Revised: February 15, 2014
Accepted: June 18, 2014
Published online: August 6, 2014
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 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 other topological features are responsible for nucleolar targeting. Partial knockdown (by 50%-70%) of mouse Tbl3 by shRNA had no discernable effects on the processing 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 finding that there was no detectable rRNA processing defects in zebra fish embryos with homozygous deletions of zebra fish 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 severely impaired their proliferation. The inhibitory effect of Tbl3 knockdown was also observed in fibroblasts, resulting in an 80% reduction in colony formation. Taken together, these results indicate that Tbl3 is a newly recognized nucleolar protein with regulatory roles at very early stages of ribosome biogenesis, perhaps at the level of rRNA gene transcription.
CONCLUSION: Tbl3 is a newly recognized nucleolar protein with important regulatory roles in ribosome biogenesis.
Core tip: The mouse gene transducin β-like 3 (Tbl3) encodes a protein with thirteen WD40 protein-protein interaction motifs and is the mammalian homologue of yeast utp13. Virtually nothing is known about the function of tbl3. In this report, we provide the first direct evidence that Tbl3 is targeted to the nucleoli and plays an important role in regulating the synthesis of the 47S pre-ribosomal RNA, i.e., at very early stages of ribosome biogenesis. This activity has never been described before and sets Tbl3 apart from all other known nucleolar proteins. TBL3 may provide an attractive target for anti-neoplastic therapy.