Deletion of the predicted cleavage sites within the second 79-nt hallmark sequence of SIDER2 retroposons prevents SIDER2-mediated mRNA decay.

<p>(A) SIDER2 retroposons are generally composed by three conserved motifs; a 18-nt thymidine-rich stretch at the 5'-end corresponding to the former recognition site for an endonuclease encoded by autonomous retroposon elements followed by two tandemly 79-nt sequences (signatures I and II) representing the hallmark of trypanosomatid retroposons. Here the nucleotide sequence of the conserved motifs of the <i>L</i>. <i>major</i> LmjF.36.3810 SIDER2 element present in the 3'UTR is shown. Deletions of 8-nt flanking the two putative cleavage sites (Cl1 and Cl2) (in red) mapped previously within the second signature sequence of the SIDER2-containing LmjF.36.3810 3'UTR [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180678#pone.0180678.ref014" target="_blank">14</a>]. (B) Northern blot hybridization of total RNA isolated from <i>L</i>. <i>major</i> LUC-3810 3'UTR and the deletion mutants LUC-3810 3'UTRΔCl1 and LUC-3810-3'UTRΔCl2 with a LUC-specific radiolabeled probe to assess the effect of cleavage 1 and 2 sites and surrounding nucleotide deletions on <i>LUC</i> mRNA decay. The blot was also hybridized with a neomycin phosphotransferase (NEO) probe to normalize for plasmid copy number and RNA loading. An ethidium bromide staining visualizing rRNA is also shown as an additional loading control. Normalized LUC-3810 3'UTRΔCl1 and LUC-3810-3'UTRΔCl2 mRNA levels relative to the full length LUC-3810 3'UTR (control) are shown below the blot. Hybridization signals were quantified by the ImageQuant 5.2 software and <i>LUC</i> mRNA values were normalized to the <i>NEO</i> mRNA. The Northern blots shown here are representative of two independent experiments yielding comparable results.