MicroRNAs (miRNAs) are well known to target 3′ untranslated regions (3′ UTRs) in mRNAs, thereby silencing gene expression at the post-transcriptional level. Multiple reports have also indicated the ability of miRNAs to target protein-coding sequences (CDS); however, miRNAs have been generally believed to function through similar mechanisms regardless of the locations of their sites of action. Here, we report a class of miRNA-recognition elements (MREs) that function exclusively in CDS regions. Through functional and mechanistic characterization of these ‘unusual’ MREs, we demonstrate that CDS-targeted miRNAs require extensive base-pairing at the 3′ side rather than the 5′ seed; cause gene silencing in an Argonaute-dependent but GW182-independent manner; and repress translation by inducing transient ribosome stalling instead of mRNA destabilization. These findings reveal distinct mechanisms and functional consequences of miRNAs that target CDS versus the 3′ UTR and suggest that CDS-targeted miRNAs may use a translational quality-control-related mechanism to regulate translation in mammalian cells.
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All deep-sequencing data generated in this study have been deposited in the Gene Expression Omnibus database under accession number GSE115146. Source data for the graphs in all figures are available online. Other data are available from the corresponding author upon reasonable request.
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This work was supported by grants from the Ministry of Science and Technology of China (2017YFA0506400, Y. Zhou and X.-D.F.), the National Natural Science Foundation of China (31670827 and 91640115, Y. Zhou), the Chinese 111 program grant (B06018, X.-D.F.), Chinese Academy of Science foundation grant (22KJZD-EW-L12, X.Z.) and NIH grants (HG004659, GM049369 and GM052872, X.-D.F.). pRL-SV40 plasmids carrying IRESs from EMCV, HCV, and CrPV were generous gifts from M. Bushell (Medical Research Council Toxicology Unit, UK).
The authors declare no competing interests.
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Integrated supplementary information
a,b, Western blotting analysis of miR-20a target genes in response to increasing doses of miR-20a mimic (a) or antagomir (b). c,d, RT-qPCR analysis of miR-20a levels in HeLa cells transfected with miR-20a mimic (c) or antagomir (d). e,f, RT-qPCR analysis of DAPK3 mRNA levels in HeLa cells transfected with miR-20a mimic (e) or antagomir (f). g, Luciferase reporter constructs containing the miR-20a MRE from DAPK3 (native) in Renilla 5’ UTR or mutated to restore base-pairing in the 5’ end (seed) coupled with progressive mismatches (3MM+seed) in the 3’ end. h,i, Results of the luciferase reporter assays (h) and quantification of luciferase mRNA (i). j,k, mRNA levels of luciferase reporters containing individual MREs from 4 indicated genes that function in both CDS and 3’UTR (j) or those that function only in CDS (k), as shown in main Fig. 2e,f. l,m, Luciferase activities from the reporters containing individual MREs from 4 indicated genes that function only in CDS in response to co-transfected Let-7b mimic (l) or antagomir (m). NC: Non-specific oligo control; SC: Scrambled RNA oligo control. n, Western blotting analysis of three additional Let-7b target genes. The three gene products were analyzed in HeLa cells transfected with non-specific RNA oligonucleotide control (NC), Let-7b mimic, or antagomir, as indicated. Actin served as loading control. The data in c,d,e,f,h,i,j,k,l,m were based on 3 independent experiments. *p<0.05; **p<0.01; ***p<0.001 (Student’s t-test).
Supplementary Figure 2 Mutation analysis of multiple Let-7b MREs in CDS regions of individual genes.
a,b,c,d, Deduced base-paring between Let-7b and its targets in individual genes that function only in CDS, as described in main Fig. 2f. A panel of mutants in each targeting site is also shown with calculated base-pairing potential (∆G). The constructs on the left of each panel contain progressive mutations in 3’ side and the constructs on the right of each panel carry progressive mutations in 3’ side with restored base-pairing in 5’ seed (red-labeled nucleotides). e,f, Luciferase activities of individual reporters containing different mutations, without (e) or with restored seed (f), as indicated. The data in e and f were based on 3 independent experiments. *p<0.05; **p<0.01; ***p<0.001 (Student’s t-test).
Supplementary Figure 3 Luciferase activities and mRNA levels of the reporters with or without restored seed.
a, Constructs containing various MREs after restoring seed base pairing (right) inserted in 3’UTR. b,c, Luciferase activities (b) and mRNA levels (c) in HeLa cells transfected with seed restored constructs in a. d, Constructs containing various ERAP2-derived MREs with decreasing base-pairing potentials (∆G) in 5’ seed inserted in CDS. e, Luciferase activities (upper panel) and mRNA levels (lower panel) in HeLa cells transfected with the constructs in d. The data in b,c,e were based on 3 independent experiments. *p<0.05; **p<0.01; ***p<0.001 (Student’s t-test).
