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The SPF27 Homologue Num1 Connects Splicing and Kinesin 1-Dependent Cytoplasmic Trafficking in Ustilago maydis

Figure 4

Reduced splicing efficiency of the rbf1-gene leads to impaired function of the Rbf1 master regulator.

(A) Plot depicting splicing efficiency of the rbf1-gene. Plotted are the FPKM values (fragments per kilobase of sequence per million fragments mapped) across the genomic region indicated (coordinates in nucleotides) of three independent RNA-Seq experiments for AB31 wild-type (blue lines) and AB31Δnum1 (red lines), respectively. Exons (E) and introns (I) are indicated. All four introns show increased intron retention rates in AB31Δnum1. (B) Western analysis showing abundance of Rbf1:3×HA and α-tubulin (loading control) from AB31 wild-type and Δnum1-deletion strains. In AB31Δnum1, Rbf1 is reduced to 30% of wild-type-level (Quantification: ImageJ [109]). (C, D) Gene expression analyses of b- and rbf1-genes (C) as well as rbf1-target genes (D) using qRT-PCR. RNA samples were isolated from strains AB31 and AB31Δnum1 eight hours after induction of the bE1/bW2-heterodimer. Gene expression is shown relative to the highest expression value, using actin and eIF2b for normalization. Shown are the mean values of three biological and two technical replicates. Error bars represent the SD. (E) Venn diagram depicting the total number of genes repressed in AB31Δnum1 and AB31Δrbf1. * RNA-Seq analysis, this study; ** Microarray analysis conducted five hours after induction of the bE1/bW2-heterodimer [2].

Figure 4