Alu Element Identification and Analysis

The Perl program “Alu_Mask” was written and used together with REPEATMASKER (http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker) to define Alu elements. The Perl program “RNA_RNA_anneal” was generated to predict intermolecular duplexes between Alu elements within lncRNAs and proven or putative SMD targets. Duplexes were then validated using RNA structure 4.6 (http://rna.urmc.rochester.edu/rnastructure.html), which provides folding free energy changes. Human HeLa or HaCaT cells were transiently transfected with the specified plasmids or siRNAs as described1 (link). For mRNA half-life measurements, HeLa Tet-Off cells (Clontech) were utilized. If formaldehyde-crosslinked, cells were sonicated six times for 30 sec to facilitate lysis, and crosslinks were subsequently reversed by heating at 65°C for 45 min after IP. IP was performed as described1 (link). Protein was purified and Western blotting was performed as noted1 (link). RNA was purified from total, nuclear or cytoplasmic cell fractions or immunoprecipitated from total-cell lysates as reported1 (link). Poly(A)⁺ RNA was extracted from total-cell RNA using the Oligotex mRNA Mini Kit (Qiagen). RT-sqPCR and RT-qPCR were as described1 (link), except when RT was primed using oligo(dT)₁₈ rather than random hexamers. The RNase protection assay employed the RPA III RNase Protection Assay Kit (Ambion) and uniformly labeled RNA probes that were generated by transcribing linearized pcDNA3.1(+)/Zeo_Chr11_66193000-66191383 in vitro using α-[P32]-UTP (Perkin Elmer) and the MAXIscript Kit (Ambion). Cells were visualized using a Nikon Eclipse TE2000-U inverted fluorescence microscope and, for phase microscopy, a 480-nm excitation spectra. Images were captured utilizing TILLVISION software (TILL Photonics). Scrape injury repair assays were essentially as published21,22. All data derive from at least three independently performed experiments that did not vary by more than the amount shown, and p values for all RT-sqPCR results were <0.05.