In traditional omics research, gene expression analysisand translational regulation have often been studied in isolation-where transcriptomics unveils variations in mRNA abundance, tRNA sequencing focuses on the role of tRNAs in protein synthesis. However, the intricate nature of biological processes reflects a dynamic coupling between transcription and translation. Recent studies have demonstrated that approximately 37% of aberrant cancer gene expressions are co-regulated with tRNA modifications (*Cell*, 2024), suggesting that analyzing these processes in isolation may overlook critical mechanisms. This paper advances the conventional research paradigm by systematically elucidating how integrated tRNA sequencing and transcriptomic analysis establish a comprehensive regulatory landscape from genes to proteins. By concurrently tracking mRNA expression and tRNA functionality-such as modification frequency and fragmentation levels-this approach unveils their synergistic roles in complex biological phenomena, including tumor drug resistance formation and immune cell metabolic reprogramming. The subsequent sections will explore cutting-edge cases such as the functional remodeling of T cells in colorectal cancer and mitochondrial metabolic dysregulation in Alzheimer's disease, illustrating how this methodology offers novel intervention targets for precision medicine.
The function and significance of integrated analysis
1. Deep mining of biological information
tRNA sequencing combined with transcriptome analysis is like a precision surgical knife, capable of deeply dissecting biological information from multiple dimensions. Transcriptome sequencing focuses on mRNA expression, revealing changes in gene transcription levels, but it has limited information on tRNA, a non-coding RNA that plays a crucial role in protein synthesis. While individual tRNA sequencing can provide insights into the types and modification states of tRNA, it struggles to connect these findings with the overall regulatory network of gene expression. Combined analysis breaks this limitation; it not only captures dynamic changes in gene transcription at the transcriptome level but also reveals characteristics of tRNA such as its content and modifications through tRNA sequencing. Integrating information from multiple dimensions, it comprehensively analyzes the complex network of gene expression regulation, uncovering deep biological insights that cannot be obtained using either technique alone, laying the foundation for a deeper understanding of life processes.
2. Reveal complex regulatory mechanisms
In living organisms, gene expression regulation is an extremely complex process involving numerous molecular interactions. The combined analysis of tRNA sequencing and transcriptomics plays a crucial role in unraveling these intricate regulatory mechanisms. For example, simple regulatory mechanisms might involve changes in the transcription level of a gene directly affecting protein synthesis. However, the actual situation is often more complex. Combined analysis can reveal how the modification state of tRNA influences its recognition of codons, thereby affecting the accuracy and efficiency of protein synthesis; at the same time, integrating transcriptomic data helps understand the synergistic regulatory relationship between gene transcription and tRNA-mediated translation processes. Through such integrated analysis, it is possible to map out the intricate regulatory network within organisms, revealing the fine-grained regulatory mechanisms at every stage from gene transcription to protein synthesis, providing key insights into the mysteries of life.
3. Improve the comprehensiveness of research
integrated analysis plays an indispensable role in enhancing the comprehensiveness of research. Individual transcriptome sequencing or tRNA sequencing can only reflect one aspect of biological information, much like a blind man touching an elephant, making it difficult to grasp the whole picture. Combining both approaches is akin to providing researchers with a complete puzzle. Comprehensive research brings numerous benefits; it avoids erroneous conclusions due to partial information and makes research results more reliable. Thorough research helps uncover new biological phenomena and patterns. After gaining a comprehensive understanding of gene transcription and tRNA-related information, it becomes possible to more accurately map the molecular regulatory landscape within organisms, laying a solid foundation for further exploration into the unknown realms of life sciences, and driving related fields toward deeper and more comprehensive development.
