Schematic representation of TTP, BRF-1, and BRF-2. The RNA-binding zinc-finger domain is shown in black (RBD-Zn). The N-terminal (NTD) and C-terminal (CTD) mRNA decay activation domains defined in this study are indicated. The percent amino acid identity to TTP, of the NTD, RNA-binding domain, and CTD of BRF-1 and BRF-2 is given. (aa) Amino acids.

The CTD of TTP and BRF-1 are mRNA decay activation domains, and overexpression of TTP NTD and CTD inhibits ARE-mediated mRNA decay. (A) Northern blots showing decay rates of β-6bs mRNA in the presence of tethered TTP and BRF-1 domains as indicated on the left. Calculated half-lives and average fold change are given on the right (four experiments). (B) Northern blots showing the decay rate of the ARE-mediated decay reporter mRNA, β-ARE, in the presence of coexpressed GFP, GFP-TTP_1-100, GFP-TTP_100-174, or GFP-TTP_176-326. Half-lives of the reporter mRNAs and average fold change are given on the right (three experiments).

The NTD of TTP and BRF-1 can activate mRNA decay when tethered via the MS2 coat protein. (A) Northern blots showing decay rates of a β-globin mRNA with six MS2 coat protein-binding sites in the 3′UTR (β-6bs), in the presence of exogenous MS2, TTP, MS2-TTP, and MS2-BRF-1 proteins indicated on the left. Calculated β-6bs mRNA half-lives and the average fold change from the assay with the MS2 protein alone are given on the right (four experiments). (B) Northern blots showing decay rates of β-6bs mRNA, in the presence of tethered NTDs of TTP and BRF-1 proteins indicated on the left. Calculated β-6bs mRNA half-lives and average fold change are given on the right (three and four experiments).

mRNA decay activation by the TTP family of ARE-binding proteins. (A) Three steps of mRNA decay. Activation of mRNA decay is poorly understood, indicated by a question mark. Regulation of mRNA decay is hypothesized to take place on the recognition or activation steps. mRNA decay enzymes are shown as “Pac-men.” (B) How TTP may activate ARE-mediated decay by recruitment of trans-acting factors, which includes mRNA decay enzymes via the NTD. The putative activation of 5′-to-3′ exonucleolytic decay is shown in brackets because it is based solely on the interaction between hXrn1 and TTP/BRF-1 shown in this study. All other indicated decay processes are based on both interaction and decay data. For details, see Discussion. The NTD is shown facing the poly-A tail based on the solution structure of the BRF-2 zinc-finger complexed with RNA (Hudson et al. 2004).

TTP and BRF-1 exist in complex with mRNA decay enzymes. (A) Western blots showing coimmunoprecipitation assays. Transiently expressed Flag-tagged TTP, BRF-1, and HuR were immunoprecipitated from HEK 293T cell extracts, and precipitates were probed for the presence of coexpressed Myc-tagged or endogenous mRNA decay proteins as indicated. (Lanes 1,3,5,7) Five percent total extract (T). (Lanes 4,6,8) Immunoprecipitate pellets (P). (Lane 2) Immunoprecipitate from cells that do not express a Flag-tagged protein. (B) Western blots showing endogenous coimmunoprecipitation assays. (Lane 3) Endogenous TTP was immunoprecipitated from marmoset T-cell extracts, and the precipitate was probed for the presence of endogenous mRNA decay proteins as indicated. (Lane 1) Five percent total cell extract (T). (Lane 2) Immunoprecipitation reaction using normal rabbit serum (NRS) in place of anti-TTP antiserum.

Overexpression of the decapping enzyme, hDcp2, activates ARE-mediated mRNA decay. (A) Northern blots showing decay rates in HeLa Tet-off cells of a β-globin reporter mRNA (β-ARE) that contains an AU-rich element from GM-CSF mRNA in the 3′UTR, in the absence of coexpressed protein (none) or in the presence of coexpressed decapping complex subunits, as indicated on the left. Time points above the panels refer to minutes after transcriptional repression. β-ARE levels were normalized to the internal control β-GAP mRNA, and β-ARE half-lives were calculated and are shown on the right. The average fold of change in half-lives (three experiments) that were significantly different from the assay with no exogenous protein is given with standard deviation on the right. (B) Northern blots showing decay rates of an NMD β-globin reporter mRNA, in the absence or presence of coexpressed hDcp2. (C) Northern blots showing decay rates of wild-type β-globin mRNA, in the absence or presence of coexpressed hDcp2. The internal control was endogenous GAPDH mRNA.

The TTP NTD is necessary and sufficient for the association with mRNA decay enzymes and is important for optimal mRNA decay stimulation. (A) Western blots showing the association of TTP deletion mutant proteins with mRNA decay enzymes. (Lanes 4,6,8,10) Transiently expressed Flag-tagged hDcp2, hRrp4, hCcr4, or hXrn1 was immunoprecipitated from HEK 293T cell extracts and probed for the presence of coexpressed Myc-tagged TTP, TTP fragments, or hnRNP A1 as indicated. (Lanes 1,3,5,7,9) Five percent total cell extract (T). (Lane 2) Immunoprecipitates from HEK 293T cells with no Flag-tagged protein. (B) Northern blots showing decay rates of the β-ARE reporter mRNA, in the absence of coexpressed protein (none), or in the presence of TTP, TTP_100-326, TTP_1-174, or TTP-F126N as indicated. Time points above the panels refer to minutes after transcriptional repression. β-ARE half-lives were calculated and are shown on the right, together with the average fold of change in half-lives, with standard deviation (three experiments) for those that were significantly different from those in the presence of TTP. (C) Western blots showing the association of TTP and TTP mutant proteins with poly-A mRNA, isolated through association with Flag-PABP. (Lane 1) Five percent total extract (T). (Lanes 2,3) Anti-Flag-PABP immunoprecipitates after treatment without (-, lane 2) or with (+, lane 3) RNase A.