Evidence providing new insights into TOB-promoted deadenylation and supporting a link between TOB's deadenylation-enhancing and antiproliferative activities

Abstract

The mammalian TOB1 and TOB2 proteins have emerged as key players in repressing cell proliferation. Accumulating evidence indicates that TOBs regulate mRNA deadenylation. A recruitment model was proposed in which TOBs promote deadenylation by recruiting CAF1-CCR4 deadenylase complex to the 3' end of mRNAs by simultaneously binding CAF1 and PABP. However, the exact molecular mechanism underlying TOB-promoted deadenylation remains unclear. It is also unclear whether TOBs' antiproliferative and deadenylation-promoting activities are connected. Here, we combine biochemical analyses with a functional assay directly monitoring deadenylation and mRNA decay to characterize the effects of tethering TOBs or their mutant derivatives to mRNAs. The results provide direct evidence supporting the recruitment model and reveal a link between TOBs' antiproliferative and deadenylation-promoting activities. We also find that TOBs' actions in deadenylation are independent of the phosphorylation state of three serines known to regulate antiproliferative actions, suggesting that TOBs arrest cell growth through at least two different mechanisms. TOB1 and TOB2 were interchangeable in the properties tested here, indicating considerable functional redundancy between the two proteins. We propose that their multiple modes of modulating mRNA turnover and arresting cell growth permit the TOB proteins to coordinate their diverse roles in controlling cell growth and differentiation.

Fig 1

Tethering TOB proteins to mRNAs promotes deadenylation and decay of the transcripts. NIH 3T3 B2A2 cells were cotransfected with Tet promoter-regulated plasmids encoding the BBB+12bs and the BBB mRNAs, along with plasmid coding for either MS2 or one of the MS2-TOB proteins as indicated. (A) Diagram showing structures of BBB and BBB+12bs mRNAs. TOB proteins with C terminus fused to RNA-binding domain derived from bacteriophage MS2 coat protein bind to the MS2-specific elements (bs [binding site]) inserted in the BBB+12bs mRNA. (B) Western blot analysis of ectopically expressed MS2, MS2-TOB1, and MS2-TOB2 proteins, with GAPDH as loading control. (C) Northern blot analyses showing that tethering MS2-TOBs (middle and right) but not MS2 alone (left) to the otherwise stable BBB+12bs mRNA triggers highly processive deadenylation and rapid decay. BBB mRNA lacking the MS2-binding sites served as control. Samples of mRNA were taken at the indicated times after tetracycline addition to terminate a short pulse of mRNA synthesis. Poly(A)⁻ RNA (A−) was prepared in vitro by treating an RNA sample from an early time point with oligo(dT) and RNase H. mRNA half-life (t1/2) determination is described in Materials and Methods. (D) Deadenylation profiles illustrating how poly(A) shortening kinetics change during BBB+12bs mRNA turnover upon tethering of TOBs.

Fig 2

The ability of TOBs to enhance deadenylation and decay of their targets is independent of PABP binding when TOBs are directly tethered to the mRNAs. (A) Co-IP–Western blot analysis showing interactions of MS2-TOB1 and MS2-TOB2 and their FF mutant derivatives with PABP and CAF1 deadenylase. Cells were transfected with plasmids encoding V5-tagged MS2-TOBs, MS2-TOB(FF) mutants, or MS2 alone. RNase A-treated total lysates were incubated with anti-V5 antibody conjugated to agarose beads, and the immunoprecipitates (I) were analyzed by SDS-PAGE and Western blotting. An aliquot (8%) of each lysate (L) was analyzed for comparison. GAPDH served as the loading and nonspecific control. (B) Western blot analysis of MS2 and MS2-TOB(FF) mutant protein levels in cells ectopically expressing BBB+12bs and BBB mRNAs. GAPDH served as loading control. (C) Northern blot analyses showing that tethering of MS2-TOB(FF) mutants (middle and right) but not of MS2 alone (left) to the otherwise stable BBB+12bs mRNA triggers highly processive deadenylation and rapid decay of the transcript. Cell transfection and poly(A)⁻ mRNA (A−) preparation were as described in the Fig. 1 legend. mRNA samples were taken at the indicated times after tetracycline addition.

