Journal: Nucleic Acids Research

Article Title: Biogenesis of mammalian microRNAs by a non-canonical processing pathway

doi: 10.1093/nar/gks026

Figure Lengend Snippet: Simtron biogenesis involves Drosha but not DGCR8. Knockdown of DGCR8 in HeLa cells using siRNA was quantitated by ( A ) RT–PCR analysis of DGCR8 mRNA and ( B ) western blot analysis of DGCR8 protein expression. The percentage of knockdown of DGCR8 was quantitated for DGCR8 mRNA using the equation 100 − [(( DGCR8 knockdown / GAPDH )/( DGCR8 control / GAPDH )) × 100], n = 5 and for DGCR8 protein using the equation 100 − [(( DGCR8 knockdown /β-actin )/( DGCR8 control / β-actin )) × 100]. (C) Changes in endogenous miRNA levels following DGCR8 knockdown were analysed by stemloop RT–PCR analysis. miR-16 is a canonical miRNA control and sno65 is a loading control. Graph shows quantitation of miRNA abundance using the equation: (miRNA experimental condition /sno65)/(miRNA control /sno65). n = 4 for all miRNAs except for miR-16, n = 5; asterisk indicates P ≤ 0.05 (Wilcoxon matched pairs signed-rank test). M indicates a synthetic size marker and filled circle indicates a non-specific primer dimer. ( D ) RT–PCR analysis of Drosha mRNA following expression of TN-Drosha in HEK-293T cells. ( E ) The effect of TN-Drosha expression on endogenous miRNA abundance was analysed by stemloop RT–PCR. Graph shows quantitation of miRNA abundance using the same equation as in C, n = 6; asterick indicates P ≤ 0.05 (Student's t -test). ( F ) Stemloop RT–PCR analysis of minigene-derived miR-877, 1226, 1225, 1228 and endogenous miR-16 isolated from HEK-293T cells transiently transfected with TN-Drosha. sno65 was used as a control. TN-Drosha mRNA expression in HEK-293T cells was analysed by radiolabelled RT–PCR. GAPDH was used as a control. ( G ) Quantitation of miRNA abundance relative to sno65 using the equation: miRNA/sno65. n = 3 for miR-877, 1226 and 1225, n = 5 for miR-1228 and n = 14 for miR-16; * P ≤ 0.05, *** P ≤ 0.0001 (). Data sets were analysed using the Student's t -test with the exception of miR-16, which was analysed using the Wilcoxon matched pairs signed-rank test. In all panels, bars represent the average ± SEM. The horizontal dotted line indicates normalized control levels.

Article Snippet: Hannon) ( ) and DGCR8 knockout (DGCR8 −/− ) cells (Novus Biologicals) were grown on a gelatin layer in Knockout Dulbecco's modified Eagle's medium (Gibco) supplemented with 15% ES cell FBS (Gibco), 1% non-essential amino acids, 1% l -glutamine, 1% penicillin/streptomycin/Amphotericin B, 0.1% ESGRO-LIF and 0.008% beta-mercaptoethanol.

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Control, Quantitation Assay, Marker, Derivative Assay, Isolation, Transfection

Journal: Nucleic Acids Research

Article Title: Biogenesis of mammalian microRNAs by a non-canonical processing pathway

doi: 10.1093/nar/gks026

Figure Lengend Snippet: Simtron biogenesis does not require DGCR8, Dicer, Ago2 or XPO5. ( A ) RT–PCR analysis of minigene-derived host gene mRNA and stemloop RT–PCR analysis of minigene-derived miRNA and endogenous miR-16 in Dicer and DGCR8 knockout mouse embryonic stem cells transfected with the wt or splicing-deficient minigene (Δss) or empty vector control (−). sno65 was used as a loading control. Graphs show quantitation of miRNA using the equation: (miRNA experimental condition /sno65)/(miRNA control /sno65). Bars represent the average ± SEM, n = 3. The horizontal dotted lines indicate normalized control levels. ( B ) Stemloop RT–PCR analysis of miR-1225 and miR-1228 immunoprecipitated from HEK-293T cell lysates that were transiently transfected with wt or Δss minigenes, or miR-877 from wt minigene along with pFLAG-Dicer (Dicer) or without (−) and immunoprecipitated with an antibody against the FLAG epitope. Input refers to cell lysates before FLAG immunoprecipitation; Un is the unbound fraction and IP is the immunoprecipitated fraction. Un is 1/20 IP and Input is 1/5 IP. The graph represents the percent of the mature miRNA found in the IP fraction versus the amount that remained in the Un fraction using the equation: (IP/(IP + (Un × 20)) × 100). ( C ) Stemloop RT–PCR analysis of minigene-derived miR-1225, miR-1228 and endogenous miR-16 from Ago2 knockout mouse embryonic fibroblasts. sno65 was used as a loading control. Cells were transiently transfected with wt or Δss minigenes or empty vector control (−). Graph shows quantitation of miRNA abundance using the same equation as in A. Bars represent the average ± SEM, n = 3 and * P ≤ 0.05 or ** P ≤ 0.01, Student's t -test. The horizontal dotted lines indicate normalized control levels. ( D ) Stemloop RT–PCR and RT–PCR analysis of miR-877 (left panel), miR-1225 (middle panel) and miR-1228 (right panel) minigene-expression in HeLa cells following siRNA-directed knockdown of XPO5 . sno65 is a loading control for miRNA using stemloop RT–PCR and GAPDH is a loading control for RT–PCR of XPO5 mRNA.

