tRNA-overlapping long non-coding RNAs repress codon-biased genes in trans

The functions of most long non-coding RNAs (lncRNAs) are not known. Here, we define a group of ‘tRNA-Overlapping LncRNA’ (tROL) genes that are upregulated during in vitro cartilage development. Deletions of tROLs result in changes in the expression of codon-biased genes, where downregulated genes are enriched in codons corresponding to tRNAs overlapping disrupted tROLs. However, tROL lncRNA expression is controlled independently of the overlapping tRNA loci. Remarkably, tROL loci are located in gene-dense regions and interact extensively in trans between chromosomes, and tROL deletions result in the upregulation of significantly overlapping subsets of genes in the vicinity of tROL loci. Taken together, the results suggest that tROL loci coalesce and are dependent on each other’s transcription to repress surrounding genes in trans. Our investigation thus sheds light on a unique role for tROLs as a regulatory bridge between the non-coding and coding genomes. To investigate lncRNAs during in vitro cartilage development, we differentiated primary human mesenchymal stem cells (MSCs) into chondrocytes over 14 days. We then performed gene expression profiling analysis of data from RNA-seq of MSCs from two donors (female and male), differentiated in replicate, at 5 time points. To investigate the function of the tROL LINC00324 in cartilage development, we generated two isogenic deletions (KOs) in C-28/I2 chondrocytes. We then performed gene expression profiling analysis of data from RNA-seq of WT and LINC00324 KO clones. To investigate the function of additional tROLs and their interdependence, we generated two isogenic deletions (KOs) for the tROLs LINC00324, LINC01962, ENSG00000284607, and ENSG00000287264 in HEK-293 cells. We then performed gene expression profiling analysis of data from RNA-seq of WT and tROL KO clones.

绝大多数长链非编码RNA（long non-coding RNAs）的功能尚未明确。本研究定义了一类‘tRNA重叠长链非编码RNA（tRNA-Overlapping LncRNA，以下简称tROL）’基因，该类基因在体外软骨发育过程中呈上调表达。敲除tROL会导致密码子偏好性基因的表达发生改变，其中下调基因富集于与被敲除tROL重叠的tRNA所对应的密码子。但tROL lncRNA的表达调控独立于其重叠的tRNA基因座。值得注意的是，tROL基因座位于基因密集区域，并在染色体间广泛发生反式相互作用；且敲除tROL会导致tROL基因座附近存在显著重叠的基因子集出现上调表达。综上，本研究结果表明，tROL基因座会发生聚集，并依赖彼此的转录以反式方式抑制其周边基因的表达。因此，本研究揭示了tROL作为非编码基因组与编码基因组间调控桥梁的独特功能。为探究体外软骨发育过程中的lncRNAs，我们将原代人间充质干细胞（mesenchymal stem cells，以下简称MSCs）诱导分化为软骨细胞，诱导周期为14天；随后，我们对两名供体（女性与男性）的MSCs在5个时间点的重复分化样本进行RNA测序（RNA-seq），并开展基因表达谱分析。为探究tROL基因LINC00324在软骨发育中的功能，我们在C-28/I2软骨细胞中构建了两株同基因敲除（knockout，以下简称KO）细胞系；随后，我们对野生型（wild type，以下简称WT）与LINC00324 KO细胞克隆进行RNA测序，并开展基因表达谱分析。为探究其他tROL的功能及其相互依赖关系，我们在HEK-293细胞中针对tROL基因LINC00324、LINC01962、ENSG00000284607及ENSG00000287264构建了两株同基因敲除细胞系；随后，我们对野生型与tROL KO细胞克隆进行RNA测序，并开展基因表达谱分析。