Warrander F et al. (2016), lin28 proteins promote expression of 17∼92 fami...

XB-ART-51424

Dev Dyn 2016 Jan 01;2451:34-46. doi: 10.1002/dvdy.24358.

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lin28 proteins promote expression of 17∼92 family miRNAs during amphibian development.

Warrander F , Faas L , Kovalevskiy O , Peters D , Coles M , Antson AA , Genever P , Isaacs HV .

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BACKGROUND: Lin28 proteins are post-transcriptional regulators of gene expression with multiple roles in development and the regulation of pluripotency in stem cells. Much attention has focussed on Lin28 proteins as negative regulators of let-7 miRNA biogenesis; a function that is conserved in several animal groups and in multiple processes. However, there is increasing evidence that Lin28 proteins have additional roles, distinct from regulation of let-7 abundance. We have previously demonstrated that lin28 proteins have functions associated with the regulation of early cell lineage specification in Xenopus embryos, independent of a lin28/let-7 regulatory axis. However, the nature of lin28 targets in Xenopus development remains obscure. RESULTS: Here, we show that mir-17∼92 and mir-106∼363 cluster miRNAs are down-regulated in response to lin28 knockdown, and RNAs from these clusters are co-expressed with lin28 genes during germ layer specification. Mature miRNAs derived from pre-mir-363 are most sensitive to lin28 inhibition. We demonstrate that lin28a binds to the terminal loop of pre-mir-363 with an affinity similar to that of let-7, and that this high affinity interaction requires to conserved a GGAG motif. CONCLUSIONS: Our data suggest a novel function for amphibian lin28 proteins as positive regulators of mir-17∼92 family miRNAs.

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Species referenced: Xenopus
Genes referenced: lin28a lin28b