Article Snippet: Hannon) ( ) and DGCR8 knockout (DGCR8 −/− ) cells (Novus Biologicals) were grown on a gelatin layer in Knockout Dulbecco's modified Eagle's medium (Gibco) supplemented with 15% ES cell FBS (Gibco), 1% non-essential amino acids, 1% l -glutamine, 1% penicillin/streptomycin/Amphotericin B, 0.1% ESGRO-LIF and 0.008% beta-mercaptoethanol.

Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Knock-Out, Transfection, Plasmid Preparation, Control, Quantitation Assay, Immunoprecipitation, FLAG-tag, Expressing, Knockdown

Journal: Nucleic Acids Research

Article Title: Biogenesis of mammalian microRNAs by a non-canonical processing pathway

doi: 10.1093/nar/gks026

Figure Lengend Snippet: Immunoprecipitation and in vitro processing of simtrons with Drosha. ( A ) Pre-miR-1225 co-immunoprecipitates with Drosha. Pre-miR-1225 derived from wt and Δss minigenes and pre-miR-877 from wt minigene were transiently transfected into HEK-293T cells with pFLAG-Drosha (Drosha) or without (−), and immunoprecipitated with an antibody against the FLAG epitope. Isolated pre-miRNAs were analysed by radiolabelled stemloop RT–PCR and products were separated by 12% native PAGE. Input (In) refers to cell lysates before FLAG immunoprecipitation; Un is the unbound fraction and IP is the immunoprecipitated fraction. Un is 1/20 IP and Input is 1/5 IP. The graph represents the percent of the pre-miRNA found in the IP fraction versus the amount that remained in the Un fraction using the equation: (IP/(IP + (Un × 20)) × 100). ( B ) Drosha-dependent in vitro simtron processing. Radiolabelled RNA transcribed from a PKD1 wt or Δss, ABCF1 wt or pri-miR-16-1 DNA template was incubated with the FLAG-immunoprecipitates from HEK-293T cells, or with HEK-293T WCEs from cells that were not transfected. FLAG-immunoprecipitates were derived from cells transfected with mock transfection (−), pFLAG-GFP (GFP), pFLAG-Drosha (Drosha), pFLAG-Drosha and pFLAG-DGCR8 (Drosha + DGCR8), pFLAG-TN-Drosha (TN Drosha), or FLAG-M2-beads that were incubated with lysis buffer but no cell lysate (−lysate). Template RNA was included as a control (RNA). Reaction products were separated by 8% denaturing PAGE. The sizes of pre-miRNAs are indicated. Asterisk indicates uncharacterized miR-16 cleavage fragments .

Article Snippet: Hannon) ( ) and DGCR8 knockout (DGCR8 −/− ) cells (Novus Biologicals) were grown on a gelatin layer in Knockout Dulbecco's modified Eagle's medium (Gibco) supplemented with 15% ES cell FBS (Gibco), 1% non-essential amino acids, 1% l -glutamine, 1% penicillin/streptomycin/Amphotericin B, 0.1% ESGRO-LIF and 0.008% beta-mercaptoethanol.

