Journal: Nucleic Acids Research
Article Title: The Hox transcription factor Ultrabithorax binds RNA and regulates co-transcriptional splicing through an interplay with RNA polymerase II
Figure Lengend Snippet: Ubx binds RNA in vivo and in vitro , partly via its Homeodomain. (A–C) RNA-immunoprecipitation (RIP-RT-qPCR) experiments of Drosophila S2R+ cells expressing GFP control (white), GFP-Ubx WT (blue) and GFP-Ubx N51A (purple) showing an enrichment of targeted exonic regions of Chas ( A ), pAbp ( B ) and Rgk1 ( C ). Values are RNA relative enrichment over GFP calculated as percentage of input. The input represents the total RNA detected in each sample and thus, normalises the enrichment to the total RNA expression level. (E + number) = exon related to JunctionSeq annotation, differentially spliced exons are underlined (purple). n = 3 independent biological duplicates. Bars represent mean ± SEM. The results exhibited a specific enrichment of differentially spliced exonic sequences in Ubx WT fraction compared to GFP and Ubx N51A . ( D–K ) Fluorescent protein-RNA interaction assay followed by UV-crosslinking and RNase digestion, performed in vitro with purified proteins namely his-MBP-GFP as control, his-Ubx (WT and N51A) full-length (FL) ( D–G ) and the HD alone his-MBP-Ubx HD (WT and N51A) ( H–K ). Interactions were detected on denaturating gels by Cy3-UTP signal (upper panel), and gels were stained by Coomassie to reveal the protein content (lower panel). Each probe is indicated relative to the genes and exons. Molecular marker is indicated showing the size of each protein. MBP fused proteins are named his-MBP-X, his-fused proteins are named his-X. (L–O) Graphical view showing the quantification of relative RNA-binding of Ubx HD compared to full-length (FL) MBP fused proteins for Chas exon cassette E5 ( L ), constitutive E3, ( M ), pAbp exon cassette E1 ( N ), constitutive E6 ( O ) normalised to Coomassie staining. n = 3 independent biological replicates. Statistical test by one-way ANOVA (* P
Article Snippet: For producing internally labelled RNA, in vitro transcription was performed using the HighYield T7 Cy3 RNA Labelling Kit (Jena Bioscience, RNT-101-CY3) in accordance to the instructions of the manufacturer.
Techniques: In Vivo, In Vitro, Immunoprecipitation, Quantitative RT-PCR, Expressing, RNA Expression, Purification, Staining, Marker, RNA Binding Assay