A Novel Drosha Isoform in Embryonic Stem Cells and Germ Line Cells (PDF)

A novel Drosha isoform specifically expressed in embryonic stem cells and germ line cells in mice ○Yuji Shimizu1, Ren Shimamoto1, Ayako Isotani1, and Katsutomo Okamura1 (1 Division of Biological Science, Nara Institute of Science and Technology) 清水 雄治 (RNA 分子医科学研究室) microRNAs (mi...

A novel Drosha isoform specifically expressed in embryonic stem cells and germ line cells in mice ○Yuji Shimizu1, Ren Shimamoto1, Ayako Isotani1, and Katsutomo Okamura1 (1 Division of Biological Science, Nara Institute of Science and Technology) 清水 雄治 (RNA 分子医科学研究室) microRNAs (miRNAs) are ~22 nt small non-coding RNAs that regulate target mRNAs, and strict regulation of miRNAs is important for normal development. The importance of cell-type specific isoforms has been reported for the core miRNA biogenesis factors including DGCR8, Dicer, and Ago2, contributing to accurate control of miRNA biogenesis during mammalian development. However, for Drosha, which initiates miRNA biogenesis, no study has reported a developmental role for a cell-type specific isoform. Through integrative analysis of publicly available transcriptome data, we identified a novel internal transcription start site (TSS) at the mouse Drosha locus, which results in the production of an isoform specifically expressed in embryonic stem cells (ESC) and germ cells (GC). The 5' UTR of the ESC/GC specific Drosha isoform is different from that of the annotated Drosha isoform, which may mediate differential regulation of its expression through upstream Open Reading Frames and actions of RNA binding proteins. In addition, translation of the ESC/GC specific Drosha isoform is expected to start at the second methionine of the annotated canonical isoform. Interestingly, while we found no evidence for internal transcription initiation of Drosha in human induced pluripotent stem cells (iPSCs), reanalysis of Ribo-seq data indicated a differential use of the first two ATG codons in HEK293T cells and iPSCs, suggesting that the preferred use of the second ATG in pluripotent cells is a conserved characteristic of the Drosha locus although the two organisms employ different mechanisms. The mouse ESC/GC specific Drosha isoform lacks the first 8-amino acids of the canonical full-length isoform, a region that contains a predicted phosphorylation site. We are currently trying to uncover functional differences and physiological roles of this Drosha isoform during differentiation using isoform-specific overexpression mouse cells