Ribosomal protein L24 mediates mammalian microRNA processing in an evolutionarily conserved manner

Abstract

To investigate the mechanism(s) underlying the expression of primate-specific microRNAs (miRs), we sought DNA regulatory elements and proteins mediating expression of the primate-specific hsa-miR-608 (miR-608), which is located in the SEMA4G gene and facilitates the cholinergic blockade of inflammation by targeting acetylcholinesterase mRNA. 'Humanized' mice carrying pre-miR-608 flanked by 250 bases of endogenous sequences inserted into the murine Sema4g gene successfully expressed miR-608. Moreover, by flanking miR-608 by shortened fragments of its human genome region we identified an active independent promoter within the 150 nucleotides 5' to pre-miR-608, which elevated mature miR-608 levels by 100-fold in transfected mouse- and human-originated cells. This highlighted a regulatory role of the 5' flank as enabling miR-608 expression. Moreover, pull-down of the 150-base 5' sequence revealed its interaction with ribosomal protein L24 (RPL24), implicating an additional mechanism controlling miR-608 levels. Furthermore, RPL24 knockdown altered the expression of multiple miRs, and RPL24 immunoprecipitation indicated that up- or down-regulation of the mature miRs depended on whether their precursors bind RPL24 directly. Finally, further tests showed that RPL24 interacts directly with DDX5, a component of the large microprocessor complex, to inhibit miR processing. Our findings reveal that RPL24, which has previously been shown to play a role in miR processing in Arabidopsis thaliana, has a similar evolutionarily conserved function in miR biogenesis in mammals. We thus characterize a novel extra-ribosomal role of RPL24 in primate miR regulation.

Keywords: DDX5; Hsa-miR-608; Pre-miRs; Pri-miRs; RPL24; miR processing.

Fig. 1

Cis-sequences regulate miR-608 expression in engineered KI mice and cultured cells. A Structure of the mouse construct: miR-608 transgenic ‘humanized’ mice were established by inserting hsa-pre-mir-608 into the third intron of the mouse Sema4g gene, with 250 flanking bases at each side. B miR-608 levels in the brain and peripheral tissues of female and male miR-608 KI mice, determined by RT-qPCR. All miR-608 levels were normalized to those of female hypothalamus (that showed the lowest expression); two-way ANOVA with Dunnet’s multiple comparisons correction, ± SD, n = 5 per group. C Experimental design: HEK293T and CT26.WT cells were seeded and 24 h later transfected with pcDNA3.1 + plasmid containing miR-608 inserted into the second intron of HBB and flanked by native sequences of varying lengths. Cells were harvested 48 h later, RNA extracted, and miR-608 levels quantified. D Symmetric bidirectional 250 base extension of the miR-608 stem-loop altered its levels in HEK293T cells; bar graph, ± SD, p = 0.0163, unpaired t-test. E Constructs of pre-miR-608 flanked by symmetric sequences ranging from 75 to 250 bases. F Levels of miR-608 expressed from these constructs in HEK293T cells. The 150 bases both upstream and downstream are critical for miR-608 expression in HEK293T cells. G The 150-base symmetrical flanks are also critical for expression in CT26.WT cells. H Constructs of pre-miR-608 flanked by asymmetric sequences ranging from 75 to 150 bases. I Levels of miR-608 expressed from these constructs in HEK293T cells were highest under control of the upstream 150 base sequence. J A similar effect is seen in CT26.WT cells transfected with these constructs. All experiments were performed in duplicate or triplicate and miR-608 levels were measured using Taqman RT-qPCR with RNU6B and snoRNA135 as normalizing genes. Results are shown relative to levels of pre-miR-608 with no flanking sequences. In all panels *p < 0.05, **p < 0.01, ***p < 0.001. In panels F, G, I, bar-graph ± SD, one-way ANOVA with Tukey’s multiple comparisons test

Fig. 2

A TATA box enabling miR-608 expression is located in the 5′ 150 bases. A The 150 bases 5′ upstream to pre-miR-608 contain a TATA box at position 111 (red). B miR-608 levels in HEK293T cells transfected with pUC57, quantified by Taqman RT-qPCR, show that the TATA box drives expression from the promoter-less bacterial vector pUC57. C. miR-608 levels in HEK293T cells transfected with the mammalian vector pcDNA3.1 + containing the TATA box T-to-A point mutation are decreased as compared to non-mutated TATA box. D Higher levels of pri-miR-608 transcripts (expressed from pcDNA3.1 +) and measured by RT-qPCR using forward primers positioned upstream (F1) vs downstream (F2) to the predicted TATA box; the reverse primer (R) is common. E Scrambling parts of the 5′ 150 bases upstream to the pre-miR-608 sequence decreased the levels of mature miR-608, as determined by Taqman RT-qPCR quantification of miR-608 relative to the intact 150 base sequence and normalized to RNU6B. Hatching represents the scrambled area. Experiments were performed in duplicate or triplicate and in all panels *p < 0.05, **p < 0.01, ***p < 0.001. In panel D unpaired t-test, in all other panels one-way ANOVA with Tukey’s correction for multiple comparisons, bar-graph ± SD

