RNA Biology Research
Overall research goal of the Kirino lab is to understand the biogenesis mechanism and molecular 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 novel and 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 transfer RNA (tRNA)-derived ncRNAs, cyclic phosphate-containing RNAs (cP-RNAs), 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).
tRNA-derived ncRNAs in Diseases
Since their discovery in the 1950s, tRNAs have been best known as abundant adapter components of the translational machinery, converting mRNA codon information into amino acid sequences. However, recent studies have established the new concept that tRNAs are further processed to generate specific tRNA-derived ncRNAs (Reviewed in Gene Regul Syst Biol 2015). 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 are produced from angiogenin-catalyzed anticodon cleavage of tRNAs and actively promote cell proliferation. Our studies have unveiled a novel tRNA-engaged pathway in tumorigenesis and implicate SHOT-RNAs as potential candidates for biomarkers and therapeutic targets in hormone-dependent cancers. While we have been continuously elucidating molecular mechanisms of SHOT-RNA function, we are further expanding our characterization of tRNA-derived ncRNAs in asthma and infectious diseases.
piRNA Pathway in Germline Development
Piwi-interacting RNAs (piRNAs) are a germline-specific class of short ncRNAs that play crucial roles in germline development. We have been utilizing mouse, Bombyx, and Drosophila systems to elucidate the biogenesis mechanism of piRNAs. Piwi proteins, bound to piRNAs, contain a conserved arginine dimethylations (sDMAs; Nat Cell Biol 2009; Methods Mol Biol 2014), which are specifically recognized by Tudor domain of proteins (RNA 2010). Our RNAi screening for Tudor proteins identified BmPapi as a novel piRNA biogenesis factor modulating piRNA maturation on mitochondrial outer membrane (RNA 2013). The “Trimmer” enzyme responsible for piRNA 3′-end maturation has been identified by analyzing BmPapi-interacting proteins (Cell 2016). Our studies further revealed how cell-cell contact regulates piRNA biogenesis (Sci Rep 2017) and clarified how piRNAs are produced from tRNAs (Nucleic Acids Res 2017).
Novel Tools for ncRNA Sequencing and Quantification
With the advent of next-generation sequencing, transcriptome profiling using RNA-seq has become a ubiquitous tool in biomedical research. However, standard RNA-seq method does not fully capture all of the cellular RNAs expressed; highly-structured or modified RNAs are often not efficiently sequenced by standard RNA-seq procedure. We have been developing methods for specific sequencing/quantification of those "difficult-to-analyze" RNAs to capture whole RNA expression profiles precisely and efficiently.
cP-RNA-seq: RNA molecules generated by ribonuclease cleavage sometimes harbor a cP at their 3'-ends, and those cP-containing RNAs (cP-RNAs) form a hidden layer of transcriptome because standard RNA-seq cannot capture them (Reviewed in Front Genet 2018). We developed cP-RNA-seq that can exclusively amplify and sequence cP-containing RNAs (Proc Natl Acad Sci 2015; Nat Protoc 2016). In the method, RNAs are treated with phosphatase and then with periodate. Only cP-RNAs survive these treatments and become substrates for later sequencing steps. Our cP-RNA-seq provides us a unique opportunity to unveil the repertoire of cP-RNAs. Our recent genome-wide identification of cP-RNAs in mouse tissues identified numerous novel cP-RNA species and their regulations in aging (PLoS Genet 2019).
YAMAT-seq: YAMAT-seq is an efficient high-throughput sequencing method of tRNAs (Nucleic Acids Res 2017). YAMAT-seq circumvents the issue of inefficient adapter ligation, a characteristic of standard RNA-seq for tRNAs, by employing the efficient and specific ligation of Y-shaped adapter to tRNAs. The method has high specificity for tRNAs and high sensitivity to detect most isoacceptors from minute amount of total RNA. Moreover, YAMAT-seq shows quantitative capability to estimate tRNA expression levels, and has high reproducibility and broad applicability for various cells and tissues. By using this method, we recently discovered the expression of novel mitochondrial ncRNAs (non-tRNAs) whose 3'-terminal CCA sequences are added by CCA-adding enzyme (RNA Biol 2019).
Dumbbell-PCR: Short ncRNAs are not always expressed as single entities with fixed terminal sequences but as multiple isoforms bearing complex heterogeneity in both length and terminal sequences, such as isomiRs, the isoforms of microRNAs. We developed Dumbbell-PCR to distinctively quantify a specific individual RNA variant (Nucleic Acids Res 2015; Methods Mol Biol 2018). In the method, stem–loop adapters are specifically hybridized and ligated to the both 5′- and 3′-ends of targeted RNAs. The resultant ‘dumbbell-like’ ligation products are quantified by TaqMan qRT-PCR. Dumbbell-PCR provides a much-needed simple method for specifically quantifying RNA variants with a single nucleotide resolution.
Four-Leaf clover qRT-PCR (FL-PCR): Standard RT-qPCR amplification of tRNAs cannot distinguish signals among mature tRNAs, their precursors, and their shorter fragments, because these RNA species have identical sequences. To circumvent the issue, we developed Four-leaf clover PCR which can specifically quantify mature tRNAs (RNA Biol 2015). In the method, a DNA/RNA hybrid stem-loop adapter is specifically hybridized and ligated to mature tRNAs, generating tRNA-adapter ligation products with a “four-leaf clover” structure which is quantified by TaqMan RT-qPCR.