tRNA-derived ncRNAs in Diseases
Since their discovery in the 1950s, tRNAs have been best known for their role in helping the cell make proteins from messenger RNA templates. However, recent studies have led to a previously-unsuspected concept that tRNAs are not always the end product; namely, they can be further processed to generate small functional ncRNAs.
We have recently discovered that a novel type of tRNA-derived RNAs, termed Sex Hormone-dependent TRNA-derived RNAs (SHOT-RNAs), are specifically and abundantly expressed in estrogen receptor (ER)-positive breast cancer and androgen receptor (AR)-positive prostate cancer (Honda et al., PNAS 2015). SHOT-RNAs are produced from aminoacylated mature tRNAs by angiogenin (ANG)-mediated anticodon cleavage, which is promoted by sex hormones and their receptors. Resultant 5′- and 3′-SHOT-RNAs, corresponding to 5′- and 3′-tRNA halves, bear a cyclic phosphate (cP) and an amino acid at the 3′-end, respectively. Importantly, SHOT-RNAs have significant functional involvement in cell proliferation. Our studies have unveiled a novel tRNA-engaged pathway in tumorigenesis of hormone-dependent cancers and implicate SHOT-RNAs as potential candidates for biomarkers and therapeutic targets.
It was only recently that the expression profiles and functions of tRNA-derived RNAs began to be explored. Since most of these studies relied on standard RNA-seq data, SHOT-RNAs (tRNA halves) have been “invisible” in these studies because of the presence of terminal modifications and therefore have been eluding us. By utilizing our original sequencing techniques (described below), we are currently characterizing the expression profiles and functions of tRNA halves in asthma and neurodegenerative disorders, as well as in cancers, which is expected to clarify the involvement of currently uncharacterized components of ncRNAs in diseases.
Biochemical tools for specific quantification and sequencing of ncRNAs
cP-RNA-seq: Although next-generation sequencings largely unveil the cellular transcriptome, the current standard RNA-seq methods, particularly those targeting small RNAs, include an adapter ligation step and thereby limit their utility for sequencing RNAs with terminal modifications. Our study revealed the presence of 3′-terminal modifications in tRNA halves, which preclude ligation of the tRNA halves to a 3′-adapter and thus exclude them from the sequence data obtained using standard RNA-seq methods. To circumvent the issues, we recently developed a “cP-RNA-seq” method that can exclusively amplify and sequence cP-containing RNAs, namely 5′-tRNA halves (Honda et al., Nat. Protoc. 2016). In the method, gel-purified RNAs are treated with phosphatase, followed by periodate treatment. The RNAs containing 3′-cP survive these treatments and therefore become the only RNAs suitable for adapter ligations, cDNA amplification, and sequencing in later steps. Our cP-RNA-seq is convenient, efficient, and specific, providing us a unique opportunity to unveil the repertoire of tRNA halves and other cP-containing RNA species in diseases.
Dumbbell-PCR (Db-PCR): Small 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 (miRNAs). We recently developed “Dumbbell PCR (Db-PCR)”, an efficient and convenient method to distinctively quantify a specific individual small RNA variant (Honda and Kirino, NAR 2015). In Db-PCR, 5′- and 3′-stem–loop adapters are specifically hybridized and ligated to the 5′- and 3′-ends of target RNAs, respectively, by T4 RNA ligase 2 (Rnl2). The resultant ligation products with ‘dumbbell-like’ structures are subsequently quantified by TaqMan qRT-PCR. Db-PCR has broad applicability for the quantification of various small RNAs in different cell types, and provides a much-needed simple method for specifically quantifying small RNA variants with a single nucleotide resolution.
Four-Leaf clover qRT-PCR (FL-PCR): The analyses of tRNA-derived ncRNAs require convenient methods to quantify mature tRNA substrates. Standard qRT-PCR amplification of the interior sequences of the mature RNAs cannot distinguish signals among mature tRNAs and their precursors and fragments, because these RNA species have identical sequences. To circumvent this issue, we recently developed “Four-Leaf clover qRT-PCR (FL-PCR)”, an efficient and convenient method to specifically quantify mature tRNAs (Honda et al., RNA Biol. 2015). In FL-PCR, a DNA/RNA hybrid stem-loop adapter (SL-adapter) is specifically hybridized and ligated to mature tRNAs by Rnl2, generating tRNA-adapter ligation products with a “four-leaf clover” structure which is amplified and quantified by TaqMan qRT-PCR. FL-PCR has broad applicability for the quantification of various tRNAs in different cell types.
We are continuously developing biochemical tools required for efficient and convenient analyses of ncRNAs. Our immediate focus is to develop an efficient sequencing method for mature tRNAs.
PIWI-interacting RNA pathway in germline development
PIWI-interacting RNAs (piRNAs) are a germline-specific class of small regulatory RNAs that are 24–31 nucleotides (nt) in length and bind to PIWI proteins. PIWI proteins are a subset of the Argonaute family proteins and are expressed predominantly in the germline. In animal gonads, the PIWI/piRNA complexes silence transposons and other targets to maintain genome integrity.
PIWI proteins contain evolutionarily-conserved arginine methylations, symmetrical dimethylarginines (sDMAs) (Kirino et al., Nat Cell Biol. 2009). Because sDMAs are recognized by the TUDOR domain of proteins, the presence of PIWI sDMAs raises the possibility of an interacting network of PIWI proteins and TUDOR domain-containing proteins (Kirino et al., RNA 2010). Our recent studies utilized a Bombyx mori ovary-derived BmN4 cell system to comprehensively investigate the function of PIWI sDMAs. RNAi screening for Bombyx genes encoding TUDOR domain proteins identified BmPAPI as a novel piRNA biogenesis factor modulating 3′-end maturation of piRNAs on mitochondrial outer membrane (Honda et al., RNA 2013). The “Trimmer” enzyme responsible for piRNA 3′-end maturation has just been identified by analyzing BmPAPI-interacting proteins (Izumi et al., Cell 2016).