Xenbase Image ID: 145213

Fig. 4. Myt1 mRNA translation is up-regulated by a 5′ variant xlmiR16. (A) Myt1 3′-UTR has seven putative xlmiR16 sites (dashes). Mutation of the site at 1,284 nt (mt3Myt1 3′-UTR) in the 528-nt or full-length Myt1 3′-UTR failed to demonstrate translation activation and was unaffected by xlmiR16 presence or depletion (SI Methods; siRNAs antisense to pre-miR16 reduced mature xlmiR16 after 6 h, likely because of interference with processing or stability instead of cleavage activity; Fig. S4A). Translation activation is restored by addition of a compensatorily mutated xlmt3miR16 that can base pair to the mutated site (SI Methods). Both B1/xlmiR16 (shown) and B3/xlmiR16 (Fig. 4C shows base pairing) are able to rescue. (B) Northern analyses of Firefly reporters containing the 528-nt Myt1 3′-UTR and mt3Myt1 3′-UTR and REN RNA levels after treatment with si-pre-miR16 with or without mt3xlmiR16 add-back. (Right) Endogenous Myt1 mRNA levels with or without si-pre-miR16 treatment; U6 RNA is a loading control. (C) Splint ligation reactions (SI Methods) containing the labeled 14-nt acceptor oligo and RNAs isolated from anti-AGO2 or -FLAG (control) immunoprecipitates (Ip). XlmiR16 variants that are successively truncated by one nucleotide at the 5′ end (B1/xlmiR16-B4/xlmiR16) were detected using DNA bridge oligos B1-B4, complementary to the first 16 nt of the predicted microRNAs. The predicted full-length form, B1/xlmiR16, is only faintly visible in the sample immunoprecipitated with anti-AGO2, suggesting that it is less abundant than B3/xlmiR16, which is revealed by the B3 bridge oligo to be an abundant microRNA in anti-AGO2 immunoprecipitates. Lanes marked B1/xlmiR16 and B3/xlmiR16 show synthetic miR16 RNAs as size controls. Underlined nucleotides are required for interaction with the Myt1 3′-UTR. Base pairing between Myt1 target site and B1/xlmiR16 or B3/xlmiR16 are shown. (D) Oocytes treated with mock (let-7a) or si-pre-miR16 were coinjected with synthetic xlmiR16 forms to assess rescue of full-length Myt1 3′-UTR reporter translation. Firefly luciferase values were normalized to REN. B1/xlmiR16 undergoes trimming to the B3/xlmiR16 form and rescues translation to a lesser extent than B3/xlmiR16. Image published in: Mortensen RD et al. (2011) Copyright © 2011. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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