Journal: bioRxiv

Article Title: Ribosomal protein L24 modulates mammalian microRNA processing and transfer RNA fragment production

doi: 10.1101/2023.05.03.539194

Figure Lengend Snippet: A . Experimental design: HEK293T cells were seeded and 24 hours later transfected with pcDNA3.1+ vector containing FLAG-RPL24 or no insert as control. Cells were harvested 48 hours 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. 2.5% of input lysate was loaded per lane, 2.5% of supernatant, and 20% of pellet. A double band is observed in the input lane; the upper band is the FLAG-RPL24 (∼24kDa) and the lower band is the endogenous RPL24 (∼23kDa). C, D . Volcano plot presenting the proteins enriched in FLAG-RPL24 IP compared to control, in the nuclear and cytoplasmic fractions. Black and red symbols denote non-significant proteins and proteins with p<0.05 and enrichment >1.5-fold. E . Venn diagram showing proteins bound to RPL24 in cytoplasmic and nuclear fractions, with common and specific proteins. F . Immunoblotted DDX5 from the nuclear fraction of HEK293T cells transfected with pcDNA3.1+ vector containing or lacking (control) 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, p =0.04 and p =0.045, respectively, but pri-miR-185 and pri-miR-126 are not expressed. Each pellet sample was normalized to its corresponding input sample and fold enrichment is determined as FLAG-RPL24 IP/NC IP.

Article Snippet: Protein concentrations were determined using the Bradford (Merck, B6916) or Lowry assay (DC Protein Assay, Bio-Rad, 5000113) and 5 μg/sample was loaded onto 4-15% gradient polyacrylamide gels (Mini-PROTEAN TGX Gels, Bio-Rad, 4561083), transferred (Bio-Rad, Trans-Blot Turbo Transfer System) to nitrocellulose membranes (Bio-Rad, 1704158) and probed with antibodies against RPL24 (Proteintech, 17082-1-AP, 1:1000), B-Actin (Santa Cruz, sc-47778, 1:1000), α-Tubulin (Merck, T5168, 1:1000), GAPDH (Cell Signaling Technology, #2118, 1:1000), Histone H3 (abcam, ab1791, 1:1000) and DDX5 (Proteintech, 10804-1-AP, 1:700).

Techniques: Transfection, Plasmid Preparation, Control, Immunoprecipitation, FLAG-tag, Western Blot, Quantitative RT-PCR

Journal: Cellular and Molecular Life Sciences

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

doi: 10.1007/s00018-023-05088-w

Figure Lengend Snippet: 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 . 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

Article Snippet: Protein concentrations were determined using the Bradford (Merck, B6916) or Lowry assay (DC Protein Assay, Bio-Rad, 5000113) and 5 μg/sample was loaded onto 4–15% gradient polyacrylamide gels (Mini-PROTEAN TGX Gels, Bio-Rad, 4561083), transferred (Bio-Rad, Trans-Blot Turbo Transfer System) to nitrocellulose membranes (Bio-Rad, 1704158) and probed with antibodies against RPL24 (Proteintech, 17082–1-AP, 1:1000), B-Actin (Santa Cruz, sc-47778, 1:1000), A-Tubulin (Merck, T5168, 1:1000), GAPDH (Cell Signaling Technology, #2118, 1:1000), Histone H3 (abcam, ab1791, 1:1000) and DDX5 (Proteintech, 10804–1-AP, 1:700).

Techniques: Mass Spectrometry, Sequencing, Western Blot, Marker, Transfection, Negative Control, Quantitative RT-PCR, Biomarker Discovery, RNA Sequencing

Journal: Cellular and Molecular Life Sciences

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

doi: 10.1007/s00018-023-05088-w

Figure Lengend Snippet: 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

Article Snippet: Protein concentrations were determined using the Bradford (Merck, B6916) or Lowry assay (DC Protein Assay, Bio-Rad, 5000113) and 5 μg/sample was loaded onto 4–15% gradient polyacrylamide gels (Mini-PROTEAN TGX Gels, Bio-Rad, 4561083), transferred (Bio-Rad, Trans-Blot Turbo Transfer System) to nitrocellulose membranes (Bio-Rad, 1704158) and probed with antibodies against RPL24 (Proteintech, 17082–1-AP, 1:1000), B-Actin (Santa Cruz, sc-47778, 1:1000), A-Tubulin (Merck, T5168, 1:1000), GAPDH (Cell Signaling Technology, #2118, 1:1000), Histone H3 (abcam, ab1791, 1:1000) and DDX5 (Proteintech, 10804–1-AP, 1:700).

Techniques: Transfection, Plasmid Preparation, Control, Immunoprecipitation, FLAG-tag, Western Blot, Quantitative RT-PCR