Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: An Exportin-1–dependent microRNA biogenesis pathway during human cell quiescence

doi: 10.1073/pnas.1618732114

Figure Lengend Snippet: Biogenesis of quiescence-induced miRNAs requires Exportin-1 but not Exportin-5. (A, Left) qRT-PCR analysis showing relative expression of primary miRNAs (light gray bars) and their corresponding mature miRNAs (dark gray bars) in proliferating HFFs 48 h after transfection with an siRNA scrambled control (dashed line) or an siRNA targeting Exportin-5 (Dharmacon) (dark and light gray bars). (Right) Western blot analysis of Exportin-1 and Exportin-5 after siRNA transfection. (B, Left) qRT-PCR analysis showing relative levels of primary miRNAs (light gray bars) and their corresponding mature miRNAs (dark gray bars) in proliferating HFFs 48 h after transfection with an siRNA scrambled control (dashed line) or an siRNA targeting Exportin-1 (s14937; Ambion) (dark and light gray bars). (Right) Western blot analysis of Exportin-1 and Exportin-5 is also shown after siRNA transfection. (C, Upper) qRT-PCR analysis of quiescence-induced miRNAs and miR-34a Northern blot 48 h after transfection of an siRNA scrambled control (black bars) or an siRNA targeting Exportin-1 (s14937; Ambion) (gray bars) into proliferating HFFs in the presence of serum (P) or into HFFs serum-starved (SS) for 24 or 72 h or for 24, 48, or 72 h for the miR-34a Northern blot (Lower). Transfection of quiescent cells was done 24 h before serum starvation. (D) Exportin-1 physically associates with pri-miR-34a in quiescent cells. qRT-PCR was used to detect U3 snoRNA, pri-miR-34a, or pri-miR-423 in control Flag-Vector (IgG, black bars) or Flag-Exportin-1 (XPO1, gray bars) immunoprecipitates from quiescent HFFs (expression data are shown in SI Appendix, Fig. S7E). An exogenous spike of C. elegans RNA was used for qRT-PCR normalization by amplifying Ama-1 mRNA. Similar results were obtained in two independent experiments. In A, B, and D, two-tailed t test results are indicated as *P < 0.05 and **P < 0.01. CTRL, control.

Article Snippet: Cells were transfected with an siRNA scrambled control, an siRNA targeting Drosha (sc-44080; Santa Cruz), or siRNAs targeting Drosha (sc-44080; Santa Cruz) and Exportin-1 (s14937; Ambion) (Drosha/XPO1).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Control, Western Blot, Northern Blot, Plasmid Preparation, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: An Exportin-1–dependent microRNA biogenesis pathway during human cell quiescence

doi: 10.1073/pnas.1618732114

Figure Lengend Snippet: Quiescence-induced primary miRNAs are (TMG)-capped by TGS1. (A) Quiescence-induced pri-miRNAs contain a (TMG)-cap. Extracts were prepared from proliferating or serum-starved quiescent HFFs transfected with control siRNA or with siRNA targeting TGS1 (s41313; Ambion). siRNAs were transfected 24 h before serum starvation. RNA was harvested 48 h after transfection of siRNAs in proliferating HFFs or 72 h after the removal of serum. Extracts then were immunoprecipitated with control antibody or with antibody recognizing the (TMG)-cap, and immunoprecipitated U3 snoRNA and pri-miRNAs were amplified by RT-PCR and visualized after gel electrophoresis. C, control anti-rabbit serum; I, input; IP, immunoprecipitate; S, supernatant; T, anti-TMG antibody. (B) TGS1 knockdown inhibits the expression of quiescence-induced miRNAs. HFFs were transfected with a siRNA scrambled control (black bars) or a siRNA targeting TGS1 (sc-45875; Santa Cruz) (gray bars). Twenty-four hours later, cells were serum starved (SS) for 24 or 72 h or were maintained in serum (P). RNA then was prepared and quantified by qRT-PCR. (C) TGS1 knockdown affects the binding of pri-miR-34a to Exportin-1 in quiescent cells. qRT-PCR was used to detect U3 snoRNA, pri-miR-34a, or pri-miR-423 in immunoprecipitate samples of control Flag-Vector (Flag) or Flag-Exportin-1 (Flag-XPO1) in quiescent HFFs. Cells were transfected with siRNA control (black bars) or siRNA against TGS1 (gray bars). In B and C, two-tailed t test results are indicated as **P < 0.01. Similar results were obtained in two independent experiments. CTRL, control.

Article Snippet: Cells were transfected with an siRNA scrambled control, an siRNA targeting Drosha (sc-44080; Santa Cruz), or siRNAs targeting Drosha (sc-44080; Santa Cruz) and Exportin-1 (s14937; Ambion) (Drosha/XPO1).