Fig 3

CAF1 is required for TOB-promoted deadenylation and decay. (A) Northern blot analyses of the effects of Caf1 knockdown (top) or control knockdown (bottom) on deadenylation and decay of BBB+12bs mRNA promoted by tethering MS2-TOBs to the transcript. NIH 3T3 B2A2 cells were cotransfected with Tet promoter-regulated plasmids encoding the BBB+12bs and the BBB control mRNAs and either the MS2 plasmid or one of the MS2-TOB plasmids as indicated, as well as either the control siRNA (Ctrl siRNA, bottom) or CAF1 siRNA (top). Samples were taken at the indicated times after tetracycline addition. The preparation of poly(A)⁻ RNA (A−) was as described in the Fig. 1 legend. (B) Western blot analysis showing expression levels of MS2, MS2-TOB1, and MS2-TOB2 proteins (top), CAF1 protein levels (middle), and GAPDH loading control (bottom) in cells transfected with either the control siRNA (Ctrl siRNA, left) or CAF1 siRNA (right).

Fig 4

Interaction with CAF1 is necessary for TOBs to elicit rapid deadenylation and decay. (A) Co-IP–Western blot analysis of interactions between CAF1 deadenylase and MS2-TOB1, MS2-TOB2, and their mutant derivatives. Cells were transfected with plasmids encoding V5-tagged MS2, MS2-TOB1, MS2-TOB2, or their TM or 3LG mutants. RNase A-treated total lysates were incubated with anti-V5 antibody conjugated to agarose beads, and the immunoprecipitates (I) were analyzed by SDS-PAGE–Western blotting. An aliquot (8%) of each lysate (L) was analyzed for comparison, and GAPDH served as loading and nonspecific control. (B) Northern blot analyses of the effects of tethering MS2-TOBs, MS2-TOB mutants, or MS2 alone (control) on deadenylation and decay of BBB+12bs mRNA. Cell transfection and poly(A)⁻ mRNA (A−) preparation were as described in the Fig. 1 legend. Samples were taken at the indicated times after tetracycline addition. (C) Western blot analysis of MS2-TOB(TM) or (3LG) mutant proteins in cells ectopically expressing BBB+12bs mRNA and BBB mRNA. GAPDH served as loading control. Wt, wild type.

Fig 5

Effects of tethering TOB proteins to mRNAs on protein production from target transcripts. (A) Top left, levels of ectopically expressed TOBs, MS2, MS2-TOB1, and MS2-TOB2; middle left, levels of GFP protein; bottom left, levels of α-tubulin (loading control); right, bar graph showing changes in the relative levels of GFP+12bs mRNA upon tethering the corresponding TOB proteins and their mutant derivatives. mRNA levels were quantified by real-time RT-PCR and are presented as the normalized percentage ± standard deviation (n = 3). (B) Top, levels of ectopically expressed MS2-TOBs detected by anti-V5 antibody, with GAPDH as loading control; bottom, levels of GFP detected by anti-Flag antibody, with α-tubulin as loading control.

Fig 6

The ability of TOBs to promote deadenylation and decay is not affected by the phosphorylation status of three conserved serines. (A and B) Co-IP–Western blot analysis of interactions of MS2-TOB1 and MS2-TOB2 and their mutant derivatives with PABP and CAF1 deadenylase. Cells were transfected with plasmids encoding V5-tagged MS2, MS2-TOB1, MS-TOB2, the 3SA mutants, or the 3SE mutants. RNase A-treated total lysates were incubated with anti-V5 antibody conjugated to agarose beads, and the immunoprecipitates (I) were analyzed by SDS-PAGE and Western blotting; the flowthrough (F) fractions were also examined. An aliquot (8%) of each lysate (L) was analyzed. GAPDH served as loading and nonspecific control. (C) Northern blot analyses of deadenylation and decay of reporter transcript (BBB+12bs mRNA) in cells expressing MS2, MS2-TOB1, MS2-TOB2, or the 3SA and 3SE mutants of the MS2-TOBs. Cell transfection and poly(A)⁻ mRNA (A−) preparation were as described in the Fig. 1 legend. Samples were taken at the indicated times after tetracycline addition. (D) Western blot analysis of protein levels for MS2-TOBs and their mutant derivatives, with GAPDH served as loading control.

Fig 7

Growth arrest actions of TOB1, TOB2, and their FF, 3SA, 3SE, and 3LG mutants. Top, cell growth in U2OS cells transfected with the indicated wild-type (Wt) or mutant TOB plasmids. Data represent the average ± standard deviation (n = 3) for cell numbers at the end of the test period, normalized to the results for the controls. The asterisks denote a significant difference analyzed by a paired t test. Bottom, Western blot analysis of protein levels for ectopically expressed TOB1, TOB2, or their mutant derivatives from one of the cell growth experiments whose results are shown in the top panel. GAPDH served as loading control.

Fig 8

Proposed modes of TOB-mediated effects on mRNA decay and growth arrest.