Multiplex quantification of 5′-tRNA halves
RNA cleavages by many ribonucleases generate RNA molecules that contain a 2′,3′-cyclic phosphate (cP) at their 3′-termini, and many cP-containing RNAs (cP-RNAs) are expressed as functional molecules in cells and tissues. Representative examples of functional cP-RNAs include the 5′-tRNA half molecules produced from tRNA anticodon cleavage by angiogenin (ANG). The resultant cP-containing 5′-tRNA half molecules play important roles in a variety of biological processes. Stress-induced 5′-tRNA halves promote stress granule formation, regulate translation, and trigger cellular stress responses and apoptosis in neurodevelopmental disorders. Sex hormone-dependent 5′-tRNA halves promote cell proliferation in hormone-dependent breast and prostate cancers. Infection-induced 5′-tRNA halves promote immune response by activating Toll-like receptor 7 (TLR7). 5′-tRNA halves further serve as direct precursors of Piwi-interacting RNAs (piRNAs) in germ cells.
To further expand cP-RNA research, it is imperative to continuously unravel expressional regulations and biological roles of cP-RNAs. cP formation is not just the consequence of specific ribonuclease digestions—cP formation itself could have a functional significance. However, many previous functional characterizations of 5′-tRNA halves relied on the transfection of cP-lacking synthetic RNAs into cultured cells. We here showed in vitro production of cP-containing 5′-tRNA half molecules that is able to prepare abundant synthetic cP-RNAs enough for functional analyses.
We further reported a multiplex TaqMan RT-qPCR method that simultaneously quantifies multiple cP-containing 5′-tRNA half species. The method enabled us to efficiently quantify 5′-tRNA halves using samples with limited amounts, such as human plasma samples, revealing drastic enhancement of 5′-tRNA half levels at approximately 1,000-fold in patients infected with Mycobacterium tuberculosis.
These in vitro production and multiplex quantification methods can be applied to any cP-RNAs, and they provide cost-effective, in-house techniques to accelerate expressional and functional characterizations of 5′-tRNA halves and other cP-RNAs.
References
- Kawamura T, Shigematsu M, Kirino Y. In vitro production and multiplex quantification of 2′,3′-cyclic phosphate-containing 5′-tRNA half molecules. Methods. 2021 May 4:S1046-2023(21)00124-9. doi: 10.1016/j.ymeth.2021.04.024. Epub ahead of print. PubMed PMID:33962012.