RBM4a-regulated splicing cascade modulates the differentiation and metabolic activities of brown adipocytes

Abstract

RNA-binding motif protein 4a (RBM4a) reportedly reprograms splicing profiles of the insulin receptor (IR) and myocyte enhancer factor 2C (MEF2C) genes, facilitating the differentiation of brown adipocytes. Using an RNA-sequencing analysis, we first compared the gene expressing profiles between wild-type and RBM4a(-/-) brown adipocytes. The ablation of RBM4a led to increases in the PTBP1, PTBP2 (nPTB), and Nova1 proteins, whereas elevated RBM4a reduced the expression of PTBP1 and PTBP2 proteins in brown adipocytes through an alternative splicing-coupled nonsense-mediated decay mechanism. Subsequently, RBM4a indirectly shortened the half-life of the Nova1 transcript which was comparatively stable in the presence of PTBP2. RBM4a diminished the influence of PTBP2 in adipogenic development by reprogramming the splicing profiles of the FGFR2 and PKM genes. These results constitute a mechanistic understanding of the RBM4a-modulated splicing cascade during the brown adipogenesis.

Figure 1. Differential expression profiles of RBM4a, PTBP1, PTBP2, and Nova1 during brown adipogenesis.

(a) Total RNA and protein extracted from embryonic day (E) 13.5 and postnatal day 0 (P0) RBM4a^+/+ (wild-type) or RBM4a^−/− interscapular brown adipose tissues were subjected to an RT-PCR and immunoblot assay using specific primer sets and antibodies. (b) Tissue lysates extracted from embryonic day (E) 13.5, 15.5 and postnatal day 0 (P0) RBM4a^+/+ (wild-type) or RBM4a^−/− interscapular brown adipose tissues were subjected to immunoblot analyses with the indicated antibodies. (c) Tissue lysates extracted from brown adipose tissues of WT or RBM4a^−/− adult mice were subjected to an immunoblot assay with the indicated antibodies. The gels or blots showed in this figure were run under the same conditions and not artificially manipulated. The bar graph represents relative levels of the indicated proteins or transcripts in three independent experiments using TotalLab Quant Software (*p < 0.05; **p < 0.01; ***p < 0.005).

Figure 2. RBM4a alters the PTBP1, PTBP2, and Nova1 protein levels during brown adipogenesis.

(a) Total RNAs and (b) cell extracts isolated from C3H10T1/2 cells cultured in growth medium (0 day) and differentiating medium (for 2 and 7 days) were subjected to RT-PCR, qRT-PCR and immunoblotting assays using specific primer sets and antibodies. (c) Total RNAs and cell extracts extracted from C3H10T1/2 cells that overexpressed RBM4a and the derived mutants were subjected to RT-PCR and immunoblotting assays using specific primer sets and antibodies. (d) Total RNAs and cell extracts extracted from RBM4a overexpressing or targeting C3H10T1/2 cells were subjected to RT-PCR, qRT-PCR and immunoblotting assays as previously described. The gels or blots showed in this figure were run under the same conditions and not artificially manipulated. The bar graph represents relative levels of the indicated proteins or transcripts in three independent experiments using TotalLab Quant Software (*p < 0.05; **p < 0.01; ***p < 0.005).

Figure 3. PTBP2 stabilizes the Nova1 transcript via directly binding to its 3′ untranslated region (UTR).

(a) Empty vector- or PTBP2 targeting vector-transfected C3H10T1/2 cells were mock-treated or treated with actinomycin D. Total RNA and cell extract prepared at indicated time points were subjected to RT-PCR, qRT-PCR, and immunoblotting assays with specific primer sets and antibodies. (b) The diagram presents the sequence of the proximal Nova1 3′ UTR. The mock eluate or recombinant His-tagged PTBP2 protein (2 μg) eluted from the Ni2+ agarose resin was incubated with 10 nM DIG-labeled probes. The mixtures were fractionated on an 8% native acrylamide gel and transferred to a nylon membrane. The membrane was probed using the horseradish peroxidase-conjugated anti-DIG Fab fragment. The gels or blots showed in this figure were run under the same conditions and not artificially manipulated. (c) The scheme shows the Renilla luciferase reporter containing the distinct fragment within the Nova1 3′ UTR. The intact pRL-Nova1 3′ UTR reporters or the derived mutant were transfected into C3H10T1/2 cells cultured under distinct conditions or cotransfected with the expressing or targeting vectors and the pGL3-basic reference vector into C3H10T1/2 cells. The luciferase assays were performed as described under “Materials and methods” and the bar graphs show relative renilla luciferase activity in three independent experiments. The statistical analyses showed the convincing difference in the activity of F1 reporter, but not other reporters in distinct experiment groups (*p < 0.05; **p < 0.01; ***p < 0.005).

Figure 4. RBM4a, PTBP2, and Nova1 possess differential effects on brown adipogenesis.

