Functional characterization of CD86. The functional effect of TTP expression on CD86 3′-UTR-mediated luciferase expression was examined in 293 and RAW cells, using wild-type (TTP), Zinc-Finger Mutant (TTP Zn-FM) (n = 4). Transfection of wild-type TTP resulted in a 24% decrease in CD86 3′-UTR nucleotides 1120–1655 in 293 cells (p < 0.05) and a 21% decrease in RAW264.7 cells (p < 0.05). The TTP Zn-FM binding mutant had no effect on CD86 3′-UTR nucleotides 1120–1655. There was no effect of TTP transfection on CD86 3′-UTR nucleotides 1656–2720.

Functional effect of TTP on MHC Class IF expression. (A) Alignment of the 3′-UTRs of the six expressed MHC Class I molecules. The 36-nt region from MHC Class IF is indicated in bold underline. The cis-element identified by RNA SCOPE is underlined in the consensus sequence. (B) Wild-type TTP significantly inhibited (p < 0.05) luciferase expression from the 99-nt MHC Class IF 3′-UTR as well as the 36-nt region from 1090 to 1126. The TTP Zn-FM mutant had no effect on luciferase expression (n = 4).

Identification of the TTP cis-element in MHC Class IB. (A) MHC Class IB 3′-UTR. Underline bold sequences are nucleotides that align with MHC Class IF. (B) MHC Class IB vectors constructs used to identify the TTP-binding site. The region of homology with MHC Class IF is indicated. (Class IB FL) Full-length Class IB 3′-UTR nucleotides 999–1517; (Class IB Delta 1) Class IB 3′-UTR with nucleotides 1104–1251 deleted. This region corresponds to the region of homology with MHC Class IF. (Class IB Delta 2) Class IB 3′-UTR with nucleotides 1251–1324 deleted; (Class IB Delta 3) Class IB 3′-UTR with nucleotides 1324–1461 deleted. (C) Wild-type TTP significantly inhibited (p < 0.05) luciferase expression from wild-type, Class IB Delta 2, and Class IB Delta 3 constructs. TTP inhibition was lost with the Delta 1 construct (n = 4). The TTP Zn-FM mutant had no effect on any of the luciferase constructs.

Functional characterization of TTP mRNA ligands. The functional effect of TTP transfection on 3′-UTR-mediated luciferase expression was examined in HEK 293 and RAW264.7 cells, using wild-type (TTP) and Zinc-Finger Mutant (TTP Zn-FM), which cannot bind mRNA (n = 4). (A) Consistent with previous work, TTP transfection had little effect on pGL3 control luciferase expression. Wild-type TTP transfection resulted in a significant decrease in full-length TNF-α 3′-UTR luciferase reporter expression in both 293 (p < 0.001) and RAW (p < 0.001) cells, while the TTP Zn-FM did not alter TNFα 3′-UTR-mediated expression. (B) Wild-type TTP significantly inhibited DUSP1 3′-UTR luciferase expression in both 293 (p < 0.001) and RAW (p < 0.01) cells. The TTP Zn-FM did not alter DUSP1 3′-UTR luciferase expression. (C) Wild-type TTP significantly inhibited IDO 3′-UTR luciferase expression in both 293 (p < 0.001) and RAW (p < 0.05) cells. The TTP Zn-FM did not alter IDO 3′-UTR luciferase expression. (D) Wild-type TTP significantly inhibited SOD2 3′-UTR luciferase expression in both 293 (p < 0.01) and RAW (p < 0.01) cells. The TTP Zn-FM did not alter SOD2 3′-UTR luciferase expression.

Phenotype of iDCs and mDCs and TTP expression. Monocyte-derived dendritic cells were generated using IL-4 and GM-CSF. (A) Flow cytometric analysis of the surface marker expression on a representative set of human iDCs and mDCs used for immunoprecipitations. Mean fluorescent intensity (MFI) for CD86, CD80, CD40, MHC Class I, and Class II increase with maturation. MFI for CD14, and mannose receptor (MR) decreased with maturation. (B) Cytoplasmic lysates generated from iDCs and mDCs were immunoblotted for TTP protein expression. TTP expression is higher in iDCs than mDCs. (C) Total cytoplasmic and total cytoplasmic lysate depleted of TTP by IP were immunoblotted for TTP protein. TTP was cleared from the iDC and mDC lysates (iDC depleted, mDC depleted). Immunoblot for GAPDH served as a loading control.

Confirmation of TTP targets by Real-Time PCR. iDC and mDC cytoplasmic lysate was pre-cleared with pre-immune serum. Total RNA was then isolated from half of the supernatant, and the other half was used for new TTP IPs and the captured RNA isolated. Total and TTP IP RNA samples were then screened for the presence of 15 putative TTP targets identified by RIP-Chip and one negative control (CD14) using Real-Time PCR. (A) Change in total mRNA level with DC maturation. The relative mRNA level of each gene is calculated using ΔΔCt. (B) The percentage of each mRNA bound by TTP in iDCs and mDCs. Values range from a low of 0.36% to a high of 18.8%. No CD14 signal was detected by real-time PCR in the TTP immunoprecipitate.

Identification of TTP targets by RIP-Chip. Messenger RNAs enriched in each RIP-Chip experiment were identified by assigning each element on the microarray a percentile rank on each of the GeneChips analyzed. These were subsequently used to calculate the mean percentile rank for both experiments. (A) TreeView image representing all 22,216 genes found on the array, sorted by percentile rank. The image's pixel settings are at 500 contrast; however, enriched genes are detected at values of greater than 10,000. The distinct band at the top represents the significantly enriched genes, as the color intensity correlates to the net intensity of the gene on the array. (B) Graphs of the distribution histograms of the mean percentile ranks for the two iDC RIP-Chip experiments (top panel) and mDC RIP-Chip experiments (bottom panel). The distribution in each is bimodal, showing a tail at the high percentile ranks (bin size = 0.006706). Putative iDC targets were defined as those that fall to the right of this trough with a percentile rank above 95.92% (the distribution of these genes is red in the histogram inset). Putative mDC targets were defined as those that fall to the right of this trough with a percentile rank above 95.90% (the distribution of these genes is red in the histogram inset). (C) Histogram showing the distribution of net intensity for all genes on the microarray (green) and the genes that have a GO biological process annotation of Protein Synthesis (pink). The Y-axis is the percentage of genes plotted at a given intensity value The percentile rank distributions are plotted on the X-axis across all four experiments (bin size = 0.33) as a percentage of all genes (green; 22,216 genes) or as a percentage of only the protein synthesis genes (pink; 78 genes).