Diagram of murine CD154 mRNA 3′-UTR. a, locations of CUREs, CAREs, polycytidines (pC), and ARE sequences are shown, numbered in reference to translational stop site. CURE and CARE nucleotide sequences are shown immediately below. b, design of pcDNA 3.1- or pTRE-based luciferase reporter plasmids and abbreviations.

hnRNP L interacts with CD154 mRNA in a 3′-UTR CARE-dependent manner. a, PTB and hnRNP L were immunoprecipitated from cytosolic and nuclear extracts from activated (PMA/ionomycin, 24 h) T cells. PTB (BB7 MoAb), hnRNP L (4D11), and P3 (irrelevant isotype control) were used. PTB and hnRNP L were coimmunoprecipitated in both extracts, although hnRNP L in PTB immunoprecipitate is not appreciated with this exposure. Immediately below is shown CD154 mRNA in hnRNP L and PTB immunoprecipitates from both cytoplasm and nucleus. No GAPDH, interleukin-2, or γ-interferon mRNA was detectable in either PTB or hnRNP L immunoprecipitates. b, immunoblot of hnRNP L immunoprecipitate from cytoplasmic extracts from HeLa cells transfected with control, CARE, or CD154 3′-UTR containing luciferase vectors or no vector (mock). c, hnRNP L immunoprecipitates contain luciferase mRNA in a 3′-UTR CARE-dependent manner, since no product is seen in extracts transfected with control vector lacking these sequences. Further specificity is shown by inability to detect GAPDH mRNA in immunoprecipitates.

Trafficking of hnRNP L to stress granules but not processing bodies. After transfection into Hep-2 cells, green fluorescent protein-labeled hnRNP L localized to the nucleus (green; a). Serum containing antibodies directed against mRNA processing body marker Ge-1 stained 5-10 dots in the cytoplasm of Hep-2 cells (red; b). After exposure to arsenite, hnRNP L localized to granules in the cytoplasm (d) and co-localized with T-cell intracellular antigen (TIA), a marker for stress granules (red; e). mRNA-processing bodies (h) localized adjacent to hnRNP L in stress granules (g). Overlap of green and red staining is shown in c, f, and i. 4′,6-Diamidino-2-phenylindole staining in f and i indicate the location of cell nuclei. White arrowheads (i) indicate the location of Ge-1-containing mRNA processing bodies adjacent to hnRNP L-containing stress granules.

The presence of the 3′-UTR-CARE is associated with differential priming of reverse transcription by oligo(dT) and random hexamers. a, total cytoplasmic RNA extracts from transient transfections of HeLa cells with specified pcDNA 3.1-based vectors were analyzed by quantitative PCR using luciferase primers following reverse transcription with either oligo(dT) or random hexamers. Poly(A) purification was omitted. The 3′-UTR CARE and the CUCARE+, but not the CURE alone, affected the measurable amount of input luciferase RNA as a function of priming with increased levels seen with random hexamers. Data are shown as a percentage of input RNA in control transfection. b, summary of effect of CARE on input luciferase mRNA measured by quantitative PCR following priming shown as random hexamers relative to oligo(dT) (n = 8).c, cytoplasmic RNA was analyzed by quantitative PCR following priming of reverse transcription with oligo(dT), RHs, or a luciferase-specific primer. Data are presented as a ratio of the effect of reverse transcription primer or luciferase-specific primer on apparent input RNA relative to that seen with oligo(dT). The 3′-UTR CARE increased the ratio of input luciferase mRNA with both luciferase-specific priming and random hexamers by 4-fold relative to that seen with oligo(dT). In contrast, little or no effect was seen on luciferase mRNA from cells transfected with control and 3′-UTR-CURE-containing plasmids.

The CURE and CARE represent separate cis-acting elements that differentially regulate the inhibitory activity of the CD154 3′-UTR. a, the murine CD154 3′-UTR limits pcDNA 3.1 reporter gene expression in transiently transfected HeLa cells at the level of cytoplasmic poly(A) mRNA as measured by oligo(dT)-primed RT-PCR. Deletion of both CURE and CARE (CUCARE-) was required to eliminate inhibitory effect of CD154 3′-UTR on poly(A) mRNA (n = 6). p values are shown relative to that seen with luciferase controls. b, the CURE and CARE alone are sufficient to limit cytoplasmic poly(A) mRNA accumulation (n = 6). c and d, pTRE-based plasmids were transiently transfected into HeLa Tet-Off cells with transcriptional inhibition triggered by the addition of doxycycline (1 μg/ml). Cytoplasmic RNA was extracted at the time of doxycycline addition and specified times thereafter with poly(A) RNA purified and analyzed by oligo(dT)-primed RT-PCR. The level of input RNA measured at time 0 for each vector is assigned a value of 100%, although both the CURE- and CARE-vectors reduced mRNA levels. Increased mRNA decay rates are only seen with reporters (c, CD154, CARE-, and CURE+) containing the CURE in their 3′-UTR. Data shown are representative of three experiments each.

