5' half of specific tRNAs feeds back to promote corresponding tRNA gene transcription in vertebrate embryos [tRNA-seq]

5'tRFls are small tRNA fragments derived from 5' half of mature tRNAs. However, it is unknown whether 5'tRFls could feed back to regulate tRNA biogenesis. Here we show that 5'tRFlGly/GCC and 5'tRFlGlu/CTC function to promote transcription of corresponding tRNA genes and are essential for vertebrate early embryogenesis. During zebrafish embryogenesis, dynamics of 5'tRFlGly/GCC and 5'tRFlGlu/CTC levels correlates with that of tRNAGly/GCC and tRNAGlu/CTC levels. Morpholino-mediated knockdown of 5'tRFlGly/GCC or 5'tRFlGlu/CTC down-regulates tRNAGly/GCC or tRNAGlu/CTC levels respectively, and causes embryonic lethality that is efficiently rescued by co-injection of properly refolded corresponding tRNA. In zebrafish embryos, tRNA:DNA and 5'tRFl:DNA hybrids commonly exist on the template strand of tRNA genes. Mechanistically, unstable 5'tRFl:DNA hybrid may prevent the formation of transcriptionally inhibitory stable tRNA:DNA hybrids on the same tRNA loci so as to facilitate tRNA genes transcription. The uncovered mechanism may be implicated in other physiological and pathological processes. Overall design: tRNAs from wildtype zebrafish eggs and early embryos were detected by high throughput sequencing. Total RNAs from mature egg, 1cell, 256c sphere, shield and 24hpf stage embryos were extracted by Trizol reagent. Then, 60-120 nt length RNA was enriched by separation and elution from denature gel. RNA was treated with a combination of ALKB protein to remove some modifications before library preparation.

5' tRNA衍生片段（5'tRFls）是源自成熟转运RNA（transfer RNA, tRNA）5'端半段的小型tRNA片段。然而目前尚不明确5'tRFls是否可通过反馈调控tRNA的生物发生过程。本研究发现，5' tRNA衍生片段Gly/GCC（5'tRFlGly/GCC）与5' tRNA衍生片段Glu/CTC（5'tRFlGlu/CTC）可促进对应tRNA基因的转录，且对脊椎动物早期胚胎发生至关重要。在斑马鱼胚胎发生过程中，5'tRFlGly/GCC与5'tRFlGlu/CTC的水平动态变化与tRNAGly/GCC及tRNAGlu/CTC的水平动态变化高度相关。通过吗啉寡聚核苷酸（Morpholino）介导敲低5'tRFlGly/GCC或5'tRFlGlu/CTC，会分别下调tRNAGly/GCC或tRNAGlu/CTC的表达水平，并引发胚胎致死表型，该表型可通过共注射适当复性的对应tRNA得到有效挽救。在斑马鱼胚胎中，tRNA:DNA与5'tRFl:DNA杂交复合物普遍存在于tRNA基因的模板链上。从机制上来说，不稳定的5'tRFl:DNA杂交复合物可阻止同一tRNA基因座上形成具有转录抑制作用的稳定tRNA:DNA杂交复合物，从而促进tRNA基因的转录。本研究揭示的机制可能也参与其他生理及病理过程。实验整体设计：通过高通量测序检测野生型斑马鱼卵子及早期胚胎中的tRNA。采用Trizol试剂（Trizol reagent）提取成熟卵子、1细胞期、256细胞囊胚期、盾期以及24小时受精后（24hpf）胚胎的总RNA。随后通过变性凝胶分离并洗脱，富集60~120 nt长度的RNA片段。在构建测序文库前，采用ALKB蛋白（ALKB protein）组合处理RNA以去除部分修饰。