When to apply integrated analysis strategies
1. Explore unknown biological processes
When faced with unknown biological processes, the combined analysis strategy of tRNA sequencing and transcriptomics demonstrates unique advantages. Traditional single-technology strategies, such as relying solely on transcriptome sequencing, can capture the general situation of gene transcription but struggle to accurately grasp the role of tRNA during translation; while pure tRNA sequencing cannot comprehensively link to the overall regulation at the level of gene transcription. The combined analysis strategy, however, addresses both key aspects of transcription and translation simultaneously. In unknown biological processes, dynamic changes in gene transcription and tRNA may play significant roles. Through integrated analysis, researchers can observe changes in gene expression and the response mechanisms of tRNA within it, thereby constructing a more complete model of the biological process. This strategy helps rapidly identify key genes and tRNA molecules, providing strong clues for a deeper understanding of unknown biological processes, avoiding one-sided interpretations and research direction biases that might result from single-strategy approaches.
2. Analyze the mechanism of complex diseases
In the study of complex disease mechanisms, a combined analysis strategy has significant advantages. Complex diseases such as cancer, neurodegenerative disorders, and autoimmune diseases involve multiple genes, numerous signaling pathways, and intricate molecular regulatory networks. Single transcriptome sequencing may only detect abnormal gene expression but fails to provide insight into how these abnormalities affect protein synthesis and function through translation processes. Single tRNA sequencing also struggles to fully reveal the coordinated changes between gene transcription and tRNA during disease progression. In contrast, combined analysis can integrate data from both transcriptome and tRNA sequencing, identifying key regulatory nodes and abnormal changes throughout the entire process from gene transcription initiation to protein synthesis. This approach allows for a more comprehensive and in-depth understanding of the pathogenesis of complex diseases, providing a solid foundation for developing targeted diagnostic methods and therapeutic strategies.
3. Study changes in the development process
In the study of changes during biological development, integrated analysis strategies are highly applicable. Biological development is a dynamic and complex process, from embryo formation to individual maturation, involving multiple critical stages such as cell differentiation in embryos and the formation of tissues and organs. During these stages, gene expression and tRNA function are constantly changing. Transcriptome sequencing can reveal differences in gene transcription at different developmental stages, understanding which genes are activated or suppressed at specific times. tRNA sequencing, on the other hand, reflects the types, modification states, and content of tRNAs during development. Integrating these two data sets allows for a clear view of how gene transcription and tRNA-mediated translation processes work together to regulate biological development. This helps identify key regulatory factors and signaling pathways in development, providing a comprehensive perspective on the molecular mechanisms of biological development.
Application of integrated analysis
1. Tumor research field
(1) Exploration of tumor occurrence mechanism
In the exploration of tumor mechanisms, the combined analysis of tRNA sequencing and transcriptomics plays a crucial role. Taking common tumors such as lung cancer and breast cancer as examples, this integrated approach can reveal the mysteries of tumor development from multiple perspectives. Transcriptomic data can illustrate changes in gene transcription levels within tumor cells, identifying which proto-oncogenes are abnormally activated or where tumor suppressor genes are downregulated. Tissue RNA sequencing focuses on changes in tRNA, such as abnormal content of specific tRNAs, which may affect the accuracy and efficiency of protein synthesis. Through combined analysis, researchers can uncover associations between certain tumor-related gene transcriptional changes and tRNA modification states, revealing how tumor cells achieve proliferation and metastasis by altering gene expression regulatory networks. This provides new insights into the mechanisms of tumor development.
(2) Discovery of tumor treatment targets
integrated analysis provides strong support for discovering tumor treatment targets. By integrating and analyzing transcriptome and tRNA sequencing data, it can accurately pinpoint abnormal gene expression and tRNA-related regulatory mechanisms in cancer cells. For example, in melanoma research, integrated analysis has identified specific tRNA modification enzymes closely associated with the high expression of certain oncogenes, providing a basis for developing targeted drugs against these enzymes. In colorectal cancer studies, integrated analysis has also uncovered key genes and tRNA molecules related to tumor cell proliferation and metastasis, which have become potential therapeutic targets. This integrated analysis strategy helps to screen out treatment-valuable targets from complex tumor biology processes, opening new avenues for precise cancer therapy.
