Nucleolin facilitates nuclear retention of an ultraconserved region containing TRA2β4 and accelerates colon cancer cell growth

// Yuzuru Satake 1 , Yuki Kuwano 1 , Tatsuya Nishikawa 1 , Kinuyo Fujita 1 , Saki Saijo 1 , Miki Itai 1 , Hiroki Tanaka 1 , Kensei Nishida 1 and Kazuhito Rokutan 1 1 Department of Pathophysiology, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima 770–8503, Japan Correspondence to: Yuki Kuwano, email: kuwanoy@tokushima-u.ac.jp Keywords: nucleolin; transcribed-UCR; TRA2β4; colon cancer; cell growth Abbreviations: TRA2β: Transformer 2-beta; UCR: ultraconserved region; T-UCR: Transcribed-ultraconserved region; lncRNA: long noncoding RNA; qPCR: quantitative real-time polymerase chain reaction Received: November 02, 2017      Accepted: May 12, 2018      Published: June 01, 2018 ABSTRACT 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 transformer 2β gene ( TRA2B ) encodes a UCR that spans exon 2 (276 bp) and its neighboring introns. Among five spliced RNA variants ( TRA2β1-5 ) transcribed from the TRA2B gene, only TRA2β4 contains the conserved exon 2. TRA2β4 is overexpressed in colon cancer cells and accelerates cell growth by blocking the transcription of CDKN1A . However, the mechanisms underlying the overexpression of TRA2β4 in colon cancer cells are unknown. Using biotinylated RNA pull-down assays followed by liquid chromatography-mass spectrometric analysis, we identified nucleolin as a TRA2β4 -binding protein. Knockdown of nucleolin reduced the nuclear retention of TRA2β4 and accelerated its degradation in the cytoplasm, whereas nucleolin overexpression increased TRA2β4 levels and its mitogenic activity. Nucleolin directly bound to TRA2β4 exon 2 via the glycine/arginine-rich (GAR) domain. Overexpression of GAR-deficient nucleolin failed to increase TRA2β4 expression and growth of colon cancer cells. RNA fluorescence in situ hybridization showed that TRA2β4 co-localized with nucleolin in nuclei but not with the mutant lacking GAR. Our results suggest that specific interactions between nucleolin and UCR-containing TRA2β4 may be associated with abnormal growth of colon cancer cells.