The presence of the 3′-UTR-CARE is associated with a shortened poly(A) tail but increased cytoplasmic mRNA stability. a, the presence of the 3′-UTR-CARE is associated with increased deadenylation of reporter gene as well as native CD154 mRNA. Cytoplasmic RNA extracts from transiently transfected HeLa cells with specified pcDNA 3.1-based plasmids were assayed for poly(A) tail length. Cytoplasmic luciferase mRNA from transient transfected HeLa cells containing the 3′-UTR CARE has a markedly shortened (<50-nt) poly(A) tail relative to control or 3′-UTR CURE as measured by the LM-PAT assay. The arrow indicates the predicted length of fully deadenylated fragments. Note the extended poly(A) tail length (∼150 A bases) from transfections with control and CURE constructs. Presence of CURE markedly reduced luciferase mRNA while not affecting poly(A) length; the data shown in this gel have twice as much PCR product in this lane loaded to make this product visible by EtBr staining. The presence of the CURE and CARE (CUCA+) yielded a short poly(A) tail as well. b, the 3′-UTR CARE results in decreased luciferase mRNA poly(A) tail length in nuclear, cytoplasmic, and polysomal fractions. Right-hand arrows show the predicted fragments following MnlI restriction enzyme digestion, establishing the identity of the amplified product. c, the presence of the CARE in the 3′-UTR resulted in increased mRNA stability, whether measured by RT-PCR following oligo(dT)- or RH-dependent priming.

The murine CD154 3′-UTR has two cis-acting elements that regulate reporter gene expression cytoplasmic poly(A)⁺ mRNA accumulation. a, murine CD154 3′-UTR limits pcDNA 3.1 reporter gene expression in transiently transfected HeLa cells. Deletion of both CURE and CARE (CUCARE-) was required to eliminate inhibitory effect of CD154 3′-UTR on luciferase gene expression. Data shown are an average of four experiments, with error bars representing the S.E. (n = 4). b, the CARE and CURE function to regulate reporter gene expression in human peripheral blood mononuclear cells under both resting and activated conditions. Transient transfection with the specified reporter vectors was performed, and luciferase activity was measured under basal (black bars) or activated (open bars) conditions, where phorbol 12-myristate 13-acetate (10 ng/ml) and ionomycin (1 μm) were added for the last 4 h. c, mutations of the polycytidine or ARE in the context of the CUCARE-deletions had no effect on gene expression. HeLa cells were transiently transfected with pcDNA 3.1-based plasmids containing the control, CD154 3′-UTR, or CUCARE-, mutation. The CUCARE-polyCmut and the CUCARE-AREmut additionally contained deletions of the polycytidine and ARE sequences, respectively. Error bars are not visible at this scale in this representative experiment (n = 4). d, the CURE and CARE alone are each sufficient to limit reporter gene expression effect on luciferase expression, whereas combination of the CURE and CARE (CUCARE+) did not enhance their activity (n = 4).

Modulation of hnRNP L levels alters the ability of the 3′-UTR CARE to regulate poly(A) tail length. a and b, immunoblotting showing modulation of nuclear hnRNP L levels by RNA interference (a) or overexpression (b). Experiments utilized transient transfection of HeLa cells with irrelevant (HuSH 303) and hnRNP L-specific (HuSH L79) short hairpin RNA plasmids (Origene) and pcDNA 3.1-hnRNP L and empty vector control on days -2 and 0 with 40-60% efficiency. Immunoblotting for hnRNP A2 is used as a loading control. The pcDNA 3.1 CARE+ plasmid was cotransfected on day 0. c, knockdown of hnRNP L resulted in loss of 3′-UTR CARE effects on luciferase activity relative to irrelevant small interfering RNA. RT-PCR analysis with either oligo(dT) or RH priming shows that hnRNP L knockdown reduces differences seen with these priming methods, indicating restoration of normal poly(A) tail length. d, overexpression of hnRNP L enhanced CARE-dependent inhibition of luciferase activity and the differential between oligo(dT) and random hexamers in detecting luciferase mRNA. e, hnRNP L knockdown is associated with increased poly(A) tail length. Note that treatment with irrelevant small interfering RNA control had little or no effect. No effect on GAPDH mRNA polyadenylation was seen. The arrows indicate predicted size generated by deadenylated mRNA. f, overexpression of hnRNP L increases 3′-UTR CARE-dependent effects on luciferase expression and shortens the poly(A) tail length in a CARE-dependent manner. Note the lack of effect on GAPDH and control luciferase mRNA. (n = 3 for each experiment in this figure.)