RNA Biology Research
Overall research goal of the Kirino lab is to understand the biogenesis and function of short non-coding RNAs (ncRNAs) and use the knowledge for development of novel biomarkers and therapeutic applications in diseases. Short ncRNAs have emerged as one of the most exciting areas of gene expression regulation. By taking advantage of RNA biology/biochemistry, molecular/cellular biology, and computational biology, the Kirino lab is particularly focused on cyclic phosphate-containing RNAs (cP-RNAs), transfer RNA (tRNA)-derived ncRNAs, and Piwi-interacting RNAs (piRNAs), which play crucial roles in various biological processes and diseases. Dr. Kirino is Associate Professor in Computational Medicine Center and in Department of Biochemistry and Molecular Biology at Thomas Jefferson University where he joined in mid-2013. Prior to joining TJU, Dr. Kirino was Assistant Professor in Department of Biomedical Sciences at Cedars Sinai Medical Center (2010-2013). Dr. Kirino received his BSc (2001), MSc (2003) and PhD (2006) from The University of Tokyo (Dr. Tsutomu Suzuki’s lab) and performed Postdoc study (2006-2010) in University of Pennsylvania School of Medicine (Dr. Zissimos Mourelatos’ lab).
Unraveling a hidden component of the cellular transcriptome
With the advent and evolution of next-generation sequencing technology, efforts to identify and catalog the expressed RNA molecules have greatly advanced our understanding of RNA biology. However, the current standard RNA-seq methods, particularly those targeting short ncRNAs, do not fully capture all of the RNAs expressed but allow for some “escapers” to slip. We have been developing methods for sequencing/quantification of those "difficult-to-analyze" escapers to capture whole RNA expression profiles precisely and efficiently.
For example, RNA molecules generated by ribonuclease cleavage sometimes harbor a 2’,3’-cyclic phosphate (cP) at their 3’-ends, and those cP-containing RNAs (cP-RNAs) cannot be captured by standard RNA-seq (Reviewed in Front Genet 2018). We developed “cP-RNA-seq” that can specifically sequence cP-RNAs (Proc Natl Acad Sci 2015; Nat Protoc 2016) and performed the first genome-wide profiling of short cP-RNA species (PLoS Genet 2019). The expression of cP-RNAs is regulated by hormones (Proc Natl Acad Sci 2015), immune response (PLoS Biol 2020), aging (PLoS Genet 2019), and stresses (RNA 2020).
We further developed “YAMAT-seq,” an efficient high-throughput sequencing method of tRNAs (Nucleic Acids Res 2017). In addition to the whole tRNA expression profiles, the method discovered the expression of novel mitochondrial ncRNAs whose 3'-terminal CCA sequences are added by CCA-adding enzyme (RNA Biol 2019).
Regarding quantification of short ncRNAs, we developed Dumbbell-PCR which can distinctively quantify a specific individual RNA variant with single-nucleotide resolution (Nucleic Acids Res 2015; Methods Mol Biol 2018). We further developed Four-Leaf clover RT-qPCR (FL-PCR) which can specifically quantify mature tRNAs (RNA Biol 2015). Moreover, our sensitive, multi-plex TaqMan RT-qPCR method enables quantification of cP-RNAs and other “difficult” short ncRNAs in various samples including human plasma or other clinical samples (Methods 2021).
Novel ncRNA molecules in diseases and germline development
Our cP-RNA research has been discovering novel ncRNA pathways which play crucial roles in various biological processes and diseases. For example, we discovered that a novel type of tRNA-derived ncRNAs, termed Sex Hormone-dependent TRNA-derived RNAs (SHOT-RNAs), are abundantly expressed in hormone-dependent breast and prostate cancers (Proc Natl Acad Sci 2015; Mol Cell Oncol 2016; RNA 2017). SHOT-RNAs belong to cP-RNAs and are produced from angiogenin-catalyzed anticodon cleavage of tRNAs. These hormone-dependent cP-RNAs actively promote cell proliferation, unveiling a novel cP-RNA-engaged pathway in tumorigenesis and implicate cP-RNAs as potential candidates for biomarkers and therapeutic targets in hormone-dependent cancers.
Our further research discovered a novel type of infection-induced cP-RNAs in innate immune response (PLoS Biol 2020). Mycobacterial infection and accompanying surface Toll-like receptor (TLR) activation upregulate the expression of specific tRNA-derived cP-RNAs in human monocyte-derived macrophages (HMDMs). Their abundant accumulation also occurs in HMDM-secreted extracellular vehicles (EVs), and the EV-contained cP-RNAs are delivered into endosomes in recipient cells and to activate endosomal TLR7. Drastic upregulation of cP-RNAs (>1,000-fold) was observed in the plasma of patients infected with Mycobacterium tuberculosis (Methods 2021). These results unveil a novel cP-RNA-engaged pathway in the innate immune response and assign the role of "immune activators" to cP-RNA molecules.
Our recent research further revealed the crucial role of cP-RNAs in piRNA biogenesis (Nat Commun 2021). In animal germlines, Piwi proteins and the associated piRNAs protect genome integrity by silencing transposons. We have been utilizing mouse, Bombyx, and Drosophila systems to elucidate the biogenesis mechanism of piRNAs (Nat Struct Mol Biol 2007; RNA 2007; Nat Cell Biol 2009; RNA 2010; J Biol Chem 2010; RNA 2013; Cell 2016; Sci Rep 2017; Nucleic Acids Res 2017). We recently discovered that, for robust piRNA production, a mitochondria-associated endoribonuclease RNase Kappa (k) cleaves single-stranded RNAs to generate short cP-RNAs, which serve as direct precursor molecules for piRNAs. Depletion of cP-RNAs by BmRNase k-knockdown elevated transposon levels and disrupted a piRNA-mediated sex determination in Bombyx embryos, indicating the crucial roles of cP-RNAs in piRNA biogenesis and embryonic development (Nat Commun 2021).