Journal: Nucleic Acids Research

Article Title: MicroRNA-Mediated Obstruction of Stem–loop Alternative Splicing (MIMOSAS) regulates long-range alternative splicing in Drosophila

doi: 10.1093/nar/gkag356

Figure Lengend Snippet: MicroRNA-dependent disruption of splice-relevant stem–loops controls Fas3 exon 5 inclusion in Drosophila . ( A ) Schematic of Fas3 pre-mRNA showing box I (red) and box II (blue), which form a splice-relevant stem surrounding exon 5. ( B ) Table listing microRNAs predicted to bind box regions with their corresponding ΔG MIMOSAS values; energetically (un)favorable candidates selected for downstream experiments are highlighted. ( C ) Model of MIMOSAS-mediated alternative splicing of Fas3 . When highlighted microRNAs disrupt stem formation, the RC splice variant that includes exon 5 is produced. In the absence of MIMOSAS activity, alternative Fas3 splice variants lacking exon 5 (RA/B/D/E/F/G) predominate. ( D, E ) ΔG MIMOSAS values and corresponding binding probabilities for miR-973 and miR-976 at box I, and miR-1000 and miR-999 at box II. microRNA binding probability was calculated for a 200-nucleotide nascent pre-mRNA segment transcribed from the 5′ end by RNA polymerase. Three colors indicate distinct transcriptional stages of the box: green, when RNA polymerase has just reached the 5′ end of the box; orange, when the 3′ end of the box has just been transcribed; and dark red, when an additional downstream segment equal in length to the box has been transcribed. Ratios of endogenous RA/B/D/E/F/G (STEM) variants to total Fas3 mRNA measured by real-time PCR in brains overexpressing microRNAs ( F ) or expressing microRNA sponges for knockdown ( G ) under the pan-neuronal driver elav C155 -GAL4 , and in a miR-999 KO loss-of-function mutant background ( H ). Ratios of STEM to Total in miR-9c and scramble sponge-expressing brains, as well as wild-type ( w 1118 ) control groups were normalized to 1, and fold changes are shown. Data are presented as mean ± s.d. Statistical significance was determined using unpaired Student’s t- test, **** P ≤ .0001, *** P ≤ .001; n ≥ 3, triplicate sampling.

Article Snippet: Eight TaqMan probes were used in this study: FAM-Dm02150883_g1 (MIMOSAS) and VIC-Dm02144515_g1 (STEM) for Nmnat mRNA variant detection; FAM-Dm01810909_g1 (STEM) and VIC-Dm01810910_m1 (MIMOSAS) for Fas3 mRNA variant detection; FAM-Dm02135667_g1 (Total), FAM-Dm02135669_g1 (RA + RH), and VIC-Dm02148683_g1 (RG) for RpL3 mRNA variant detection; and VIC-Dm02151827_g1 for rp49 detection as an internal control.

Techniques: Disruption, Alternative Splicing, Variant Assay, Produced, Activity Assay, Binding Assay, Real-time Polymerase Chain Reaction, Expressing, Knockdown, Mutagenesis, Control, Sampling