moontag system demonstrated the translation of cytb-187aa mRNAs in the cytoplasm. A ribosome probe showed its translation on cytoplasmic ribosomes, further verifying its unique translation pattern using the standard genetic code.

3. Subcellular Localization and Functional Investigation: Microscopic imaging revealed that the cytb-187aa protein mainly localizes in the mitochondrial matrix, with its mitochondrial signal peptide located at the N-terminal region of 1-37 amino acids. The protein is highly expressed in pluripotent cells and plays a key role in the necessary inducible transition of pluripotent stem cells.

4. Functional Verification: By overexpressing or knocking down cytb-187aa, its significant role in the ATP-dependent transition to pluripotency was discovered. Further exploration of its interaction with the mitochondrial phosphate carrier Slc25a3 confirmed that cytb-187aa promotes the state transition of pluripotent cells by regulating ATP levels.

5. Mouse Model Validation: In a cytb-187aa knockdown mouse model, the protein was found to play an important role in female fertility and the number of ovarian follicles, with higher expression levels in ovarian tissues.

Conclusion and Significance

This paper is the first to reveal that the mitochondrial cytb gene encodes not only the Cytb protein of complex III but also a new 187-amino-acid-long protein, cytb-187aa, which is translated in the cytoplasm using the standard genetic code. This new finding expands the understanding of mitochondrial genome translation and its functions. Additionally, the protein shows important regulatory roles in mammalian early development and female reproductive capability, suggesting potential applications in maintaining and transforming pluripotent stem cells.

Research Highlights

1. Revealing a New Translation Pattern: For the first time, it was discovered that the mRNA of the mitochondrial gene cytb can be translated in the cytoplasm using cytoplasmic ribosomes with the standard genetic code, elucidating a new mechanism for the mitochondrial genome in the cytoplasm.

2. Discovery of a New Functional Protein: A new mitochondrial-encoded protein, cytb-187aa, was discovered and confirmed to play an important role in the transition of pluripotent stem cells and female mouse fertility.

3. Application of Novel Experimental Techniques: Systematic methods based on LC-MS/MS and PRM-MS, combined with gene-editing technology, were used to precisely verify the existence and function of the new protein.

Limitations and Future Directions

Although this paper reveals the regulatory role of cytb-187aa in pluripotency transitions and fertility, its specific regulatory mechanisms require further exploration. Future research should further uncover the detailed mechanisms of cytb-187aa in regulating cell fate and gene expression, with the aim of providing new theoretical foundations and practical references for stem cell therapy and reproductive medicine.