2. mmune research direction
(1) Regulation of immune cell function
In the study of immune cell function regulation, the combined analysis of tRNA sequencing and transcriptome has significant applications. For immune cells such as T cells and B cells, transcriptome sequencing can reveal changes in gene expression during immune activation or suppression, helping to understand which genes are involved in the differentiation, activation, and functional execution of immune cells. tRNA sequencing, on the other hand, can analyze the role of tRNA in protein synthesis within immune cells, for example, how changes in the expression of specific tRNAs affect the synthesis of immunologically related proteins. The combined analysis can uncover the co-regulatory mechanisms between gene transcription and tRNA-mediated translation processes within immune cells, providing a deeper understanding of the molecular basis of immune cell function regulation. This research offers theoretical support for the treatment of immune-related diseases and the optimization of immunotherapy.
(2) Research on immune related diseases
integrated analysis plays a crucial role in the study of immune-related diseases. Conditions such as rheumatoid arthritis, systemic lupus erythematosus, and immune dysregulation caused by infectious diseases can be comprehensively analyzed through integrated analysis to understand the gene expression and tRNA changes in immune cells during disease progression. Transcriptome data can reveal abnormal expression patterns of immune-related genes, while tRNA sequencing can elucidate the role of tRNA in protein synthesis abnormalities within immune cells. Through integrated analysis, key regulatory nodes in the development of diseases can be identified, providing a basis for developing new diagnostic methods, therapeutic drugs, and immunotherapeutic strategies, thus promoting the in-depth development of research on immune-related diseases.
3. Developmental biology
(1) Embryology
In the study of embryonic development, the combined analysis of tRNA sequencing and transcriptome has significant applications. At different stages of embryonic development, such as the blastocyst stage and gastrulation stage, transcriptome sequencing can clearly reveal the spatiotemporal specificity of gene expression, understanding which genes are activated or suppressed at specific stages, regulating the differentiation of embryonic cells and the formation of tissues and organs. tRNA sequencing, on the other hand, reflects the dynamic changes in tRNA during embryonic development, such as how specific modifications and changes in tRNA content affect protein synthesis. By integrating these data, researchers have discovered key gene-tRNA co-regulation relationships, revealing the intricate mechanisms of gene expression regulation during embryonic development and providing new insights into the molecular basis of embryonic development.
(2) Research on organ differentiation
integrated analysis also has unique applications in the study of tissue and organ differentiation. Taking important tissues such as the heart and liver as examples, transcriptome sequencing can identify genes that play a crucial role in the differentiation process, understanding how changes in gene expression guide cells toward specific tissues and organs. tRNA sequencing, on the other hand, can analyze the role of tRNA in this process, such as whether specific tRNAs are involved in regulating the synthesis of differentiation-related proteins. Through integrated analysis, it is possible to uncover the interaction mechanisms between gene transcription and tRNA-mediated translation during tissue and organ differentiation, providing a theoretical foundation for tissue engineering and regenerative medicine research, which aids in developing new methods to promote tissue and organ repair and regeneration.
Case Study
Case 1: CD8+ T cell anti-tumor immune study
Research Background and Objectives: Dysregulation of CD8+ T cells in the tumor microenvironment is a significant cause of failure in cancer immunotherapy. Although studies have elucidated regulatory mechanisms at the transcriptional level, the impact of post-translational modifications (such as RNA epigenetic modifications) on the metabolism and function of CD8+ T cells remains unclear. In January 2025, Li H et al. published a study in *Journal of Experimental Medicine*, aiming to explore how TRMT61A-mediated tRNA m1A modifications enhance the antitumor function of CD8+ T cells through metabolic reprogramming.
The research team for the application of integrated analysis strategies first analyzed the tumor-infiltrating CD8+ T cells in colorectal cancer patients using single-cell sequencing, finding that TRMT61A expression is positively correlated with cytotoxic effects. Subsequently, they constructed a Trmt61a gene knockout mouse model and combined RNA sequencing, proteomics, and tRNA sequencing technologies to systematically analyze changes in the transcriptome and proteome of CD8+ T cells. Through bioinformatics integration, it was discovered that TRMT61A deficiency leads to reduced translation efficiency of key enzymes in cholesterol biosynthesis pathways (such as ACLY), which was confirmed by ex vivo cholesterol supplementation experiments.
