Satake Yuzuru, Kuwano Yuki, Nishikawa Tatsuya, Fujita Kinuyo, Saijo Saki, Itai Miki, Tanaka Hiroki, Nishida Kensei, Rokutan Kazuhito
Department of Pathophysiology, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima 770-8503, Japan.
Oncotarget. 2018 Jun 1;9(42):26817-26833. doi: 10.18632/oncotarget.25510.
Transcribed-ultraconserved regions (T-UCRs), which contain conserved sequences with 100% identity across human, rat and mouse species, are a novel category of functional RNAs. The human gene () encodes a UCR that spans exon 2 (276 bp) and its neighboring introns. Among five spliced RNA variants () transcribed from the gene, only contains the conserved exon 2. is overexpressed in colon cancer cells and accelerates cell growth by blocking the transcription of . However, the mechanisms underlying the overexpression of in colon cancer cells are unknown. Using biotinylated RNA pull-down assays followed by liquid chromatography-mass spectrometric analysis, we identified nucleolin as a -binding protein. Knockdown of nucleolin reduced the nuclear retention of and accelerated its degradation in the cytoplasm, whereas nucleolin overexpression increased levels and its mitogenic activity. Nucleolin directly bound to exon 2 via the glycine/arginine-rich (GAR) domain. Overexpression of GAR-deficient nucleolin failed to increase expression and growth of colon cancer cells. RNA fluorescence hybridization showed that co-localized with nucleolin in nuclei but not with the mutant lacking GAR. Our results suggest that specific interactions between nucleolin and UCR-containing may be associated with abnormal growth of colon cancer cells.
转录超保守区域（T-UCRs）是一类新型的功能性RNA，其包含在人类、大鼠和小鼠物种中具有100%同一性的保守序列。人类基因（）编码一种跨越外显子2（276 bp）及其相邻内含子的UCR。在从该基因转录的五种剪接RNA变体（）中，只有包含保守的外显子2。在结肠癌细胞中过表达，并通过阻断的转录来促进细胞生长。然而，结肠癌细胞中过表达的潜在机制尚不清楚。通过生物素化RNA下拉实验，随后进行液相色谱-质谱分析，我们鉴定出核仁素是一种结合蛋白。敲低核仁素可减少在细胞核中的滞留，并加速其在细胞质中的降解，而核仁素过表达则增加水平及其促有丝分裂活性。核仁素通过富含甘氨酸/精氨酸（GAR）的结构域直接与外显子2结合。缺乏GAR的核仁素过表达未能增加结肠癌细胞的表达和生长。RNA荧光杂交显示，与核仁素在细胞核中共定位，但与缺乏GAR的突变体不共定位。我们的结果表明，核仁素与含UCR的之间的特定相互作用可能与结肠癌细胞的异常生长有关。