Fig. 3

RPL24 KD alters the levels of diverse miRs in addition to miR-608. A Pull-down analysis: mass spectrometry identified 15 proteins bound to the 5′ 150 base-upstream sequence with p < 0.05 and enrichment > 1.5-fold (marked in red), the most enriched being RPL24 (p = 0.039, fold enrichment = 8). B Immunoblot of subcellular fractions identified RPL24 in both the nuclear and the cytoplasmic fractions. Fraction purity was validated with GAPDH, a cytoplasmic marker, and H3, a nuclear marker; each of which was localized solely to its expected compartment. C Experimental design: HEK293T cells were seeded, transfected with 50 nM of siPOOLs targeting RPL24 or a non-targeting negative control pool (NC) 24 h later, then transfected with pcDNA3.1 + containing miR-608 48 h later. RNA and proteins were extracted 24 h post-transfection. D RPL24 depletion in HEK293T cells elevates miR-608 levels. E RPL24 depletion in Caco2 cells shows the same effect. Quantification of miR-608 by Taqman RT-qPCR, with results normalized to RNU6B and relative to NC siPOOLs; for D, one-way ANOVA with Tukey’s correction for multiple comparisons ± SD. For E, unpaired t-test ± SD. F Heatmap showing the 22 DE miRs increased or decreased after RPL24 KD, analyzed by DESeq2, adjusted p-value < 0.05, Benjamini–Hochberg correction [27]. See Supp. Table 3 for full information on the DE miRs. G RT-qPCR validation confirming the RNA-seq results with data shown relative to the NC and normalized to SNORD47 and SNORD48; unpaired t-test for each miR ± SD, in panels D, E, G, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

RPL24 binds pri-miRs and inhibits their processing through direct interaction with DDX5. A Experimental design: HEK293T cells were seeded and 24 h later transfected with pcDNA3.1 + vector containing FLAG-RPL24 or no insert as control. Cells were harvested 48 h after transfection, fractionated, and nuclear and cytoplasmic fractions immunoprecipitated separately with an antibody against the FLAG-tag, with subsequent MS analysis. B Immunoblot of nuclear fraction from cells transfected with a pcDNA3.1 + vector containing FLAG-RPL24 insert or empty pcDNA3.1 + vector as control, showing pull-down of the tagged-RPL24. 2.5% of input lysate, 2.5% of supernatant, and 20% of pellet were loaded per lane. Of the double band observed in the input lane, the upper and lower bands are the FLAG-RPL24 (~ 24kDa) and the endogenous RPL24 (~ 23kDa). C, D Volcano plot presenting the proteins enriched in FLAG-RPL24 IP compared to control, in the nuclear (C) and cytoplasmic (D) fractions. Red symbols denote proteins with p < 0.05 and enrichment > 1.5-fold and black symbols denote non-significant proteins. E Venn diagram showing proteins bound to RPL24 in cytoplasmic and nuclear fractions, with common and specific proteins. F Immunoblot showing the presence of DDX5 in RPL24 IP from the nuclear fraction of HEK293T cells transfected with pcDNA3.1 + vector containing ( +) vs lacking (−) a FLAG-RPL24 insert, confirming RPL24-DDX5 interaction. 2.5% of input lysate and 90% of pellet were loaded per lane. G RT-qPCR quantification of pri-miRs in nuclear pellet samples of FLAG-RPL24 IP. Pri-miR-608 and pri-miR-196b are enriched, but pri-miR-126 and pri-miR-185 are not. Each pellet sample was normalized to its corresponding input sample and fold enrichment is determined as FLAG-RPL24 IP/NC IP. H RT-qPCR quantification of miRs following DDX5 KD. miR-608 and miR-196b-5p are downregulated, but miR-126-3p and miR-185-5p levels are unchanged. For Panels G and H, experiments were performed in triplicate. Unpaired t-test, bar-graph ± SD, *p < 0.05