STUDY OF NON-CO-LINEAR EVENTS IN HUMAN PLURIPOTENT STEM CELL

CAROLINA COLLI; GUADALUPE AMIN; MARIA AGUSTINA SCARAFÍA; ALAN MIQUEAS MÖBBS; LUCÍA NATALIA MORO; ARIEL WAISMAN; CARLOS DANIEL LUZZANI; ALEJANDRO DAMIÁN LA GRECA; SANTIAGO GABRIEL MIRIUKA

Congreso; REUNIÓN ANUAL DE SOCIEDADES DE BIOCIENCIA 2021; 2021

RNA sequences topologically inconsistent with the correspondentDNA sequence in the reference genome are known as “non-co-linearevents” (NCLe). These events can be linear (trans-splicing) orcircular (circRNA) and both are post-transcriptional events. In humanpluripotent stem cells (iPSC), NCLe were described to contributeto the regulation of early lineage differentiation; in particularcircRNAs formed by quaking protein (QKI) 5 were described as necessaryfor cardiac differentiation. Trans-splicing events are formedby separate pre-mRNA with inverted and repeated sequences (Alu)while circRNA originate from a backspliced junction in a pre-mRNA.The aim of this work was to characterize NCLe and the role of QKI5/6/7 in circRNA formation in iPSC. RNAseq data from an iPSC linewas analyzed with NCLscan pipeline, revealing 1109 NCLe, amongwhich 3 occurred between different genes. To validate these intergenicjunctional events, we amplified them by RTq-PCR with specificprimers and sequenced the product, corroborating that these alternativejunctional organizations were not informatic artifacts. PCRon purified DNA showed they are not genomic rearrangements. Furthermore,using magnetic oligo dT beads we also demonstrated thatthe 3 events are polyadenylated and they are sensitive to degradationwith RNase R, thus linearly conformed. In parallel, we designedRNA guides to knock out QKI in FN2.1 and H9 using CRISPR/Cas9.PCR and immunofluorescence analysis revealed the absence of thetarget, indicating that the strategy was successful. In conclusion,we identified NCLe in an iPSC line and characterized 3 differenttrans-splicing. We were also successful in preparing knockout linesfor QKI to assess its role in differentiation as well as the circRNAsdependent on its function. In the future we plan to assess theseknockout lines by RNAseq and functionality of the trans-splicing withCRISPR/Cas13 and characterize using northern blot.