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

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

Mar del Plata

Congreso; LXVI Reunion Anual de Sociedades de Biociencias; 2021

Sociedad Argentina de Investigacion Clinica (SAIC)

RNA sequences topologically inconsistent with the correspondent DNA sequence in the reference genome are known as “non-co-linear events” (NCLe). These events can be linear (trans-splicing) or circular (circRNA) and both are post-transcriptional events. In human pluripotent stem cells (iPSC), NCLe were described to contribute to the regulation of early lineage differentiation; in particular circRNAs formed by quaking protein (QKI) 5 were described as necessary for cardiac differentiation. Trans-splicing events are formed by 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 QKI 5/6/7 in circRNA formation in iPSC. RNAseq data from an iPSC line was analyzed with NCLscan pipeline, revealing 1109 NCLe, among which 3 occurred between different genes. To validate these intergenic junctional events, we amplified them by RTq-PCR with specific primers and sequenced the product, corroborating that these alternative junctional organizations were not informatic artifacts. PCR on purified DNA showed they are not genomic rearrangements. Furthermore, using magnetic oligo dT beads we also demonstrated that the 3 events are polyadenylated and they are sensitive to degradation with RNase R, thus linearly Conformed. In parallel, we designed RNA guides to knock out QKI in FN2.1 and H9 using CRISPR/Cas9. PCR and immunofluorescence analysis revealed the absence of the target, indicating that the strategy was successful. In conclusion, we identified NCLe in an iPSC line and characterized 3 different trans-splicing. We were also successful in preparing knockout lines for QKI to assess its role in differentiation as well as the circRNAs dependent on its function. In the future we plan to assess these knockout lines by RNAseq and functionality of the trans-splicing with CRISPR/Cas13 and characterize using northern blot.