Techniques: Transfection, Control, Immunoprecipitation, Amplification, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Knockdown, Expressing, Quantitative RT-PCR, Binding Assay, Plasmid Preparation, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: An Exportin-1–dependent microRNA biogenesis pathway during human cell quiescence

doi: 10.1073/pnas.1618732114

Figure Lengend Snippet: Detection of cytoplasmic pri-miR-34a is dependent on Exportin-1 expression. (A) Detection of pri-miR-34a in the cytoplasmic fraction of Drosha knock-down HFFs. Proliferating (P) and quiescent (Q) HFFs were transfected with control siRNA or siRNA targeting Drosha (sc-44080; Santa Cruz) and after 48 h were subjected to cytoplasmic and nuclear RNA fractionation. RT-PCR was used to detect pri-miR-34a, pri-miR-29b, and pri-miR-423. U6 snRNA is a nuclear control, and mitochondrial (mt) tRNA-val is a cytoplasmic marker. (B) qRT-PCR of cytoplasmic fractions as in A was used to quantify the levels of pri-miR-34a. Two-tailed t test results are indicated as **P < 0.01. (C and D) Exportin-1 knockdown affects the distribution of pri-miR-34a in quiescent HFFs. Cells were transfected with an siRNA scrambled control, an siRNA targeting Drosha (sc-44080; Santa Cruz), or siRNAs targeting Drosha (sc-44080; Santa Cruz) and Exportin-1 (s14937; Ambion) (Drosha/XPO1). Twenty-four hours later, cells were serum starved for 72 h. RNA then was prepared from the cytoplasmic (C) or nuclear (D) fraction for the quantification of pri-miR-34a and pri-miR-423 by qRT-PCR. Normalization was done using C. elegans total RNA as an exogenous spike for the amplification of the worm-specific ama-1 gene. Similar results were obtained in two independent experiments. CTRL, control.

Article Snippet: Cells were transfected with an siRNA scrambled control, an siRNA targeting Drosha (sc-44080; Santa Cruz), or siRNAs targeting Drosha (sc-44080; Santa Cruz) and Exportin-1 (s14937; Ambion) (Drosha/XPO1).

Techniques: Expressing, Knockdown, Transfection, Control, Fractionation, Reverse Transcription Polymerase Chain Reaction, Marker, Quantitative RT-PCR, Two Tailed Test, Amplification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: An Exportin-1–dependent microRNA biogenesis pathway during human cell quiescence

doi: 10.1073/pnas.1618732114

Figure Lengend Snippet: Detection of a small cytoplasmic isoform of Drosha during quiescence. (A) Western blot showing the reduction of full-length Drosha and the appearance of a more rapidly migrating form of Drosha (Droshashort) during the induction of quiescence by serum starvation. (B) A small form of Drosha (Droshashort) is found in the cytoplasmic fraction of quiescent cells. Western blot analysis of Drosha and DGCR8 in total, nuclear, and cytoplasmic fractions in proliferating (P) and quiescent (Q) HFFs. Ten percent of the total loading was used for the nuclear fraction in the Drosha Western blot. Histone deacetylase 1 (HDAC1) was used as the nuclear marker, and β-tubulin was used as the cytoplasmic marker. (C) Cytoplasmic localization of Drosha in quiescent HFFs. Immunofluorescence of Drosha (green) in proliferating and quiescent HFFs. Nuclear fluorescence (DAPI, blue) and cytoskeleton immunofluorescence (actin, red) were used to visualize the localization of Drosha. (Scale bars, 20 μm.) (D) Quantification of cytoplasmic Drosha immunofluorescence. Proliferating and quiescent HFFs were incubated with digitonin (to permeabilize selectively only the plasma membrane) to diminish masking of cytoplasmic Drosha staining with nuclear Drosha signal. Confocal microscopy was used to visualize the cells, and fluorescent images were used to obtain relative pixel area quantification using ImageJ software. Two-tailed t test results are indicated as *P < 0.05. Similar results were obtained in three independent experiments. (E) Drosha mRNA exon skipping during quiescence. Illustration and sequencing data show two alternative splicing events (red lines) between exons 3 and 4 and between exons 5 and 7 in Drosha mRNA and the affected regions in Drosha protein. Numbers represent the nucleotide sequence of Drosha mRNA. dsRBD, dsRNA-binding domain; P-rich, the proline-rich region; RIIIDa and RIIIDb, RNase III domains a and b, respectively; RS-rich, the arginine- and serine-rich region. (F) The short isoform of Drosha is responsible for the processing of pri-miR-34a. qRT-PCR of pri-miR-34a and pri-miR-423 was conducted in quiescent HFFs transfected with control siRNA or siRNA against the full-length Drosha (s26491; Ambion) or both Drosha isoforms (both) (sc-44080; Santa Cruz). Reduced accumulation of pri-miR-34a in cells treated with siRNA targeting full-length Drosha compared with siRNA against both Drosha isoforms (both) suggests the short Drosha isoform can process pri-miR-34a. (G) qRT-PCR of mature miR-34a and miR-423-3p was conducted from the experiments shown in F. Two-tailed t test results are indicated as *P < 0.01 in F and G. Similar results were obtained in two independent experiments. CTRL, control; Prolif, proliferating; Quies, Quiescent.

Article Snippet: Cells were transfected with an siRNA scrambled control, an siRNA targeting Drosha (sc-44080; Santa Cruz), or siRNAs targeting Drosha (sc-44080; Santa Cruz) and Exportin-1 (s14937; Ambion) (Drosha/XPO1).

Techniques: Western Blot, Histone Deacetylase Assay, Marker, Immunofluorescence, Fluorescence, Incubation, Clinical Proteomics, Membrane, Staining, Confocal Microscopy, Software, Two Tailed Test, Sequencing, Alternative Splicing, Binding Assay, Quantitative RT-PCR, Transfection, Control