Prediction of RNA secondary structures with Mfold

As the Mfold program (48) does not allow to predict the folding of long RNA sequences (maximum 800 bases), we used chimeric sequences of variable length that included the two regions regulated by DDX5 and DDX17, i.e. the exon 6 and the pri-mir-26a2. The default Mfold parameters were used. The picture presented in Figure ​Figure22 shows a part of a typical structure predicted from a 500 nt-long sequence composed of: (i) a 200-nt fragment that includes full exon 6 and the beginning of intron 6; (ii) a 300-nt fragment centered on the miRNA hairpin. Note that a competing base-pairing between the proximal intronic sequence and the lower part of the pri-mir-26a2 hairpin was predicted whatever the length of the tested sequence (up to 800 nt).

DDX5 and DDX17 ensure the correct processing of CTDSP2/miR-26a2 transcripts. (A) Genomic organization of CTDSP2 and CTDSPL genes (NCBI Gene screenshots) showing the position of miR-26a2 and miR-26a1 within CTDSP2 intron 6 and CTDSPL intron 8, respectively. Exon numbering is based on the FasterDB database (78), which reports exons (in brackets) not annotated by the NCBI. (B) RT-PCR analysis of CTDSP2 and CTDSPL transcripts. The inclusion of the exon preceding the two miR-26a loci was monitored following treatment with control siRNA or siDDX5/DDX17. The size of the different PCR products (exon inclusion and exon skipping) is indicated. (C) RT-PCR analysis of CTDSP2 exon 6 inclusion in stable MCF7 cell lines. Cells were treated with siRNA to silence endogenous DDX5 and DDX17 genes, and treated or not with doxycyclin to induce the expression of recombinant DDX17 (iDDX17) or DDX5 (iDDX5). (D) RT-qPCR analysis of pri-miR-26a2 in stable MCF7 cell lines. Experimental details are as in C. The ratio of pri-mir-26a2 precursor to total CTDSP2 mRNA is represented as the mean value + S.E.M. of independent experiments (n = 3), normalized to the control condition (siCtrl without Doxycyclin). *P-value < 0.05; **P-value < 0.01 (Student's t-test). (E) Predicted folding of the RNA region encompassing CTDSP2 exon 6 and pri-mir-26a2. The framed inset on top of the figure represents a linear view of the region. The black arrowhead indicates the site of the junction between the 2 sequence fragments used for the Mfold prediction (see Materials and Methods for details). Exon 6 (E6) and the pri-mir-26a2 hairpin are highlighted by thick blue lines. The thick black element indicates the 5′-located intronic sequence predicted to base-pair (double-headed arrow in the inset) with the lower stem of the pri-miRNA. The antisense competitor RNA (AON-26a2, in red) was designed to perfectly hybridize with the repressor element, in order to restore the correct folding of the pri-miRNA hairpin. (F) Competing with the intronic inhibitory structure rescues the pri-mir-26a2 processing defect in absence of DDX5/DDX17. The AON-26a2 (or a control AON) was transfected in cells along with control siRNA (siCtrl) or siDDX5/DDX17. The pri-mir-26a2 precursor and the CTDSP2 mRNA were quantified by RT-qPCR. Data are represented as the mean values + S.E.M. of independent experiments (n = 4), normalized to the control condition (siCtrl + Ctrl AON), which was set to 1. *P-value < 0.05; **P-value < 0.01 (Student's t-test). (G) Competing with the intronic inhibitory structure rescues the inclusion defect of CTDSP2 exon 6 in absence of DDX5/DDX17. Experimental design is as in panel D. Inclusion of CTDSP2 exon 6 was analysed as in panel B. The percentage of spliced-in sequence (PSI) is indicated. (H) Inhibition of pri-mir-26a2 processing upon silencing of Drosha (RNASEN). Details are as in panel D. ( I) Silencing of Drosha (RNASEN) does not induce the skipping of CTDSP2 exon 6 (measured as in panel B), showing that splicing and pri-mir-26a2 processing are intrinsically uncoupled processes.