Journal: Nucleic Acids Research

Article Title: Discovery of novel microRNA mimic repressors of ribosome biogenesis

doi: 10.1093/nar/gkad1235

Figure Lengend Snippet: The MIR-28 siblings, hsa-miR-28-5p and hsa-miR-708-5p, elicit highly-similar transcriptomes in human MCF10A and hTERT RPE1 cells, including a potent reduction in RPS28 levels. ( A ). Comparison of our results with the MIR-28 family members hsa-miR-28-5p and hsa-miR-708-5p, in MCF10A cells. The full mature microRNA sequence is shown for each sibling, with the shared AGGAGCU seed sequence indicated in blue. The table compares the RB phenotypes observed in MCF10A cells following treatment with either microRNA mimic. ( B ). Regression comparing log 2 -scale RNAseq differential expression profiles of MCF10A cells treated with either hsa-miR-28-5p or hsa-miR-708-5p, relative to siNT negative control. Each dot represents one mRNA, with RPS28 labeled. The line-of-best-fit shown in blue, with equation, R 2 value, and p value for non-zero slope indicated in top left. The data from 3 biological replicates were graphed and analyzed in JMP. ( C ). MIR-28-induced RPS28 downregulation in MCF10A cells, measured by normalized RNAseq read counts or RT-qPCR analysis. For RNAseq reads, the mean ± SEM are shown alongside individual data points, colored by replicate (3 replicates). For RT-qPCR, data were normalized to 7SL RNA abundance as an internal control, then to siNT for comparison using the ΔΔC T method. Mean ± SEM are shown alongside individual data points, colored by replicate (5 replicates). ( D ). Regression comparing log 2 -scale RNAseq differential expression profiles of hTERT RPE-1 cells treated with either hsa-miR-28-5p or hsa-miR-708-5p, relative to siNT negative control. Details are as written in (B). ( E ). MIR-28 sibling-induced RPS28 downregulation in hTERT RPE-1 cells, measured by normalized RNAseq read counts or RT-qPCR analysis. Details are as written in (C). ( F ). Bioanalyzer analysis of total RNA isolated from control- or MIR-28 sibling-treated hTERT RPE-1 cells. Left, the 28S/18S mature rRNA ratio; right, 18S mature rRNA/total RNA ratio. Mean ± SEM are shown alongside individual data points, colored by replicate. Data were graphed and analyzed by ordinary one-way ANOVA with multiple comparisons against siNT (non-targeting negative control) and Holm-Šídák correction in GraphPad Prism 8. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The FLAG-tagged RPS28 plasmid is available on Addgene (plasmid #203156).

Techniques: Comparison, Sequencing, Expressing, Negative Control, Labeling, Quantitative RT-PCR, Isolation

Journal: Nucleic Acids Research

Article Title: Discovery of novel microRNA mimic repressors of ribosome biogenesis