Techniques: Immunoprecipitation, In Vitro, Derivative Assay, Transfection, FLAG-tag, Isolation, Reverse Transcription Polymerase Chain Reaction, Clear Native PAGE, Incubation, Lysis, Control

Journal: Nucleic Acids Research

Article Title: Biogenesis of mammalian microRNAs by a non-canonical processing pathway

doi: 10.1093/nar/gks026

Figure Lengend Snippet: Simtron processing is context independent. ( A ) Diagram comparing intronic and intergenic pre-miRNA expression. ( B ) Control, Dicer (Dicer −/− ) or DGCR8 (DGCR8 −/− ) knockout mouse embryonic stem cells were transiently transfected with the intergenic wt minigene, or intergenic splicing-deficient minigene (Δss). Minigene-derived miRNAs and endogenous miR-16 were analysed by stemloop RT–PCR. Left panel: simtron miR-1225. Right panel: mirtron miR-877. sno65 was analysed as a loading control. ( C ) Graph shows quantitation of miR-1225 abundance using the equation: (miRNA Dicer−/− or DGCR8−/− /sno65)/(miRNA control /sno65). Bars represent the average values ±SEM, n = 4 for Dicer −/− and n = 3 for DGCR8 −/− . The horizontal dotted line indicates normalized control cell levels.

Article Snippet: Hannon) ( ) and DGCR8 knockout (DGCR8 −/− ) cells (Novus Biologicals) were grown on a gelatin layer in Knockout Dulbecco's modified Eagle's medium (Gibco) supplemented with 15% ES cell FBS (Gibco), 1% non-essential amino acids, 1% l -glutamine, 1% penicillin/streptomycin/Amphotericin B, 0.1% ESGRO-LIF and 0.008% beta-mercaptoethanol.

Techniques: Expressing, Control, Knock-Out, Transfection, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Quantitation Assay

Journal: Nucleic Acids Research

Article Title: Biogenesis of mammalian microRNAs by a non-canonical processing pathway

doi: 10.1093/nar/gks026

Figure Lengend Snippet: Proposed model of simtron biogenesis compared to other miRNA processing pathways. The pathways shown begin with the primary transcript and end with the mature product. Left: simtron pathway, Middle: mirtron pathway, Right: canonical miRNA pathway. Exons are depicted as boxes and introns and miRNAs as lines. Each protein or protein complex is labelled. Proteins labelled with question marks are proposed but not known. Simtrons (such as miR-1225 and miR-1228) processing from the intron involves Drosha and possibly an unknown binding partner. Simtrons are further processed by unknown factors and enter the RISC complex with any of the four human Argonaute proteins. Mirtrons (such as miR-877 and miR-1226) are excised from the host gene by the spliceosome, are debranched, exported from the nucleus by exportin5 (XPO5), cleaved by Dicer and enter the RISC complex. Canonical miRNAs (such as miR-16) are processed by Drosha and DGCR8, exported from the nucleus by XPO5, cleaved by Dicer and enter the RISC complex. All three pathways result in functional miRNAs.

Article Snippet: Hannon) ( ) and DGCR8 knockout (DGCR8 −/− ) cells (Novus Biologicals) were grown on a gelatin layer in Knockout Dulbecco's modified Eagle's medium (Gibco) supplemented with 15% ES cell FBS (Gibco), 1% non-essential amino acids, 1% l -glutamine, 1% penicillin/streptomycin/Amphotericin B, 0.1% ESGRO-LIF and 0.008% beta-mercaptoethanol.

Techniques: Binding Assay, Functional Assay

Journal: Scientific Reports

Article Title: DGCR8 regulates multiple processes of transcription coupled nucleotide excision repair

doi: 10.1038/s41598-026-38338-5

Figure Lengend Snippet: UV-induced interactions between DGCR8 and TC-NER factors require S153-phosphorylation. ( a ) Schematic overview of the experimental design and proximity ligation assay (PLA). ( b–f ) Quantification and representative PLA images showing interactions between DGCR8 and the specified TC-NER proteins. PLA signals (red) were detected in nuclei counterstained with DAPI (blue). U2-OS cells expressing either wild-type DGCR8 (left) or the S153A mutant (right) were irradiated with 20 J/m 2 UV-C and allowed to recover for 1, 2, or 4 h (purple dots); non-irradiated controls are shown in gray (UV-). Horizontal black bars indicate the median of each group. Asterisks indicate statistically significant differences relative to non-irradiated controls or between genotypes (* p < 0.05, ** p < 0.01, *** p < 0.001). White scale bars, 10 μm.

Article Snippet: Dgcr8 -knockout MEFs were purchased from Novus Biologicals and cultured according to the manufacturer’s instructions.