(a) C3H10T1/2 cells were transfected with the expression vectors of RBM4a, PTBP2, Nova1, or targeting vectors of PTBP2 and Nova1. Total RNAs and cell extracts were extracted from the transfectants cultured in growth or differentiating medium, followed by RT-PCR, qRT-PCR and immunoblotting analyses. (b) C3H10T1/2 cells were transfected with expressing vectors of PTBP2 and Nova1, or targeting vectors of PTBP2 and cultured in differentiating medium 24 h post-transfection. After 48 h, total RNAs and cell extracts were isolated from the transfectants and subjected to RT-PCR, qRT-PCR and immunoblotting analyses. The bar graph presents results of the qRT-PCR in three independent experiments. The gels showed in this figure were run under the same conditions and not artificially manipulated. (c) Parallel experiments were performed as described in the last section and then subjected to oil-red-O staining. The bar graph shows the spectrophotometric analysis of the extracted oil-red-O optical density (OD) at 550 nm and numbers of oil-red-O-stained cells in 100 cells (*p < 0.05; **p < 0.01; ***p < 0.005).

Figure 5. RBM4a and PTBP2 proteins exhibit differential effects on splicing profiles of the FGFR2 and PKM genes.

(a) C3H10T1/2 cells were transfected with the expression vectors of RBM4a or PTBP2. Total RNAs and cell extracts extracted from the mock or actinomycin D-treated cells were subjected to an RT-PCR and immunoblotting analysis. (b) C3H10T1/2 cells were transfected with the expression vectors of RBM4a and derived mutants. Total RNAs extracted from the transfectants were subjected to an RT-PCR analysis, followed by digestion with restriction enzymes. (c) Total RNAs were isolated from C3H10T1/2 cells transfected with the expression or targeting vectors of RBM4a and PTBP2, followed by an RT-PCR assay and enzyme digestion. The gels showed in this figure were run under the same conditions and not artificially manipulated.The bar graph presents relative levels of PCR-amplified transcripts in three independent experiments using TotalLab Quant Software (*p < 0.05; ** p < 0.01; ***p < 0.005).

Figure 6. RBM4a-modulated splicing profile of FGFR2 correlates with brown adipogenesis-related signaling.

(a) Total RNA extracted from the embryonic day (E) 13.5, and postnatal day 0 (P0) RBM4a^+/+ (wild-type) or RBM4a^−/− interscapular brown adipose tissues was subjected to RT-PCR, enzyme digestion and qRT-PCR analyses. (b,c) Total RNAs and cell extract were isolated from the proliferating and differentiating cells at different time points, followed by RT-PCR, enzyme digestion, qRT-PCR and immunoblotting assay with specific primer sets and antibodies. The bar graph presents relative levels of the PCR-amplified transcripts (FGFR2) or a qRT-PCR analysis (Ucp1 and Prddm16) using TotalLab Quant Software (*p < 0.05; **p < 0.01; ***p < 0.005). (d) C3H10T1/2 cells were transfected with an empty vector, expression vectors, or targeting vectors of RBM4a and PTBP2. Before being harvested, transfected cells were mock-treated or treated with FGF10 for 6 h. Cell extracts were isolated from transfectants, followed by immunoblotting assays with anti-ERK1/2, anti-phospho ERK1/2, and anti-Actin antibodies. The gels or blots showed in this figure were run under the same conditions and not artificially manipulated.

Figure 7. RBM4a-regulated splicing profile of PKM gene modulates the mitochondrial activity of brown adipocytes.

(a) Total RNA extracted from embryonic day (E) 13.5 and postnatal day 0 (P0) RBM4a^+/+ (wild-type) or RBM4a^−/− interscapular brown adipose tissues were subjected to RT-PCR analyses and enzyme digestion. (b) Total RNAs and cell extract were isolated from proliferating and differentiating cells at different time points, followed by RT-PCR assay and enzyme digestion. The gels showed in this figure were run under the same conditions and not artificially manipulated. The bar graph presents relative levels of the PCR-amplified transcripts using TotalLab Quant Software (*p < 0.05; **p < 0.01; ***p < 0.005). (c) C3H10T1/2 cells were transfected with expression vectors of RBM4a or PTBP2 for 24 h. Transfectants were then cultured in growth medium or differentiating medium for an additional 48 h. The bar graph presents the basal and maximal oxygen consumption rates and spare respiratory capacity that were measured using an XF20 Bioanalyzer (n = 4). (d) Parallel experiments were performed as described in the last section. Mitochondrial content were visualized by epifluorescence in living cells with the Mitotracker Red FM dye. (e) The RBM4a-regulated splicing cascade correlated with brown adipogenesis. Elevated RBM4a induced alternative splicing-coupled nonsense-mediated decay toward PTBP1/2 transcripts in differentiating brown adipocytes. The interplay between RBM4a and PTBP2 programmed the splicing profiles of the FGFR2 and PKM1 genes which manipulated the differentiation signaling and energy expenditure of brown adipocytes.