Auto- and cross-regulation of the hnRNPs D and DL

Figures

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  FIGURE 1.. The ultraconserved element in the 3′UTR of hnRNP DL contains an NMD-sensitive exon. (A) Exon cluster of the hnRNP DL gene. Constitutive exons are shown in black, the alternative exon 8 in blue. Lines indicate introns, big boxes show coding exons, while smaller boxes show untranslated exons. Arrows indicate the location of oligonucleotides used for RT-PCR in B. The sequence conservation of 100 vertebrates is shown below. An ultraconserved element (uc. 144) is located in the 3′UTR and covers exon 8. (B) RT-PCR after puromycin treatment (puro) in HeLa cells. Cells were treated for 4 h with 200 µg/mL puromycin. The short isoform 7/9 corresponds to the exon 8 exclusion isoform, the long isoform 7/8/9 to exon 8 inclusion. RPLP0 (P0) is shown as loading control. n = 2. (C) Transient knockdown of the NMD factor UPF1. (Left) RT-qPCR quantification of UPF1 mRNA after siRNA-mediated knockdown. (Right) RT-qPCR quantification of isoforms hnRNP DL 7/9 and hnRNP DL 7/8/9 after UPF1 knockdown. Expression values are normalized to the housekeeping gene RPLP0. n = 4, (**) P-value <0.01.

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  FIGURE 2.. HnRNP DL promotes inclusion of the poison exon in a minigene system. (A) Scheme of the luciferase-hnRNPDL-3′UTR minigene. The entire 3′UTR of hnRNP DL was fused to the luc2 gene in a dual luciferase vector (Luc_DL_UTR). The cassette exon 8 is indicated in blue. The big box indicates the protein coding region, small boxes indicate untranslated regions, lines indicate intronic sequences. Arrows indicate the location of oligonucleotides used for RT-PCR in C. (B) Western blot against hnRNP DL (DL). HnRNP DL or GFP as a control were transiently transfected into HeLa cells. Anti-β-actin was used as a loading control. n = 2. (C) RT-PCR of Luc_DL_UTR after overexpression of hnRNP DL (DL) or GFP as control (GFP). The short isoform 7/9 corresponds to the exon 8 exclusion isoform, the long isoform 7/8/9 to exon 8 inclusion. n = 2. (D) Luciferase activity of Luc_DL_UTR after overexpression of hnRNP DL (DL) or GFP (GFP) as a control. Firefly luciferase activity was normalized to Renilla luciferase as an internal control. Values are normalized to an empty vector control, without hnRNP DL 3′UTR sequences. n = 4, (**) P-value <0.01.

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  FIGURE 3.. HnRNP DL regulates its own expression in a negative feedback loop. (A) Western blot of endogenous hnRNP DL protein levels after integration of a GFP-hnRNP DL fusion protein. Anti-hnRNP DL was used to simultaneously detect endogenous hnRNP DL (DL) and the integrated GFP-hnRNP DL fusion protein (GFP-DL). Anti-β-actin was used as a loading control. n = 3. (B) RT-qPCR quantification of isoforms hnRNP DL 7/9 and hnRNP DL 7/8/9 in GFP-hnRNP DL expressing cells (GFP-DL). A GFP expression cell line (GFP) was used as control. Expression values are normalized to the housekeeping gene RPLP0. n = 3. (C) RNA immunoprecipitation using antibodies against the GFP-hnRNP DL fusion protein and GFP or U1-70K as controls. Detected by RT-qPCR were fully spliced, mature hnRNP DL mRNA (DL mRNA), hnRNP DL pre-mRNA (DL pre-mRNA_1: oligonucleotides located in exon 8 and intron 8, DL pre-mRNA_2: both oligonucleotides located in intron 8), and U1 snRNA. n = 5. (**) P-value <0.01; (*) P-value <0.05.

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  FIGURE 4.. Cross-regulation between hnRNP D and DL. (A) Western blot of hnRNP DL and hnRNP D overexpression. GFP, hnRNP DL, or hnRNP D isoforms (D45, D42, D40, D37) were transiently transfected into HeLa cells. Anti-hnRNP DL (above) and anti-hnRNP D (middle) were used to verify the respective overexpression. Anti-HSP60 (below) was used as loading control. (B) Luciferase activity of Luc_DL_UTR and Luc_D_UTR after overexpression of GFP as a control (GFP), hnRNP DL (DL), or hnRNP D isoforms (D45, D42, D40, D37). Luciferase activity was normalized to Renilla luciferase as internal control. Values are normalized to an empty vector control, without hnRNP D or DL 3′UTR sequences. n = 3. (C) RT-PCR of Luc_DL_UTR (left) and Luc_D_UTR (right) after overexpression with GFP as a control (GFP), hnRNP DL (DL), or hnRNP D isoforms (D45, D42, D40, D37). The short isoforms 7/9 and 8/10 correspond to exon exclusion isoforms, the long isoforms 7/8/9 and 8/9/10 to exon inclusion isoforms of hnRNP DL and D, respectively. n = 2. (D,E) RT-qPCR quantification of isoforms (D) hnRNP D 8/10 and hnRNP D 8/9/10 or (E) hnRNP DL 7/9 and hnRNP DL 7/8/9 after siRNA-mediated knockdown of hnRNP DL or D, respectively.…