BioProject

MicroRNA (miRNA) biogenesis begins with Drosha cleavage, the fidelity of which is critical for downstream processing and mature miRNA target specificity. More...

MicroRNA (miRNA) biogenesis begins with Drosha cleavage, the fidelity of which is critical for downstream processing and mature miRNA target specificity. To understand how pri-miRNA sequence and structure influence Drosha cleavage, we studied the processing of three pri-miR-9 paralogs (pri-miR-9-1, pri-miR-9-2 and pri-miR-9-3), which encode the same miRNA but differ in the surrounding scaffold. We show that pri-miR-9-1 has a unique Drosha cleavage profile due to its kinked tertiary structure. Cleavage of pri-miR-9-1, but not pri-miR-9-2 or pri-miR-9-3, generates an alternative-miR-9 with a shifted seed sequence that expands the scope of its target RNAs. Here, we report the the miRNA-seq data of HEK293T and HeLa cell lines transfected with different pri-miR-9 constructs. Overall design: Briefly, HEK293 and HeLa cells were transfected with several wild-type and chimeric pri-miR-9 constructs. Transfections were performed using PolyJet™ DNA Transfection Reagent (SignaGen) according to the manufacturer's instructions. 48 hours after transfection total RNA was obtained using a chloroform-isopropanol extraction. 5µg of total RNA was ligated with RNA 3’ adaptor using T4 RNA Ligase 2 - truncated (NEB), in the presence of RNase Inhibitor (NEB). RNA 5’ adaptor was ligated using T4 RNA Ligase 1 - high concentration (NEB) and 10 mM ATP. Ligated small RNAs were reverse transcribed using SuperScript® IV Reverse Transcriptase (Thermo-Fisher). Small RNA library cDNA was amplified and indexed using Phusion® High-Fidelity DNA polymerase (NEB). 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 Reagent Kit v3 (Illumina) according to the manufacturer specifications. Adaptors, primers sequences and detailed protocol temperatures can be found at Supplementary Table S2. Fastq files were analyzed using a Python-based custom software, QuagmiR (https://github.com/Gu-Lab-RBL-NCI/QuagmiR) designed for the optimal mapping and analysis of isomiRs. Less...