Supplementary Figure 4 Functional test for Ago2 or GW182 dependency and for CDS-targeted miRNA to induce translation abortion.
a, One miR-20a MRE and four Let-7b MREs were tested in wild-type or Ago2 knockout MEFs. Note that Ago2 knockout MEFs contain a small population of undeleted cells, which likely contributed to the basal level of Ago2, as shown by western blotting on top. The MREs inserted in both CDS (black bar) or 3’UTR (grey bar) were tested. b, One miR-20a MRE and four Let-7b MREs were tested in non-specific siRNA-treated (NC) or siGW182-treated HeLa cells. Knockdown efficiency was determined by western blotting, as shown on top. The MREs inserted in both CDS (black bar) or 3’UTR (gray bar) were tested. The data shown in a and b were based on 3 independent experiments. *p<0.05; **p<0.01; ***p<0.001 (Student’s t-test). c, Western blotting analysis of endogenous proteins in non-specific siRNA-treated (-) or siGW182-treated (+) HeLa in response to miR-20a mimic (left) or antagomir (right). d, Repeated experiments as in main Fig. 4b. Western blotting analysis of Ago2 and GW182 associated with the Renilla luciferase reporter carrying perfect seed base-pairing (“seed”) or imperfect seed with or without additional 3nt mismatches in 3’ end (“3p3MM+seed” and “3p3MM”) in CDS or 3’UTR. Equal amounts of captured reporter mRNAs were detected by RT-PCR and the Firfly reporter served as a negative control. e, Repeated experiments as in main Fig. 5b. Western blotting of the FLAG-tagged luciferase reporter proteins expressed in transfected HeLa cells. The second to last lane was from cells co-transfected with the reporter containing the miR-20a MRE and the miR-20a antagomir. f, Repeated experiments as in main Fig. 5c. Western blotting of GFP-2A-MRE-2A-mCherry reporter proteins expressed in transfected HeLa cells. Co-transfected FLAG-tagged Renilla served as a loading control.
a, The Pearson coefficient between two biological replicates of Ago2 eCLIP-seq on HeLa cells before (left) and after miR-20a overexpression (right). b, Percentage of sequences underlying Ago2 eCLIP peaks in 3’UTR that can base pair with miRNA seed. Left, Enriched Ago2 peaks in 3’UTR for top 20 mostly expressed miRNAs in comparison with non-enriched and random sites. Right, miR-20a induced Ago2 peaks in 3’UTR compared to uninduced and random sites. c, Meta-profiles of Ago2 binding on miR-20a induced (left) and uninduced (right) sites. d, Boxplots of ΔG between miR-20a and sequences underlying miR-20a induced Ago2 peaks (red) in CDS and 3’UTR in comparison with uninduced (blue) and random (gray) sites. Data from two independent eCLIP replicates are separately displayed. The median value for each group is shown with a horizontal gray line. Filled boxes extend from the first to the third quartile. The upper/lower whisker extends from the hinge to the highest/lowest value that is within 1.5 * IQR of the hinge, where IQR is the inter-quartile range. Data beyond the end of the whiskers are outliers and plotted as points. *p<0.05; **p<0.01; ***p<0.001 (Student’s t-test). e, The Pearson coefficient among three Ribo-seq and two RNA-seq experiments under before (WT) and after miR-20a overexpression (OE). f, Length distribution of ribosome protected fragments (RPFs) from three repeats under WT and OE conditions. g, The 3nt periodicity of RPFs with different lengths. h,i, Meta-gene analysis of RPFs on miR-20a induced Ago2 peaks (left) in comparison with uninduced Ago2 peaks (middle) and random sites (right). Data from two independent Ago2 eCLIP experiments (h, Repeat 1; i, Repeat 2) were used to perform the meta-gene analysis. See the data with combined data in main Fig. 6d.
a, Luciferase activities (upper panel) and relative mRNA levels determined by real-time RT-PCR (lower panel) in three stable HeLa cell lines expressing the luciferase reporter carrying the miR-20a MRE in different locations (note that, as shown in Fig. 2a, the native MRE is non-functional in 3’UTR, which served as control). b,c,d, Polysome profiling by sucrose gradients on the three stable HeLa cell lines. OD260 traces are shown below individual constructs and the distribution of mRNAs from individual reporters and the endogenous Actin gene is shown at bottom. The mRNA levels were determined by real-time RT-PCR on individual fractions and expressed as percentage of the sum. The data shown in a,b,c,d were based on 3 independent experiments. *p<0.05; **p<0.01; ***p<0.001 (Student’s t-test).
Supplementary Figures 1–6 and Supplementary Table 1
Western blotting and original gel images
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Zhang, K., Zhang, X., Cai, Z. et al. A novel class of microRNA-recognition elements that function only within open reading frames. Nat Struct Mol Biol 25, 1019–1027 (2018). https://doi.org/10.1038/s41594-018-0136-3
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