Project description:As HNRNPD regulates the alternative splicing of hundreds of genes, we sought to investigate whether HNRNPD could regulate the biogenesis of circRNAs. To identify the effect of HNRNPD on circRNAs and linear RNAs, we performed circRNA and linear RNAs (ployA enrichment RNA and lncRNA) sequencing in HNRNPD knockout and wild-type HEK293T cells. We also inserted 3FHBH (3xFLAG, Histidine, Biotin, and Histidine) into the HNRNPD genome to obtain HEK293T_HNRNPD_3FHBH cells by using CRISPR-Cas9 technology, which was used to ascertain the direct HNRNPD targeting RNA sequence with the help of FLASH sequencing (Fast Ligation of RNA after some sort of Affinity Purification for High-throughput Sequencing). To evaluate the impacts of HNRNPD deficiency on circRNA formation, we captured nascent RNA in HEK293T and HNRNPD knock-out HEK293T cells by immunoprecipitation of 5-ethyluridine (EU) labeling. Comparative analysis of these data, we found the higher propensity of binding to introns of HNRNPD was related to inhibiting circRNA biogenesis.
Project description:As HNRNPD regulates the alternative splicing of hundreds of genes, we sought to investigate whether HNRNPD could regulate the biogenesis of circRNAs. To identify the effect of HNRNPD on circRNAs, we performed circRNA sequencing in HNRNPD knockout and wild-type SW839 cells. In addition, to evaluate the impacts of HNRNPD deficiency on circRNA formation, we captured nascent RNA in SW839 and HNRNPD knock-out SW839 cells by immunoprecipitation of 5-ethyluridine (EU) labeling. Comparative analysis of these data, we found the higher expression level of circRNAs in HNRNPD KO SW839 cells.The HNRNPD could influence the circRNA biogenesis.
Project description:Goal: elucidate transcriptomic changes upon knock-out of components of the FERRY complex Methods: RNA extraction from HeLa wildtype and fy-1, fy-2, fy-4 and fy-5 knock-out celllines and subsequent RNASeq Results: We observed differences in the transcriptome of all four knock-out cell lines Conclusions: In the Analysis we focused on genes that were differentially expressed in all four KO cell lines or upon KO of fy-1 and fy-2.