Fig. 1. Northern blots from T-cell, monocytic and tumour cell line RNAs revealed the presence, in addition to the canonical APRIL mRNA (white arrow), of a variant transcript (black arrow). (A) Northern blot with cytoplasmic RNAs from resting (lane 1) and 4 day-activated human primary T cells (lanes 2–4: 5, 10 and 20 µg). (B) Northern blot from total RNAs of CD4⁺ and CD8⁺ cells, purified from a population of 4 day-activated T cells. In (A) and (B), the upper panel shows methylene blue staining of the membrane; the middle panel shows two signals detected following hybridization with an RNA probe specific for the hAPRIL ORF; and the lower panel shows hybridization of the same filter with an RNA probe specific for the hTWEAK ORF. In addition to the TWEAK mRNA signal (grey arrow), a lower mobility signal was detected following longer exposure. (C) Northern blot analysis of cytoplasmic RNAs from human monocytic cell lines U937 and Mono Mac 1 activated with LPS (10 µg/ml) for 0, 60 or 90 min. Hybridization with the hAPRIL probe reveals signals for APRIL (white arrow) and the variant transcript (black arrow). (D) Northern blot of cytoplasmic RNAs from various colon carcinoma cell lines, as indicated above. Upper panel: hybridization with the hAPRIL probe shows APRIL (white arrow) and the variant (black arrow) transcripts. Lower panel: hybridization of the same filter with the hTWEAK probe reveals, in addition to TWEAK mRNA (grey arrow), a lower mobility signal upon longer exposure. 28S and 18S rRNAs are indicated.

Fig. 2. APRIL and the variant transcripts are engaged in protein synthesis in human primary T cells and monocytes. Cytoplasmic extracts from human primary T lymphocytes (A) and human primary monocytes (B) were fractionated on a sucrose gradient to separate mRNAs according to ribosome loading. Upper panels show rRNA distribution in gradients following methylene blue staining of the membranes. (A) Resting T lymphocytes were compared with lymphocytes activated for 4 days with anti-CD3 and anti-CD28. Middle (resting) and lower (activated) panels show the distribution of APRIL (white arrow) and variant (V, black arrow) transcripts in the sucrose gradient fractions from resting and activated cells, visualized by hybridization with the hAPRIL RNA probe. The same membranes, hybridized with the hTWEAK probe, show the distribution of TWEAK mRNA (grey arrow) and of a lower mobility mRNA. The quantification of signals for APRIL, variant and TWEAK mRNAs in the sucrose gradients is plotted below. (B) The middle (resting) and lower (LPS-stimulated) panels show the distribution of APRIL (white arrow) and variant (V, black arrow) transcripts in sucrose gradient fractions from human primary monocytes, resting or LPS stimulated for 1.5 h, as visualized following hybridization with the hAPRIL RNA probe. The quantification of signals in the sucrose gradients is plotted below.

Fig. 3. The variant transcript is a TWEAK–APRIL hybrid mRNA. (A) Sequence of hTWE-PRIL mRNA and the protein encoded. Primers used for PCR experiments are indicated by an arrow. The putative transmembrane region (deduced from that of hTWEAK) is shown as a black box, and the putative furin cleavage site (deduced from that of hAPRIL) as a dark grey box with white letters. The white box shows the portion of the sequence identical to that of hTWEAK mRNA; the grey box shows that corresponding to hAPRIL mRNA. (B) RNase protection assay using a probe encompassing the TWEAK–APRIL junction of the TWE-PRIL hybrid mRNA shows four specific protected fragments. One corresponds to TWE-PRIL mRNA, one to APRIL, and the doublet to TWEAK mRNA. The asterisk indicates a non-specific band, visible in yeast tRNA; molecular weights are shown on the left.

Fig. 4. Organization of the human TWEAK and APRIL gene loci. (A) TWEAK and APRIL genes are in the same orientation and separated by <1 kb. (B) TWEAK mRNA is composed of TWEAK exons 1–7, APRIL mRNA of six APRIL exons, whereas the TWE-PRIL transcript comprises TWEAK exons 1–6 fused to APRIL exons 2–6.

Fig. 5. TWE-PRIL is expressed at the cell surface of HEK 293T transfectants. (A) Protein expression by HEK 293T cells transfected with expression vector, empty or encoding hAPRIL, hTWEAK or TWE-PRIL, was analysed by western blotting of NP-40 cell lysates separated by 15% SDS–PAGE under non-reducing conditions, using polyclonal anti-APRIL and anti-TWEAK antibodies. White arrows indicate signals corresponding to APRIL; the asterisk shows intracellularly processed APRIL. Black and grey arrows indicate TWE-PRIL- and TWEAK-specific signals, respectively. (B) FACS analysis of HEK 293T transfected cells as in (A), using anti-APRIL and -TWEAK antibodies. In each panel, staining with secondary antibody alone is shown in grey as a reference. One representative staining of three is shown.

Fig. 6. TWE-PRIL is expressed at the cell surface of HeLa and NRK transfectants. (A) Protein expression of HeLa cells transfected with expression vector, either empty (control) or encoding hAPRIL, hTWEAK or hTWE-PRIL, was analysed by western blotting of cell lysates, using polyclonal anti-APRIL antibody, as for Figure 5A. White arrows indicate signals corresponding to hAPRIL; the asterisk shows intracellularly processed APRIL. The black arrow indicates the TWE-PRIL-specific signal. (B and C) Immunofluorescence analysis of HeLa (B) or NRK (C) cells transfected with APRIL, TWE-PRIL or TWEAK. Cells were exposed to the indicated antibodies prior to fixing, revealing cell surface expression of the corresponding antigens. Bar = 200 µm.

Fig. 7. Endogenous APRIL and TWE-PRIL are detected in human primary T cells and monocytic cells. Membrane-enriched lysates were prepared from resting and activated primary T cells, the U937 monocytic cell line and, as controls, 293T cells transfected with empty vector or vector encoding APRIL and TWE-PRIL, as indicated. Western blot analysis of these extracts using an anti-APRIL antibody shows endogenous expression of full-length (white arrow) and processed APRIL (white arrow, *) in all cells tested. A 48 kDa protein corresponding to TWE-PRIL was also detected in activated T cells and U937 (black arrow) cells. Note that although a longer exposure was required to visualize TWE-PRIL in T cells, no signal corresponding to APRIL or TWE-PRIL was observed in empty vector cell lysates.

Fig. 8. TWE-PRIL is a biologically active protein. CSFE-labelled Jurkat (A) and Ramos (B and C) cells were cultured on monolayers of 293T cells transfected with empty expression vector or vector encoding APRIL or TWE-PRIL, as indicated. Cell distribution in 2-fold decreasing peaks (as indicated by gates) analysed by FACS after 4 (A) or 3 (B and C) days of co-culture, are indicated below the FACS profiles. In (C), the culture supernatant of the 293T cells was replaced by fresh medium before addition of Ramos cells, to remove any fibroblast-secreted protein. Data shown are representative of five independent experiments.