IDENTIFICATION OF INTERGENIC TRANS-SPLICING IN HUMAN INDUCED PLURIPOTENT STEM CEL

COLLI CAROLINA; MARIA AGUSTINA SCARAFÍA; ALAN MIQUEAS MÖBBS; NATALIA LUCÍA SANTÍN VELAZQUE; CYNTIA ABAN; LUCÍA NATALIA MORO; CARLOS DANIEL LUZZANI; ALEJANDRO DAMIÁN LA GRECA; SANTIAGO GABRIEL MIRIUKA

Congreso; REUNIÓN ANUAL DE SOCIEDADES DE BIOCIENCIA 2019; 2019

“Non-co-linear” (NCL) events are known RNA sequences, topologically inconsistent with their correspondent DNA sequences in the reference genome. Trans-splicing events are a linear type of NCL transcripts, which are formed post-transcriptionally by junction of separate pre-mRNAs. Trans-splicing has been described for various species, even though, their functions remain unclear. However, in human pluripotent stem cell, one RNA trans-splicing event (tsRMST) was described to contribute to the regulation of early lineage differentiation. We aim to identify intergenic trans-splicing events in induced pluripotent stem cell (iPSCs). First, the RNAseq of FN2.1 (iPSCs line developed in our laboratory) was subjected to NCLscan (a pipeline to discover NCL transcripts in human transcriptome) and we observed 1109 NCL events in iPSCs; of these, only 3 events occur between different genes: TIAM2-SCAFA8-1, TIAM2-SCAFA8-2 and ZRANB1-AL731577.2. Then, based on NCL scan data, we designed primers to amplify the junction for these three intergenic events. For this, total iPSCs RNA was treated with or without RNase R for selectively degrade linear RNA, and cDNA was synthesized using random primers or oligo(dT) primers for identifier events poly(A) and non-poly(A). Negative control was made treating total RNA without MMLV, to discard permanent fixture of the genome at the DNA level. Trans-splicing intergenic events were validated by RT-qPCR and its subsequent corroboration in agarose gel. Starting from this we could observe that TIAM2-SCAFA8-1 and TIAM2-SCAFA8-2 were detected by RT-qPCR either random primers or oligo(dT) primers but not remained stable after RNase R treatment. ZRANB1-AL731577.2 was only detected by RT-qPCR using random primers and not remained stable after RNase R treatment. We observed that TIAM2-SCAFA8-1 and TIAM2-SCAFA8-2 linear polyadenylated events, while ZRANB1-AL731577.2 is a non-polyadenylated linear event. In summary, we found potentially but scarce relevant transplicing events in PSC.