doi: 10.1093/nar/gkad1235

Figure Lengend Snippet: The MIR-28 siblings target tandem binding sites in the RPS28 3′ UTR to interrupt pre-18S processing. ( A ). Schematic for 3′ UTR luciferase reporter assay for testing the targeting of RPS28 by the MIR-28 siblings, miR-28-5p and hsa-miR-708-5p. Diagram for the RPS28 mRNA transcript, indicating untranslated regions (UTR; dark blue) or coding sequence (CDS; light blue). Putative MIR-28 binding sites 1 and 2 are shown in red, and their wild-type (WT) sequence is given, along with the design for scrambled MIR-28 sites (SCR) rescue construct. Antisense pairing of the 6mer MIR-28 seed is shown with the putative WT binding sites. Note the perfect complementarity between the seed sequence and the predicted target. The WT or SCR regions of the RPS28 3′ UTR containing both putative MIR-28 sites were cloned into the miR test site in the 3′ UTR of the Renilla luciferase expression cassette in the psiCHECK2 plasmid. ( B ). Quantification of 3′ UTR luciferase reporter assays in (A), testing targeting of RPS28 by the MIR-28 siblings. The vector construct is indicated as wild-type (WT) or scrambled (SCR) above the transfection treatment labels. Luciferase data were collected for each treatment and construct combination, then normalized to the siNT treatment on a per-construct basis. RLU stands for relative light units. Mean ± SEM are shown alongside individual data points, colored by replicate (5 replicates). The data were analyzed by unpaired two-sided Welch's t -tests between WT and SCR constructs in GraphPad Prism 8. * P < 0.05. ( C ). Schematic for rescue experiment conditionally expressing an RPS28 mRNA lacking the MIR-28 family binding sites to recover normal 28S/18S mature rRNA ratio in the presence of MIR-28 sibling overexpression. (Top) Diagram of the engineered HEK 293 Flp-In T-REx cells containing a tetracycline-inducible FLAG-tagged RPS28 cassette lacking the WT 3′ UTR harboring tandem MIR-28 binding sites. (Bottom) Experimental outline indicating steps for cell seeding, simultaneous MIR-28 mimic transfection and engineered RPS28 tetracycline induction, incubation, and mature rRNA analysis by electropherogram. ( D ). Bioanalyzer analysis of the 28S/18S mature rRNA ratio from engineered HEK cells, treated as indicated. Treatments include siNT siRNA or each of the MIR-28 sibling mimics, hsa-miR-28-5p or hsa-miR-708-5p. Induction of RPS28 expression with 1 μg/ml tetracycline (Tet) is indicated with a +. Cell line type was either the parental non-engineered line (empty vector, EV) or engineered line containing FLAG-tagged RPS28 lacking WT 3′ UTR MIR-28 binding sites (FLAG-RPS28). Mean ± SEM are shown alongside individual data points, colored by replicate ( n = 3 biological replicates). Data were graphed and analyzed by ordinary one-way ANOVA with multiple comparisons against siNT (non-targeting negative control) and Holm-Šídák correction in GraphPad Prism 8. ns, not significant; *** P < 0.001.

Article Snippet: The FLAG-tagged RPS28 plasmid is available on Addgene (plasmid #203156).

Techniques: Binding Assay, Luciferase, Reporter Assay, Sequencing, Construct, Clone Assay, Expressing, Plasmid Preparation, Transfection, Over Expression, Incubation, Negative Control

Journal: Nucleic Acids Research

Article Title: Discovery of novel microRNA mimic repressors of ribosome biogenesis

doi: 10.1093/nar/gkad1235

Figure Lengend Snippet: Model of the mechanism of MIR-28 sibling-mediated inhibition of ribosome biogenesis. The RPS28 mRNA harbors two tandem, primate-specific binding sites for the MIR-28 microRNA family seed sequence. The MIR-28 siblings, hsa-miR-28-5p and hsa-miR-708-5p, can target RPS28 at these sites, reducing RPS28 mRNA and RPS28 protein levels. Lack of RPS28 protein in the nucleolus causes defects in pre-18S rRNA processing that lead to a reduction in levels of the mature 18S rRNA and subsequently 40S (small) ribosomal subunits.

Article Snippet: The FLAG-tagged RPS28 plasmid is available on Addgene (plasmid #203156).

Techniques: Inhibition, Binding Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: Discovery of novel microRNA mimic repressors of ribosome biogenesis

doi: 10.1093/nar/gkad1235

Figure Lengend Snippet:

Article Snippet: The FLAG-tagged RPS28 plasmid is available on Addgene (plasmid #203156).

Techniques:

Journal: Nucleic Acids Research

Article Title: Discovery of novel microRNA mimic repressors of ribosome biogenesis

doi: 10.1093/nar/gkad1235

Figure Lengend Snippet:

Article Snippet: The FLAG-tagged RPS28 plasmid is available on Addgene (plasmid #203156).

Techniques: