ORIGINAL ARTICLE Tbl3 encodes a WD40 nucleolar protein with regulatory roles in ribosome biogenesis Jindong Wang, Schickwann Tsai Jindong Wang, Schickwann Tsai, Department of Medicine, University of Utah School of Medicine, Salt Lake City, UT 84132, United States Author contributions: Wang J designed and constructed vari- ous expression vectors, performed localization and ribosome profling, analyzed the data, prepared the fgures and co-wrote the manuscript; Tsai S designed the project, performed cell culture experiments, analyzed the data, prepared the fgures and co-wrote the manuscript. Supported by In part by a grant from the St. Perres Fund, No. 11-02011 Correspondence to: Schickwann Tsai, MD, PhD, Depart- ment of Medicine, University of Utah School of Medicine, 5C402, 30 North 1900 East, Salt Lake City, UT 84132, United States. schickwann.tsai@hsc.utah.edu Telephone: +1-801-5850495 Fax: +1-801-5850496 Received: October 27, 2013 Revised: February 15, 2014 Accepted: June 18, 2014 Published online: August 6, 2014 Abstract AIM: To investigate the subcellular localization and the function of mouse transducin β-like 3 (Tbl3). METHODS: The coding sequence of mouse Tbl3 was cloned from the cDNAs of a promyelocyte cell line by reverse transcription-polymerase chain reaction. Fusion constructs of Tbl3 and enhanced green fluorescent protein (EGFP) were transfected into fibroblasts and examined by fluorescence microscopy to reveal the subcellular localization of tbl3. To search for nucleolar targeting sequences, scanning deletions of Tbl3-EGFP were constructed and transfected into fibroblasts. To explore the possible function of Tbl3, small hairpin RNAs (shRNAs) were used to knock down endogenous Tbl3 in mouse promyelocytes and fbroblasts. The ef- fects of Tbl3 knockdown on ribosomal RNA (rRNAs) synthesis or processing were studied by labeling cells with 5,6- 3 H-uridine followed by a chase with fresh medium for various periods. Total RNAs were purifed from treated cells and subjected to gel electrophoresis and Northern analysis. Ribosome profling by sucrose gradient centrifugation was used to compare the amounts of 40S and 60S ribosome subunits as well as the 80S monosome. The impact of Tbl3 knockdown on cell growth and proliferation was examined by growth curves and colony assays. RESULTS: The largest open reading frame of mouse Tbl3 encodes a protein of 801 amino acids (AA) with an apparent molecular weight of 89-90 kilodalton. It con- tains thirteen WD40 repeats (an ancient protein-protein interaction motif) and a carboxyl terminus that is highly homologous to the corresponding region of the yeast nucleolar protein, utp13. Virtually nothing is known about the biological function of Tbl3. All cell lines surveyed expressed Tbl3 and the level of expression correlated roughly with cell proliferation and/or biosyn- thetic activity. Using Tbl3-EGFP fusion constructs we obtained the frst direct evidence that Tbl3 is targeted to the nucleoli in mammalian cells. However, no previ- ously described nucleolar targeting sequences were found in Tbl3, suggesting that the WD40 motif and/or other topological features are responsible for nucleolar targeting. Partial knockdown (by 50%-70%) of mouse Tbl3 by shRNA had no discernable effects on the pro- cessing of the 47S pre-ribosomal RNA (pre-rRNA) or the steady-state levels of the mature 28S, 18S and 5.8S rRNAs but consistently increased the expression level of the 47S pre-rRNA by two to four folds. The results of the current study corroborated the previous fnding that there was no detectable rRNA processing defects in zebra fish embryos with homozygous deletions of zebra fsh Tbl3. As ribosome production consumes the bulk of cellular energy and biosynthetic precursors, dysregulation of pre-rRNA synthesis can have negative effects on cell growth, proliferation and differentiation. Indeed, partial knockdown of Tbl3 in promyelocytes se- verely impaired their proliferation. The inhibitory effect of Tbl3 knockdown was also observed in fbroblasts, re- sulting in an 80% reduction in colony formation. Taken World Journal of Hematology WJ H Online Submissions: http://www.wjgnet.com/esps/ Help Desk: http://www.wjgnet.com/esps/helpdesk.aspx doi:10.5315/wjh.v3.i3.93 World J Hematol 2014 August 6; 3(3): 93-104 ISSN 2218-6204 (online) © 2014 Baishideng Publishing Group Inc. All rights reserved. 93 August 6, 2014|Volume 3|Issue 3| WJH|www.wjgnet.com