In-cell RNA secondary structure analysis of functional SINE transcripts

SINEUPs are long non-coding RNAs which contain a functionally crucial SINE repeat element and positively regulate target mRNA translation at post-transcription level. To study secondary (2D) structure of these functional SINE-derived RNAs, various SINEUP-GFP plasmids containing different mouse SINEB2 repeat elements or human SINE FRAM repeat were transfected along with sense EGFP plasmid in HEK293T/17 cells. Total RNA in transfected cells were probed at flexible regions with NAI-N3 reagent and icSHAPE (in-vivo click selective hydroxyl acylation and profiling experiment) libraries were prepared. To further understand the structure dynamics of SINE transcripts in subcellular locations, cells were fractionated in nuclear and cytoplasmic fractions after 24 h of transfection and fraction-specific icSHAPE libraries were constructed. Overall design: In-vivo icSHAPE libraries for mouse (SINEB2) and human (FRAM) SINE-derived transcripts from whole cell lysate. Two biological replicates (rep) of each NAI-N3 and DMSO (mock) treated samples. For RNA structure study in cellular compartments, mouse SINEB2 and human FRAM containing functional SINEUP-GFPs were transfected in HEK293T/17 cells and pooled together (SINE) to treat with either NAI-N3 or DMSO and then separated in nuclear (Nuc) and cytoplasmic (Cyto) fractions followed by standard icSHAPE library preparation.