Figure 1.
hnRNP F and CAD form RNA-independent complexes with TTP and BRF1.
(A) Silver-stained SDS-polyacrylamide gels showing proteins that co-purify with stably expressed Flag-tagged TTP (left gel) or BRF1 (right gel) from extracts of T-REx HEK 293 cell lines after RNase-treatment. Proteins co-purified from cells expressing no Flag-tagged protein are run alongside as controls (lanes labeled ‘none’). (B) Western blots of anti-Flag IP reactions from RNase-treated extracts of HEK 293T cells transiently expressing Flag-tagged TTP (lane 6), BRF1 (lane 7), HuR (lane 8), or empty vector control (lane 5). Precipitates were probed for the presence of co-expressed Myc-tagged CAD, hnRNP F, 14-3-3ε, or hnRNP A1 as indicated. Lanes 1–4 shows 5% of the total extract for each IP reaction.
Figure 2.
hnRNP F complexes with TTP/BRF1 in LPS-stimulated RAW macrophages.
(A) Western blots for endogenous proteins that co-IP with anti-TTP (lanes 4–6) or pre-immune sera (lanes 7–9) from RNase-treated extracts of RAW264.7 cells stimulated with 100 ng/ml LPS for the indicated times. Precipitates are compared with 1% of the input (lanes 1–3). The anti-hnRNP F/H antibody recognizes both hnRNP F (bottom band) and hnRNP H proteins (top band). (B) Same as panel A, except the IP was performed with anti-hnRNP F/H (lanes 4–6) or anti-Myc (lanes 7–9).
Figure 3.
hnRNP F binds to and stimulates the decay mediated by the BRSK1 ARE-containing 3′UTR.
(A) Northern blot showing reporter mRNAs that co-precipitate with transiently expressed Flag-tagged hnRNP F (lanes 4, 5), TTP (lanes 6, 7), or empty vector (lanes 8, 9) in HEK 293T cells. Precipitates and 5% of total extract (lanes 1–3) were probed for the presence of β-globin and β-BRSK1 mRNAs. Bottom, A cartoon schematic of the β-BRSK1 mRNA reporter with the 3′UTR from BRSK1 gene containing an ARE (black bar) and G-rich sequence repeats (grey bars) downstream of the β-globin coding region. (B) Northern blots showing mRNA decay of the β-BRSK1 mRNA reporter in HeLa Tet-off cells transfected with siRNAs targeting hnRNP F, TTP/BRF1/BRF2 (TTP/BRF), or luciferase (Luc.) as indicated. Numbers above lanes refer to times in minutes after transcriptional shut-off by tetracycline addition (Chase). Levels of the reporter mRNA was normalized to the constitutively expressed β-globin control mRNA and the average half-life (t1/2) and the standard error of the means was determined from 6 independent experiments. P-value was calculated with Students t-test (paired two-tailed). (C) HeLa Tet-Off cells were transfected with the corresponding siRNAs and knockdown of hnRNP F was assessed by Western Blot. Upf1 served as a loading control. Numbers above lanes with Luc siRNA samples refer to the percent of extract loaded.
Figure 4.
hnRNP F stimulates the decay of TTP/BRF-target mRNAs that show low binding to hnRNP F.
(A) Northern blots showing mRNA decay of the β-GMCSF-ARE mRNA reporter in HeLa Tet-off cells transfected with siRNAs targeting hnRNP F or luciferase (Luc.) as indicated. Levels of reporter mRNA was normalized to the constitutively transcribed control mRNA (β-GAP mRNA) and half-life (t1/2) and standard error of the mean was determined from two independent experiments. Cartoon of the β-ARE mRNA reporter with the ARE from GMCSF inserted in the 3′ UTR is shown below the panel. (B) Same as panel A, but monitoring the decay of a reporter mRNA containing the ARE from the TNFα mRNA. Average half-life and standard error of the mean was calculated from two independent experiments. (C) Same as panel A, but monitoring decay of the β-TNFα mRNA reporter, containing the entire 3′ UTR of TNFα mRNA. Average half-life and standard error of the mean was calculated from 4 independent experiments.
Figure 5.
hnRNP F stimulates decay of the endogenous TTP-target LIF mRNA in NIH 3T3 cells.
(A) Western blots showing levels of TTP, BRF1, and HuR in NIH 3T3 cells incubated with serum for the indicated time after 24 hours of serum starvation. (B) Quantification of the enrichment relative to GAPDH mRNA of known TTP-target mRNAs (listed below the graph) that co-precipitate with an antibody against hnRNP F/H from extracts of NIH 3T3 cells incubated for 2 hours with serum after 24 hours of serum starvation. mRNA levels were determined by qRT-PCR. The fold enrichment for each ARE-mRNA was calculated by dividing the level of ARE-mRNA relative to that of GAPDH mRNA in each IP sample, after subtracting background levels from IP reactions with anti-Myc. Indicated standard error of the mean values were calculated from three experiments. Asterisks represent P-values <0.1 (*) and P-value <0.05 (**). The number of hnRNP F/H consensus binding sites (DGGGD) within the 3′ UTR of each mRNA is listed below the graph. (C) mRNA decay assays for LIF, cFOS, and IER3 mRNAs in NIH 3T3 cells transfected with 80 nM of the indicated siRNAs and incubated for 3 hours in full media after 24 hours of serum starvation. Samples were collected at 0, 40, 80, and 120 minutes after addition of 10 µg/ml Actinomycin D. ARE mRNA was normalized to GAPDH mRNA by qRT-PCR to determine relative abundance and to calculate mRNA decay half-lives. Average half-life, standard error of the mean and P-values for LIF and cFOS were determined from three biological repeat experiments and two biological repeats for IER3. (D) Western blots showing depletion of hnRNP F and TTP in NIH 3T3 cells transfected with 80 nM of the indicated siRNA and incubated with serum for 0 or 2 hours after 24 hours of serum starvation. PABP serves as a loading control.