Dumbbell-PCR: a method to quantify specific small RNA variants with a single nucleotide resolution at terminal sequences.

Recent advances in next-generation sequencing technologies have revealed that cellular functional RNAs 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. Unraveling the biogenesis and biological significance of heterogenetic RNA expression requires distinctive analysis of each RNA variant. Here, we report the development of dumbbell PCR (Db-PCR), an efficient and convenient method to distinctively quantify a specific individual small RNA variant. In Db-PCR, 5\u27- and 3\u27-stem-loop adapters are specifically hybridized and ligated to the 5\u27- and 3\u27-ends of target RNAs, respectively, by T4 RNA ligase 2 (Rnl2). The resultant ligation products with \u27dumbbell-like\u27 structures are subsequently quantified by TaqMan RT-PCR. We confirmed that high specificity of Rnl2 ligation and TaqMan RT-PCR toward target RNAs assured both 5\u27- and 3\u27-terminal sequences of target RNAs with single nucleotide resolution so that Db-PCR specifically detected target RNAs but not their corresponding terminal variants. Db-PCR had broad applicability for the quantification of various small RNAs in different cell types, and the results were consistent with those from other quantification method. Therefore, Db-PCR provides a much-needed simple method for analyzing RNA terminal heterogeneity

YAMAT-seq: an efficient method for high-throughput sequencing of mature transfer RNAs.

Besides translation, transfer RNAs (tRNAs) play many non-canonical roles in various biological pathways and exhibit highly variable expression profiles. To unravel the emerging complexities of tRNA biology and molecular mechanisms underlying them, an efficient tRNA sequencing method is required. However, the rigid structure of tRNA has been presenting a challenge to the development of such methods. We report the development of Y-shaped Adapter-ligated MAture TRNA sequencing (YAMAT-seq), an efficient and convenient method for high-throughput sequencing of mature tRNAs. YAMAT-seq circumvents the issue of inefficient adapter ligation, a characteristic of conventional RNA sequencing methods for mature tRNAs, by employing the efficient and specific ligation of Y-shaped adapter to mature tRNAs using T4 RNA Ligase 2. Subsequent cDNA amplification and next-generation sequencing successfully yield numerous mature tRNA sequences. YAMAT-seq has high specificity for mature tRNAs and high sensitivity to detect most isoacceptors from minute amount of total RNA. Moreover, YAMAT-seq shows quantitative capability to estimate expression levels of mature tRNAs, and has high reproducibility and broad applicability for various cell lines. YAMAT-seq thus provides high-throughput technique for identifying tRNA profiles and their regulations in various transcriptomes, which could play important regulatory roles in translation and other biological processes

The biogenesis pathway of tRNA-derived piRNAs in Bombyx germ cells.

Transfer RNAs (tRNAs) function in translational machinery and further serves as a source of short non-coding RNAs (ncRNAs). tRNA-derived ncRNAs show differential expression profiles and play roles in many biological processes beyond translation. Molecular mechanisms that shape and regulate their expression profiles are largely unknown. Here, we report the mechanism of biogenesis for tRNA-derived Piwi-interacting RNAs (td-piRNAs) expressed in Bombyx BmN4 cells. In the cells, two cytoplasmic tRNA species, tRNAAspGUC and tRNAHisGUG, served as major sources for td-piRNAs, which were derived from the 5\u27-part of the respective tRNAs. cP-RNA-seq identified the two tRNAs as major substrates for the 5\u27-tRNA halves as well, suggesting a previously uncharacterized link between 5\u27-tRNA halves and td-piRNAs. An increase in levels of the 5\u27-tRNA halves, induced by BmNSun2 knockdown, enhanced the td-piRNA expression levels without quantitative change in mature tRNAs, indicating that 5\u27-tRNA halves, not mature tRNAs, are the direct precursors for td-piRNAs. For the generation of tRNAHisGUG-derived piRNAs, BmThg1l-mediated nucleotide addition to -1 position of tRNAHisGUG was required, revealing an important function of BmThg1l in piRNA biogenesis. Our study advances the understanding of biogenesis mechanisms and the genesis of specific expression profiles for tRNA-derived ncRNAs

Increasing cell density globally enhances the biogenesis of Piwi-interacting RNAs in Bombyx mori germ cells.

Piwi proteins and their bound Piwi-interacting RNAs (piRNAs) are predominantly expressed in the germline and play crucial roles in germline development by silencing transposons and other targets. Bombyx mori BmN4 cells are culturable germ cells that equip the piRNA pathway. Because of the scarcity of piRNA-expressing culturable cells, BmN4 cells are being utilized for the analyses of piRNA biogenesis. We here report that the piRNA biogenesis in BmN4 cells is regulated by cell density. As cell density increased, the abundance of Piwi proteins and piRNA biogenesis factors was commonly upregulated, resulting in an increased number of perinuclear nuage-like granules where Piwi proteins localize. Along with these phenomena, the abundance of mature piRNAs also globally increased, whereas levels of long piRNA precursor and transposons decreased, suggesting that increasing cell density promotes piRNA biogenesis pathway and that the resultant accumulation of mature piRNAs is functionally significant for transposon silencing. Our study reveals a previously uncharacterized link between cell density and piRNA biogenesis, designates cell density as a critical variable in piRNA studies using BmN4 cell system, and suggests the alteration of cell density as a useful tool to monitor piRNA biogenesis and function

