1Strand selection is a critical step in microRNA (miRNA) biogenesis. 2Although the dominant strand may alter depending on cellular contexts, 3 the molecular mechanism and physiological significance of such 4 alternative strand selection (or "arm switching") remain elusive. Here we 5 find mir-324 as one of the strongly regulated miRNAs by arm switching, 6 and identify terminal uridylyl transferases TUT4 and TUT7 as the key 7 regulators. Uridylation of pre-mir-324 by TUT4/7 re-positions DICER on 8 the pre-miRNA and shifts the cleavage site. This alternative processing 9 produces a duplex with a different terminus, from which the 3 ′ strand (3p) 10 is selected instead of the 5 ′ strand (5p). In glioblastoma, the TUT4/7 and 11 3p levels are upregulated while the 5p level is reduced. Manipulation of 12 the strand ratio is sufficient to impair glioblastoma cell proliferation. This 13 study uncovers a role of uridylation as a molecular switch in alternative 14 strand selection and implicates its therapeutic potential.
Keywords16 miRNA, miRNA biogenesis, arm switching, strand selection, miRNA 17 tailing, uridylation, TUTase, DICER, miR-324, glioblastoma 18 2/51 103In this study, we aimed to identify miRNAs that undergo conserved arm 104 switching and uncover its molecular mechanism. We find that mir-324 is 105 the most prominently regulated miRNA through arm switching and that 106 the arm switching is actively controlled by uridylation enzymes.
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RESULTS
108Arm switching of miR-324 109 In order to gain molecular insights into miRNA arm switching, we first 110 searched for miRNAs that exhibit significant alterations in their strand 111
5/51We are grateful to Eunji Kim for cloning and mutagenesis of expression 685 constructs. We also thank Yoonseok Jung, Dongwan Kim, Hyunjoon 686