Tbl3 encodes a WD40 nucleolar protein with regulatory roles in ribosome biogenesis

• 作者: Jindong Wang Schickwann Tsai
• 来源期刊: 世界血液学杂志
• 学科: 医学
• 关键词: NUCLEOLUS NUCLEOLAR protein RIBOSOME biogenesis RIBOSOMAL RNA Pre-ribosomal RNA
• 年，卷（期）: 2014,（3）
• 页码范围: 93-104
• 页数: 12页
• 分类号: R

摘要

AIM:To investigate the subcellular localization and the function of mouse transducinβ-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 fibroblasts.The effects of Tbl3 knockdown on ribosomal RNA(rRNAs)synthesis or processing were studied by labeling cells with 5,6-3H-uridine followed by a chase with fresh medium for various periods.Total RNAs were purified from treated cells and subjected to gel electrophoresis and Northern analysis.Ribosome profiling 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 contains 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 biosynthetic activity.Using Tbl3-EGFP fusion constructs we obtained the first direct evidence that Tbl3 is targeted to the nucleoli in mammalian cells.However,no previously described nucleolar targeting sequences were found in Tbl3,suggesting that the WD40 motif and/or