Figure 1. Expression of RBMS3 in HSCs and fibrotic livers. (a) Expression of RBMS3 in quiescent (Q) and culture activated rat HSCs (A). HSCs were harvested two days after isolation (lane 1, Q) or eight days after isolation (lane 2, A) and the level of RBMS3 mRNA was estimated by RT-PCR. Expression of neurotrimin (NTRM) and β-actin (ACTIN) is shown as control. (b) The time-course of RBMS3 expression during culture activation of rat HSCs, which were cultured for the indicated time-points and expression of RBMS3 was estimated as in (a). (c) Expression of RBMS3 in animal model of liver fibrosis. Five rats were treated with CCl₄ for four weeks to induce liver fibrosis and expression of RBMS3 mRNA in fibrotic livers (lanes 3–7) was compared to that in normal livers (lanes 1 and 2) by RT-PCR. Actin is shown as a loading control.

Figure 2. RBMS3 interacts with a sequence within the 3′ UTR of Prx1 mRNA. (a) A representation of RNA probes used to map the RBMS3 binding site. Relationship between the full-size 3′ UTR and various probes is shown (not to scale): 60 nt of the RBMS3 binding site is shown as a black box. The length of probes is given in nucleotides. (b) Binding of RBMS3 to the 566 nt probe. HA-tagged RBMS3 was over-expressed in human fibroblasts by adenoviral delivery. Cytosolic extract of RBMS3-expressing cells was used in gel mobility-shift with 566 nt probe (RBMS3, lane 2) and compared to shift with extracts of cells infected with control virus (CON, lane 3). Lane 1 is probe alone. Before loading onto the gel, the samples were digested with RNase T₁. Migration of RNase-resistant RNA/protein complex is indicated, as well as that of undigested probe. (c) Experiment as in (b), except the 240 nt probe was used and RNase digestion was omitted (lanes 1–3). Migration of the RNA/protein complex is indicated. Lanes 4–6 show a Western blot of the gel shown in lanes 1–3, probed with anti-HA antibody. Arrow indicates position of RBMS3 protein in the slot of the gel.

Figure 3. Recombinant RBMS3 interacts with 60 nt within the 3′ UTR of Prx1 mRNA. (a) RBMS3 was purified as a GST fusion protein after expression in E. coli. The purified protein was analyzed by SDS-PAGE and staining with Coomassie brilliant blue (lane 3). Lane 2 is GST alone and lane 1 is a size marker (M). Sizes (in kDa) are shown to the left. (b) Binding of the recombinant protein to the 240 nt probe. Gel mobility-shift with purified GST-RBMS3 fusion protein (RBMS3, lane 2) and GST alone (CON, lane 3). Lane 1 is probe alone. Migration of the RNA/RBMS3 complex and the free probe is indicated. In lanes 4 and 5, a probe derived from the mouse GAPDH sequence was used. This probe shows spontaneous formation of a dimer (lane 4). In lanes 7 and 8, a probe encompassing the 5′ stem–loop of mouse collagen α1(I) was used (5′SL). (c) Binding of recombinant RBMS3 to 150 nt probes. Two probes from the 5′ end (150A) and 3′ end (150B) of the 240 nt probe sequence, respectively, were derived. These probes have a 60 nt overlap. The gel mobility-shift experiment was done as in (a). Migration of the RNA/RBMS3 complexes and the free probe is indicated. (d) Binding of recombinant RBMS3 to the 60 nt overlapping sequence shared by probes 150A and 150B. Lane 1 is the 60 nt probe alone. This probe forms a dimer spontaneously, which is indicated. Lane 2 is binding of recombinant RBMS3 to the probe. In lanes 3 and 4, two different non-specific competitor RNAs were added at the molar excess indicated, while in lane 5 10 μg of total liver RNA was added. In lane 6, specific competitor RNA was added at the molar excess indicated. Migration of the RBMS3/RNA complex is indicated. (e) Poly(A)⁺ RNA from the liver competes binding of RBMS3 to the Prx1 sequence. Experiment as in (d), but 0.5 μg of poly(A)⁺ RNA from mouse liver (lane 4) or 2.5 μg of poly(A)⁺ RNA from mouse liver (lane 5) was used as the competitor. Lane 3 is competition with 10 μg of total liver RNA and lane 2 is binding without competitor. Lane 1 is probe alone, and the migration of RNA and RBMS3/RNA complex is shown. (f) Sequence comparison of the RBMS3 binding site in vertebrate Prx1 mRNAs.

Figure 4. Ectopic expression of RBMS3 upregulates Prx1 and collagen α1(I) mRNA. Quiescent HSCs at day 2 after isolation were transduced with the control adenovirus (CON, lane 1) or adenovirus expressing RBMS3, which was done in duplicate (RBMS3, lanes 2 and 3). At day 5, expression of Prx1 and collagen α1(I) (COL) was analyzed by RT-PCR. Arrows indicate migration of the specific PCR products. β-Actin was used as a loading control.

Figure 5. RBMS3 increases expression of Prx1 protein when encoded by the mRNA containing the RBMS3 binding site. (a) A representation of reporter constructs. The genes were driven by the CMV promoter (white box), the 5′ UTR was derived from the vector, followed by the Prx1 open reading frame (gray box) with an HA tag at the N terminus (black box). The reading frame was followed by the complete Prx1 3′ UTR (WT) or by the 3′ UTR from which the RBMS3-binding site (stippled box) was deleted (MUT). All Prx1 sequences were derived from the mouse gene. (b) Prx1 protein expression from the reporter constructs. WT reporter together with expression plasmid for RBMS3 (RBMS, lane 1) or control protein expression plasmid (CON, lane 2) were transiently transfected into HEK293 cells. Lanes 3 and 4 are identical, except the mutant reporter (MUT) was transfected. All transfections were normalized to expression of β-gal internal standard gene. Western blot was performed with the anti-HA antibody. Expression of RBMS3 was visualized (indicated by arrow), because it contained an HA tag. The control protein cannot be seen, because it did not have a tag. The expression of Prx1 is indicated in the bottom part by the arrow. Lanes 5–9 represent an identical independent experiment and correspond to lanes 1–4.

Figure 6. Steady-state level and stability of reporter mRNAs is affected by RBMS3. (a) The steady-state mRNA level. RNA was extracted from samples shown in (a) and expression of reporter mRNAs (REP) was analyzed by RT-PCR. The amounts of RNA used in analysis were normalized to the transfection efficiencies of individual samples, which were within 20%. Lane 1 shows non-transfected cells. The expression of reporter mRNAs (REP) is indicated. (b) The stability of mRNAs. WT and MUT reporter genes were cotransfected with RBMS3 expression plasmid and 48 h after transfections the cells were treated with actinomycin D for the indicated lengths of time. RNA was extracted and analyzed as in (a). For better comparison, the amount of total RNA in MUT samples was increased to give the Prx1 signal at time zero comparable to that of WT samples.