Study of TUT tailing on miRNA

Many microRNAs (miRNAs) exist alongside abundant miRNA isoforms (isomiRs), most of which arise from post-maturation sequence modifications, such as 3' uridylation and adenylation. However, the ways in which these sequence modifications affect miRNA function remain poorly understood. To this aim, we have generated single knock-out cell lines of TUT4, TUT7 and TENT2 (TUT2), as well as double knock-out (DKO) and triple knock-out (TKO) cell lines. Here, using these different cell lines, we have discovered that some of the redundant functions and specific functions of each tailing enzyme. Our study provides a comprehensive characterization of tailing on miRNAs. Overall design: Briefly, HEK293 cells and corresponding knock-outs were transfected with several wild-type and catalytic-dead constructs. Transfections were performed using PolyJet™ DNA Transfection Reagent (SignaGen) according to the manufacturer's instructions. 48 hours after transfection, either total or AGO-bound (IP) RNA was obtained using a chloroform-isopropanol extraction. RNA was used to prepare small RNA-seq libraries using QIAseq miRNA Library Kit (Qiagen). Constructs were purified in a 6% (w/v) native acrylamide gel based on the expected product size and purified by ethanol precipitation. Libraries quality was assessed by using Qubit dsDNA HS Assay Kit (ThermoFisher) and Agilent High Sensitivity DNA kit (Agilent). Libraries were pulled together, denaturalized and prepared at a final concentration of 12pM and run on MiSeq or NovaSeq 500 according to the manufacturer specifications. Fastq files were analyzed using a Python-based custom software, QuagmiR (https://cgc.sbgenomics.com/public/apps#nikola_tesic/quagmir/quagmir-1-0/) designed for the optimal mapping and analysis of isomiRs.