Division of Genomics and RNomics, Biocenter, Medical University of Innsbruck, 6020, Innsbruck, Austria.
Helmholtz Institute for Metabolic, Obesity and Vascular Research (HI-MAG) of the Helmholtz Zentrum München at the University of Leipzig and University Hospital Leipzig, Philipp-Rosenthal-Str. 27, 04103, Leipzig, Germany.
Genome Biol. 2020 Dec 8;21(1):299. doi: 10.1186/s13059-020-02199-6.
The presence of nuclear mitochondrial DNA (numtDNA) has been reported within several nuclear genomes. Next to mitochondrial protein-coding genes, numtDNA sequences also encode for mitochondrial tRNA genes. However, the biological roles of numtDNA remain elusive.
Employing in silico analysis, we identify 281 mitochondrial tRNA homologs in the human genome, which we term nimtRNAs (nuclear intronic mitochondrial-derived tRNAs), being contained within introns of 76 nuclear host genes. Despite base changes in nimtRNAs when compared to their mtRNA homologs, a canonical tRNA cloverleaf structure is maintained. To address potential functions of intronic nimtRNAs, we insert them into introns of constitutive and alternative splicing reporters and demonstrate that nimtRNAs promote pre-mRNA splicing, dependent on the number and positioning of nimtRNA genes and splice site recognition efficiency. A mutational analysis reveals that the nimtRNA cloverleaf structure is required for the observed splicing increase. Utilizing a CRISPR/Cas9 approach, we show that a partial deletion of a single endogenous nimtRNA within intron 28 of the PPFIBP1 gene decreases inclusion of the downstream-located exon 29 of the PPFIBP1 mRNA. By employing a pull-down approach followed by mass spectrometry, a 3'-splice site-associated protein network is identified, including KHDRBS1, which we show directly interacts with nimtRNA by an electrophoretic mobility shift assay.
We propose that nimtRNAs, along with associated protein factors, can act as a novel class of intronic splicing regulatory elements in the human genome by participating in the regulation of splicing.
核线粒体 DNA（numtDNA）已在几个核基因组中被报道存在。除了线粒体蛋白编码基因外，numtDNA 序列还编码线粒体 tRNA 基因。然而，numtDNA 的生物学功能仍然难以捉摸。
通过计算机分析，我们在人类基因组中鉴定出 281 个线粒体 tRNA 同源物，我们将其称为 nimtRNAs（核内含子线粒体衍生的 tRNA），它们位于 76 个核宿主基因的内含子中。尽管 nimtRNAs 与它们的 mtRNA 同源物相比存在碱基变化，但仍保持着典型的 tRNA 三叶形结构。为了研究内含子 nimtRNAs 的潜在功能，我们将其插入组成型和选择性剪接报告基因的内含子中，并证明 nimtRNAs 依赖于 nimtRNA 基因的数量和位置以及剪接位点识别效率来促进前体 mRNA 的剪接。突变分析表明，nimtRNA 的三叶形结构是观察到的剪接增加所必需的。利用 CRISPR/Cas9 方法，我们表明在 PPFIBP1 基因的内含子 28 中单个内源性 nimtRNA 的部分缺失会降低下游 PPFIBP1 mRNA 中exon29 的包含。通过采用下拉法结合质谱法，鉴定出一个 3'-剪接位点相关蛋白网络，包括 KHDRBS1，我们通过电泳迁移率变动分析表明，它可以直接与 nimtRNA 相互作用。
我们提出，nimtRNAs 及其相关蛋白因子可以通过参与剪接调节，成为人类基因组中一类新的内含子剪接调节元件。