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	Figure 1. A: Western blot analysis of endogenous lin28a and lin28b expression in embryos injected with a total of 12.5 ng/embryo of lin28 MOs in the compound knockdown compared with CMO‐injected and uninjected control embryos at stage 16. GAPDH was used as a loading control. B: Scale diagram showing the genomic organisation of Xenopus mir‐17∼92 and mir‐106∼363 clusters. Coloured boxes indicate pre‐miRNA sequences, with each colour corresponding to paralog groupings based on seed sequence. Where known, the black and grey boxes, respectively, represent the major and minor forms derived from a common precursor. C: qRT‐PCR was performed on embryos injected with 10 ng each/embryo of lin28a1, a2 and b MOs and control embryos, at stage 10.5. Fold change in expression of miRNAs is shown compared with controls and normalised using U6 by the 2‐ΔΔCt method. Fold change is given as average of 3 biological replicates, with error bars representing SE. D: Predicted secondary structure of Xenopus pre‐mir‐363. The sequences of mature mir‐363‐5p, mir‐363‐3p, and the putative lin28 binding site are indicated.
	Figure 2. A: Western blot analysis of lin28 proteins at stage 10.5 in control embryos and embryos injected with 1 ng of mRNAs coding for either lin28a1, lin28a2 or lin28b. GAPDH was used as a loading control. B: Phenotype of control embryos and embryos overexpressing either lin28a1, lin28a2, or lin28b at stage 38. C: qRT‐PCR was performed on embryos injected with control embryos and either 1 ng of mRNAs coding for lin28a1, a2 or b MOs at stage 10.5. Fold change in expression of miRNAs is shown compared with controls and normalised using U6 by the 2‐ΔΔCt method. Fold change is given as average of 3 biological replicates, with error bars representing SE.
	Figure 3. A: Scale diagram of the Xenopus proteins used in this study. Cold shock domains are shaded magenta and zinc knuckles green. B: EMSA performed with 32P‐labelled L‐let‐7g and indicated concentrations of human recombinant LIN28A protein, either full‐length or truncated (rt). Arrows indicate labelled RNA (blue) and LIN28A‐RNA complex (red). C: EMSA performed with 32P‐labelled L‐let‐7g and indicated concentrations of Xrt‐lin28a. Gel shown is representative of n = 3. Arrows indicate labelled RNA (blue) and lin28‐RNA complex (red). Band intensities were quantified from three independent experiments and the proportion bound was calculated. Data were fit by nonlinear regression as described in Materials and Methods. Bmax = 1.017. D: EMSA performed with 32P‐labelled L‐mir‐138 and indicated concentrations of Xrt‐lin28a. Arrows indicate RNA and lin28a‐RNA complex. Gel shown is representative of n = 3. Arrows indicate labelled RNA (blue) and lin28‐RNA complex (red).
	Figure 4. A: EMSA performed with 32P‐labelled L‐mir‐363 and indicated concentrations of Xrt‐lin28a. Gel shown is representative of n = 3. Band intensities were quantified from three independent experiments and the proportion bound was calculated. Data were fitted by nonlinear regression as described in Materials and Methods. Bmax = 0.962. B: EMSA performed with 32P‐labelled L‐mir‐363 and 1 μM of Xrt‐lin28a (except for RNA only lane). Arrows indicate RNA and lin28a‐RNA complex. Reactions were competed with unlabelled RNA of L‐mir‐363 or L‐mir‐138 in excess levels as indicated. Band intensities were quantified and proportion of RNA bound was calculated. Gel shown is representative of n = 2. Arrows indicate labelled RNA (blue) and lin28‐RNA complex (red). C: EMSA performed with 32P‐labelled mL‐mir‐363 and indicated concentrations of Xrt‐lin28a. Gel shown is representative of n = 3.
	Figure 5. A,B: EMSAs performed with 32P‐labelled L‐mir‐363 and embryo extract from uninjected controls or embryos injected with 1 ng of either (A) lin28a1 or (B) lin28a2. Embryo extract was used at 1/16 dilution for lanes 2–3, 5–6, and at 1/32 dilution for lower concentration of overexpressing extract in lane 4. Arrows indicate unbound RNA (blue), lin28‐RNA complex (red), and supershift complex of antibody‐lin28‐RNA (green). + Ab = 1/20 dilution α‐lin28a, + ser = 1/20 dilution preimmune bleed serum, both incubated with protein on ice for 20 min before addition of probe. C,D: EMSAs performed with 32P‐labelled pre‐mir‐363 and embryo extract from uninjected controls or embryos injected with 1 ng of either (C) lin28a1 or (D) lin28a2. Embryo extract was used at 1/8 dilution for lanes 2–3, 5–6, and at 1/16 dilution for lower concentration of overexpressing extract in lane 4. Arrows indicate unbound RNA (blue), lin28‐RNA complex (red), and supershift complex of antibody‐lin28‐RNA (green). + Ab = 1/20 dilution α‐lin28a, + ser = 1/20 dilution preimmune bleed serum, both incubated with protein on ice for 20 min before addition of probe.
	Figure 6. A: Developmental time course for expression of pri‐miR‐17∼92 and pri‐miR‐106∼363 was undertaken using RT‐PCR. L8 was used as a loading control. Image is representative of n=2. miR‐17‐92 = 669 bp, miR‐106‐363 = 639 bp, L8 = 435 bp. B: In situ hybridisations showing expression of pri‐miR‐17∼92 and pri‐miR‐106∼363 RNAs in early development. Vegetal views of early gastrula stage 10.5 embryos, with the dorsal side is to the top. Arrows indicate the dorsal blastopore lip. C: In situ hybridisation using an anti‐sense LNA probe showing mir‐363‐3p expression in early development. Vegetal views of gastrula stages 10 and 10.5 are shown, with dorsal‐side to the top. An animal to vegetal bisect of a stage 10 embryo is shown with the animal hemisphere to the top and dorsal to the right. A dorsal view of a late neurula stage 19 embryo, anterior to the left. Plane of bisection (black line), dorsal blastopore lip (bl) and neural plate (np) are indicated. D: In situ hybridisation using an anti‐sense LNA probe showing mir‐363‐5p expression in early development. Vegetal views of gastrula stages 10 and 11 are shown, with dorsal‐side to the top. An animal to vegetal bisect of a stage 10 embryo is shown with the animal hemisphere to the top and dorsal to the right. A dorsal view of a late neurula stage 19 embryo, anterior to the left. Plane of bisection (black line) is indicated.

References [+] :

Branney, Characterisation of the fibroblast growth factor dependent transcriptome in early development. 2009, Pubmed, Xenbase