肺動脈性肺高血圧症におけるプロスタサイクリン受容体作動薬セレキシパグの作用

13301甲第5558号博士（創薬科学）金沢大学博士論文本文Full 以下に掲載：PLOS ONE 15(10) 2020. PLOS. 共著者：本田 洋平, 古杉 圭司, 渕上 千晶, 倉本 和也, 沼倉 佑樹, 桑野 敬

In vitro production of L-cysteine using thermophilic enzymes

L-Cysteine (L-Cys) is a commercially important amino acid and widely used in food, pharmaceutical, and cosmetic industries. Commercial production of L-Cys has long been done by an acid-hydrolysis of human hair and animal feather, leading to the generation of a large quantity of hazardous wastes. Although several biotechnology companies have recently launched a fermentative production of L-Cys using engineered bacteria, these processes suffer from the low product titer mainly due to the cytotoxic effect of L-Cys. To provide an alternative approach for the commercial production of L-Cys, we aimed at the development of a non-fermentative, in vitro manufacturing system using thermophilic enzymes. In this system, enzymes from (hyper)thermophilic bacteria and archaea were assembled to construct an in vitro synthetic pathway for the one-pot conversion of glucose to L-Cys (Figure 1). By using experimentally optimized concentrations of enzymes, L-Cys could be produced at a rate of 0.9 g/L/h with a molar conversion yield of 25%. Please click Additional Files below to see the full abstract

Fogarty catheter for OLV of a neonate

Here, we report two cases involving a neonate and child in which a slip joint section was used to thread a Fogarty catheter into the endotracheal tube for one-lung ventilation (OLV). Both the neonate and infant required OLV, and were placed under general anesthesia. A Fogarty catheter was used for OLV. The Fogarty catheter was passed into the intraluminal side of the endotracheal tube through a slip joint section. OLV was maintained successfully without severe air leakage or Fogarty catheter displacement. The neonate had been intubated pre-operatively with a 3.5-mm inner diameter endotracheal tube, and we used that tube. These cases indicate that the technique can be applied to pre-operatively intubated patients and does not require surgeons to exchange endotracheal tubes. Use of the slip joint section technique facilitates Fogarty catheter fixation without additional dead space

Identifying Potential Problems and Risks in GQM+Strategies Models Using Metamodel and Design Principles

Although GQM+Strategies®1 assures that business goals and strategies are aligned throughout an organization and at each organizational unit based on the rationales to achieve the overall business goals, whether the GQM+Strategies grid is created correctly cannot be determined because the current definition of GQM+Strategies allows multiple perspectives when aligning goals with strategies. Here we define modeling rules for GQM+Strategies with a metamodel specified with a UML class diagram. Additionally, we create design principles that consist of relationship constraints between GQM+Strategies elements, which configure GQM+Strategies grids. We demonstrate that the GQM+Strategies grids can be automatically determined with the help of design principles described in OCL. In fact, an experiment is implemented using these approaches in order to show that this method helps identify and improve potential problems and risks. The results confirm that our approaches help create a consistent GQM+Strategies grid

Sex hormone-dependent tRNA halves enhance cell proliferation in breast and prostate cancers.

Sex hormones and their receptors play critical roles in the development and progression of the breast and prostate cancers. Here we report that a novel type of transfer RNA (tRNA)-derived small RNA, 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 cell lines. SHOT-RNAs are not abundantly present in ER(-) breast cancer, AR(-) prostate cancer, or other examined cancer cell lines from other tissues. ER-dependent accumulation of SHOT-RNAs is not limited to a cell culture system, but it also occurs in luminal-type breast cancer patient tissues. SHOT-RNAs are produced from aminoacylated mature tRNAs by angiogenin-mediated anticodon cleavage, which is promoted by sex hormones and their receptors. Resultant 5\u27- and 3\u27-SHOT-RNAs, corresponding to 5\u27- and 3\u27-tRNA halves, bear a cyclic phosphate (cP) and an amino acid at the 3\u27-end, respectively. By devising a cP-RNA-seq method that is able to exclusively amplify and sequence cP-containing RNAs, we identified the complete repertoire of 5\u27-SHOT-RNAs. Furthermore, 5\u27-SHOT-RNA, but not 3\u27-SHOT-RNA, has significant functional involvement in cell proliferation. These results 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

Genome-wide identification of short 2\u27,3\u27-cyclic phosphate-containing RNAs and their regulation in aging.

RNA molecules generated by ribonuclease cleavage sometimes harbor a 2\u27,3\u27-cyclic phosphate (cP) at their 3\u27-ends. Those cP-containing RNAs (cP-RNAs) form a hidden layer of transcriptome because standard RNA-seq cannot capture them as a result of cP\u27s prevention of an adapter ligation reaction. Here we provide genome-wide analyses of short cP-RNA transcriptome across multiple mouse tissues. Using cP-RNA-seq that can exclusively sequence cP-RNAs, we identified numerous novel cP-RNA species which are mainly derived from cytoplasmic tRNAs, mRNAs, and rRNAs. Determination of the processing sites of substrate RNAs for cP-RNA generation revealed highly-specific RNA cleavage events between cytidine and adenosine in cP-RNA biogenesis. cP-RNAs were not evenly derived from the overall region of substrate RNAs but rather from specific sites, implying that cP-RNAs are not from random degradation but are produced through a regulated biogenesis pathway. The identified cP-RNAs were abundantly accumulated in mouse tissues, and the expression levels of cP-RNAs showed age-dependent reduction. These analyses of cP-RNA transcriptome unravel a novel, abundant class of non-coding RNAs whose expression could have physiological roles