RNA-seq profiling following a 5' isomiR  transfection
Annotated canonical miRNAs (anomiRs) are critical regulators of COVID-19 pathogenesis. 5' isomiRs, which exhibit a shifted 5' end and distinct seed region compared to their cognate anomiRs, represent an essential component of the miRNAome. However, little is known about their repertoire and expression dynamics following SARS-CoV-2 infection. Herein, we performed an integrative analysis to investigate the expression and regulation of 5' isomiRs in SARS-CoV-2-infected human lung cells. Using Argonaute (AGO) immunoprecipitated small-RNA sequencing data, we established a repertoire of AGO-loaded 5' isomiRome encompassing 826 5' isomiRs from SARS-CoV-2-infected lung cells. A total of 54 5' isomiRs were differentially expressed, with the majority showing dysregulation 24 hours after SARS-CoV-2 infection. Compared to anomiRs, 5' isomiRs were preferentially induced at 24 hours post SARS-CoV-2 infection, including six 5' isomiRs derived from miR-4485 precursor. By focusing on a 5' isomiR of miR-4485-3p exhibiting one nucleotide downstream shift of the 5' end (miR-4485-3p|+1), we demonstrated that miR-4485-3p|+1 overexpression significantly repressed genes involved in cellular processes relating to cell cycle, cytoplasmic translation, and carbohydrate derivative metabolic process. Importantly, transcriptome integration further revealed that these cellular processes repressed by miR-4485-3p|+1 overexpression were strongly downregulated at 24 hours following SARS-CoV-2 infection. Moreover, 26 out of 43 (60.5%) hub genes co-repressed by miR-4485-3p|+1 and SARS-CoV-2 infection in these three cellular processes have reported association with COVID-19 in the literature. Taken together, our findings reveal the repertoire and expression alterations of AGO-loaded 5' isomiRs in SARS-CoV-2-infected lung cells. The identified regulatory effects of miR-4485-3p|+1 highlight potential roles of 5' isomiRs in COVID-19 pathogenesis. Overall design: RNA-Seq profiling from Calu-3 cells  transfected with miR-4485-3p|+1 mimics or control mimics