Nucleolin HITS-CLIP, RNA-Seq and Ribo-Seq upon inhibition of 5'-tRFCys

Expression of 5'-tRFCys is significantly upregulated during breast cancer metastasis and suppression of 5'-tRFCys significantly reduced metastatic lung colonization of breast cancer. We identified Nucleolin (Ncl) as the direct binding partner of 5'-tRFCys. To identify the molecular mechanism underlying 5'-tRFCys promotion of breast cancer, we performed Ncl HITS-CLIP, RNA-Seq, Ribosome profiling (Ribo-Seq) upon inhibition of 5'-tRFCys. These analyses reveal that 5'-tRFCys enhances Ncl binding to a subset of transcripts, resulting in increased transcript stability of those transcripts and expression. To identify tRNA fragments (tRFs) that regulate metastasis, tRFs were profiled from non-metastatic 67NR, poorly-metastatic 4TO7 or highly-metastatic 4T1 breast cancer cells using small RNA-Seq (sRNA-Seq). To identify the direct targets of 5'-tRFCys, Ncl HITS-CLIP was performed upon 5'-tRFCys suppression. To determine the impact of 5'-tRFCys on its target transcripts, 4T1 cells were transfected with a scrambled control or 5'-tRFCys antisense LNA oligo and RNA-Seq as well as Ribo-Seq were performed.