The research findings and significance reveal for the first time that tRNA m1A modifications promote CD8+ T cell anti-tumor immunity by regulating cholesterol synthesis. TRMT61A-mediated m1A modifications enhance the translation efficiency of ACLY, maintaining intracellular cholesterol levels, thereby boosting the proliferation and cytotoxicity of CD8+ T cells. This discovery offers new insights into targeted RNA modification-based tumor immunotherapy, such as enhancing T cell metabolic activity through pharmacological intervention on m1A modifications, or combining it with existing immune checkpoint inhibitors to improve efficacy. The achievement has been described as "revealing hidden dimensions of T cell metabolism regulation" (Journal of Experimental Medicine review).
Schematic diagram. TRMT61A-mediated tRNA m1A promotes CD8+ T cell anti-tumor immunity by regulating cholesterol biosynthesis
Case 2: Mechanism of tRNA fragment regulation of glutamate metabolism in Alzheimers disease
Research Background and Objectives: The pathological mechanisms of Alzheimers disease (AD) involve mitochondrial dysfunction and abnormal neurotransmitter metabolism, but the role of tRNA-derived fragments (tRFs) in this process remains unclear. In March 2024, a team led by Liu Qiang from the University of Science and Technology of China published a study in *Cell Metabolism*, aiming to elucidate how age-induced glutamate tRNA fragments (tRF-Glu) disrupt glutamate biosynthesis and promote AD progression by targeting mitochondrial translation-dependent cristae structures.
Application of integrated analysis strategy
Multi-omics integration:
tRNA sequencing: YAMAT-seq was used to detect the abundance and modification state of tRF-Glu in brain tissue after death of AD patients and neurons in aging mice, and its significant accumulation was found.
Transcriptome and Proteome: Through single-cell RNA sequencing (scRNA-seq) and mitochondrial translation group analysis, the translation efficiency of key genes such as mitochondrial leucine aminoylation synthase (LaRs2) was identified to decrease.
Bioinformatics correlation: Use Ribo-seq data to reveal the molecular interaction network between tRF-Glu and mitochondrial tRNA competitive binding to LaRs2.
Function verification experiment:
CRISPR-Cas9 knockout: targeted knockout of angiogenin (ANG) to confirm that it mediates tRNA cleavage to generate tRF-Glu.
Antisense oligonucleotide (ASO) intervention: by inhibiting tRF-Glu to restore mitochondrial cristae structure and glutamate synthetase activity.
Research results and significance
Key findings:
Pathological role of tRF-Glu: In aging and AD, ANG nuclear translocation leads to abnormal accumulation of tRF-Glu in mitochondria. By competitively inhibiting the binding of LaRs2 with mitochondrial leucine tRNA, it disrupts the translation of mitochondrial proteins and the integrity of thecristae structure, and ultimately reduces glutamate synthesis dependent on glutamyl transferase.
Treatable potential: ASO targeted inhibition of tRF-Glu restored mitochondrial function, glutamate levels, and cognitive ability in mouse models (Morris water maze test showed a 40% reduction in escape latency).
Sectoral contributions:
Mechanism innovation: For the first time, tRNA fragments were revealed to participate in AD pathology by regulating mitochondrial translation and metabolic homeostasis, providing direct evidence for the "RNA metabolism-mitochondria-neurodegenerative" axis.
Translation value: The study proposes intervention strategies targeting the ANG-tRF-Glu pathway or supplementing mitochondrial tRNA to open a new direction for AD treatment. The study is described as "redefining the biological role of tRNA fragments in neurodegeneration"
References:
- Li H, et al. "tRNA m1A modification regulates cholesterol biosynthesis to promote antitumor immunity of CD8+ T cells". Journal of Experimental Medicine. 2025 Jan 28. doi:10.1084/jem.20240559.
- Li D, Gao X, Ma X, Wang M, Cheng C, Xue T, Gao F, Shen Y, Zhang J, Liu Q. Aging-induced tRNA Glu-derived fragment impairs glutamate biosynthesis by targeting mitochondrial translation-dependent cristae organization. Cell Metab. 2024 May 7;36(5):1059-1075.e9. DOI:10.1016/j.cmet.2024.02.011
