Yohei Kirino

Schizophrenia patients’ olfactory epithelial cells contain abundant immune-activating tRNA fragments, linking small RNA biology to inflammation and suggesting avenues for diagnostics.

TRNA halves are among the most abundant short non-coding RNAs in the cellular transcriptome. Here we report that in androgen receptor-positive LNCaP prostate cancer cells, the hormone-dependent 5′-tRNA^LysCUU half promoted cell proliferation by facilitating cell cycle progression. Global mRNA profiling upon the 5′-tRNA^LysCUU half depletion revealed that the mRNA of p21, a negative regulator of the cell cycle, is post-transcriptionally destabilized via a 5′-tRNA^LysCUU half-driven mechanism. YBX1, identified as a protein interacting with 5′-tRNA^LysCUU half in the cytosol, was shown to stabilize p21 mRNA. Specific sequences resembling the 5′-tRNA^LysCUU half, located in the 3′-UTR of p21 mRNA and termed LL588, were identified as the binding site for YBX1 and are required for p21 mRNA stability. In vitro binding assays demonstrated that the 5′-tRNA^LysCUU half is capable of displacing YBX1 from LL588. Collectively, our findings suggest that the 5′-tRNA^LysCUU half directly binds to and displaces YBX1 from p21 mRNA, leading to the destabilization of p21 mRNA and the promotion of cell cycle progression in hormone-dependent cancers. Our study illuminates the role of tRNA halves in regulating mRNA stability and suggests that this may be part of broader regulatory networks affecting mRNA levels, orchestrated by various tRNA halves and their interacting proteins.

Angiogenin (Ang), an endoribonuclease belonging to the RNase A superfamily, cleaves the anticodon-loops of tRNAs to produce tRNA-half molecules. Although previous studies have demonstrated the involvement of Ang in the pathobiology of neurodegenerative disorders, the characterization of Ang-generated tRNA halves in neuronal cells remains limited. This is partly due to the technical limitations of standard RNA-seq methods, which cannot capture Ang-generated RNAs containing a 2′,3′-cyclic phosphate (cP). In this report, we established an Ang-treatment model using SH-SY5Y, a human neuroblastoma cell line, and demonstrated Ang-dependent accumulation of tRNA halves. By performing cP-RNA-seq, which selectively captures cP-containing RNAs, we identified Ang-generated tRNA halves and the specific cleavage positions within tRNA anticodon-loops responsible for their generation. Our results provide insights into the anticodon-loop cleavage and the selective production of a specific subset of tRNA halves by Ang.

Chronic obstructive pulmonary disease (COPD) is the most prevalent lung disease, and macrophages play a central role in the inflammatory response in COPD. We here report a comprehensive characterization of circulating short non-coding RNAs (sncRNAs) in plasma from patients with COPD. While circulating sncRNAs are increasingly recognized for their regulatory roles and biomarker potential in various diseases, the conventional RNA-seq method cannot fully capture these circulating sncRNAs due to their heterogeneous terminal structures. By pre-treating the plasma RNAs with T4 polynucleotide kinase, which converts all RNAs to those with RNA-seq susceptible ends (5′-phosphate and 3′-hydroxyl), we comprehensively sequenced a wide variety of non-microRNA sncRNAs, such as 5′-tRNA halves containing a 2′,3′-cyclic phosphate. We discovered a remarkable accumulation of the 5′-half derived from tRNA ^ValCAC in plasma from COPD patients, whereas the 5′-tRNA ^GlyGCC half is predominant in healthy donors. Further, the 5′-tRNA ^ValCAC half activates human macrophages via Toll-like receptor 7 and induces cytokine production. Additionally, we identified circulating rRNA-derived fragments that were upregulated in COPD patients and demonstrated their ability to induce cytokine production in macrophages. Our findings provide evidence of circulating, immune-active sncRNAs in patients with COPD, suggesting that they serve as inflammatory mediators in the pathogenesis of COPD.

Toll-like receptors (TLRs) are crucial components of the innate immune system. Endosomal TLR7 recognizes single-stranded RNAs, yet its endogenous ssRNA ligands are not fully understood. We previously showed that extracellular (ex-) 5′-half molecules of tRNA ^HisGUG (the 5′-tRNA ^HisGUG half) in extracellular vesicles (EVs) of human macrophages activate TLR7 when delivered into endosomes of recipient macrophages. Here, we fully explored immunostimulatory ex-5′-tRNA half molecules and identified the 5′-tRNA ^ValCAC/AAC half, the most abundant tRNA-derived RNA in macrophage EVs, as another 5′-tRNA half molecule with strong TLR7 activation capacity. Levels of the ex-5′-tRNA ^ValCAC/AAC half were highly up-regulated in macrophage EVs upon exposure to lipopolysaccharide and in the plasma of patients infected with Mycobacterium tuberculosis . The 5′-tRNA ^ValCAC/AAC half-mediated activation of TLR7 effectively eradicated bacteria infected in macrophages. Mutation analyses of the 5′-tRNA ^ValCAC/AAC half identified the terminal GUUU sequence as a determinant for TLR7 activation. We confirmed that GUUU is the optimal ratio of guanosine and uridine for TLR7 activation; microRNAs or other RNAs with the terminal GUUU motif can indeed stimulate TLR7, establishing the motif as a universal signature for TLR7 activation. These results advance our understanding of endogenous ssRNA ligands of TLR7 and offer insights into diverse TLR7-involved pathologies and their therapeutic strategies.