Journal: Nucleic Acids Research

Article Title: CFIm-mediated alternative polyadenylation remodels cellular signaling and miRNA biogenesis

doi: 10.1093/nar/gkac114

Figure Lengend Snippet: CFIm KD increases the activity of miRNAs. ( A ) Genome browser tracks showing the coverage of DICER1 TE (bottom track) by RNA-seq reads from two replicate experiments for each condition, with the two PAS that were quantified for this gene marked by black lines. The conditions are color-coded (as in Figure ) and also indicated on the y-axis. Y-axis shows the smoothened number of reads mapping along the TE, calculated by the GViz R package. ( B ) RNA fluorescence in situ imaging of DICER1 isoforms in Control and CFIm25 KD HEK293 cells with probes corresponding to the common region of the long and short 3′ UTRs (red) or to the region between the proximal and distal cleavage sites, thus present exclusively in the long 3′ UTR (green). Nuclei are marked with DAPI. Zoom-ins of the regions marked with dashed boxes are further shown both with the individual and merged channels. A snapshot of a digital representation of the actual image as processed in IMARIS is also depicted for reference. ( C ) Quantification of the copy number of the long and short 3′ UTR isoforms of DICER1 in the nucleus (left plot) and cytoplasm (right plot) of Control, CFIm25 and CFIm68 KD cells. Colocalization of the red and green signals reveals the presence of the long 3′ UTR isoform (yellow) whereas the signal from the red probe only reveals the presence of the shorter 3′ UTR isoform. mRNA copy numbers were estimated separately from the nucleus (overlapping with DAPI) and cytosol. Segregation of the signal was performed with IMARIS (see Methods). ( D ) Representative western blot showing the DICER1 expression in the Control, CFIm25 and CFIm68 KD cells. The quantification is relative to GAPDH. ( E ) qPCR measurements of let-7, miR-92a, miR-16 and miR-19b expression in CFIm25/68 KD cells relative to Control. ΔΔct values were calculated relative to U6 snRNA and then relative to the Control cells (where the ratio was set to 1). ( F ) Normalized Renilla luciferase expression of reporter mRNAs carrying binding sites for miR-16 and miR-92a in their 3′ UTRs, in Control, CFIm25 and CFIm68 KD cells, respectively. The Firefly luciferase expressed from the same construct was used as normalization control.

Article Snippet: Detection and analysis of spots were performed using automated pipelines developed in image analysis software IMARIS (BITPLANE).

Techniques: Activity Assay, RNA Sequencing, Fluorescence, In Situ, Imaging, Control, Western Blot, Expressing, Luciferase, Binding Assay, Construct