Techniques: Phospho-proteomics, Proximity Ligation Assay, Expressing, Mutagenesis, Irradiation

Journal: Scientific Reports

Article Title: DGCR8 regulates multiple processes of transcription coupled nucleotide excision repair

doi: 10.1038/s41598-026-38338-5

Figure Lengend Snippet: UV-induced TC-NER complex formation requires S153-phosphorylation. ( a–f ) Quantification and representative PLA images showing interactions between the specified TC-NER proteins. PLA signals (red) detected in nuclei counterstained with DAPI (blue). U2-OS cells expressing either wild-type DGCR8 (left) or the S153A mutant (right) were irradiated with 20 J/m² UV-C and allowed to recover for 1, 2, or 4 h (purple dots); non-irradiated controls are shown in gray (UV-). Horizontal black bars indicate the median of each group. Asterisks indicate statistically significant differences relative to non-irradiated controls or between genotypes (* p < 0.05, ** p < 0.01, *** p < 0.001). White scale bars, 10 μm. ( g ) Schematic model illustrating the proposed DGCR8-centered protein interaction network in response to UV.

Article Snippet: Dgcr8 -knockout MEFs were purchased from Novus Biologicals and cultured according to the manufacturer’s instructions.

Techniques: Phospho-proteomics, Expressing, Mutagenesis, Irradiation

Journal: Scientific Reports

Article Title: DGCR8 regulates multiple processes of transcription coupled nucleotide excision repair

doi: 10.1038/s41598-026-38338-5

Figure Lengend Snippet: S153A mutation disrupts UV-induced interactions between DGCR8 and TC-NER factors in MEFs. (a, b) Quantification and representative PLA images showing CSB–CSA ( a ) and DGCR8–CSB ( b ) interactions in MEFs. Wild-type, heterozygous (wt/S153A), and homozygous (S153A/S153A) MEFs were irradiated with 10 J/m 2 UV-C (purple dots) or left untreated (gray dots), followed by 2 hours of recovery. PLA signals (red) were detected in nuclei counterstained with DAPI (blue). Horizontal black bars indicate the median of each group. Asterisks denote significant differences relative to non-irradiated or between genotypes (*** p < 0.001). White scale bars, 10 μm.

Article Snippet: Dgcr8 -knockout MEFs were purchased from Novus Biologicals and cultured according to the manufacturer’s instructions.

Techniques: Mutagenesis, Irradiation

Journal: Scientific Reports

Article Title: DGCR8 regulates multiple processes of transcription coupled nucleotide excision repair

doi: 10.1038/s41598-026-38338-5

Figure Lengend Snippet: DGCR8 interacts with chromatin remodelers and may act as a molecular switch in response to UV irradiation. (a, b) Representative PLA images showing interactions between DGCR8 and SPT16 or SMARCA5 ( a ) and interactions between phosphorylated S153-DGCR8 (pS153) and Drosha or CSA ( b ). U2-OS cells were irradiated with 20 J/m 2 UV-C (purple) or left untreated (gray), followed by 1, 2, or 4 h of recovery. White scale bars, 10 μm. ( c ) Quantification of PLA signals shown in (a) and (b). PLA signals (red) were detected and quantified in nuclei counterstained with DAPI (blue). Horizontal black bars indicate the median of each group. Asterisks denote significant differences relative to untreated cells (*** p < 0.001).

Article Snippet: Dgcr8 -knockout MEFs were purchased from Novus Biologicals and cultured according to the manufacturer’s instructions.

Techniques: Irradiation

Journal: Scientific Reports

Article Title: DGCR8 regulates multiple processes of transcription coupled nucleotide excision repair

doi: 10.1038/s41598-026-38338-5

Figure Lengend Snippet: DGCR8-pS153 contributes to R-loop regulation. ( a ) R-loop detection by S9.6 immunostaining in Dgcr8 −/− MEFs transduced with wild-type human DGCR8, the S153A mutant, or an empty vector. Note that the vertical axis scale differs due to microscope camera settings (see Methods). White scale bars, 10 μm. ( b ) S9.6 immunostaining in wild-type and S153A U2-OS cells. ( c ) Schematic of the R-loop probe: a catalytically inactive RNase H1 mutant (D210N) fused to EGFP (mRNH) was used to visualize R-loops in live or fixed cells. ( d ) Colocalization of R-loops with nucleoli in non-irradiated U2-OS cells, with nucleoli labeled using Nucleolus Bright Red (Dojin). (e, f) PLA analyses of interactions between DGCR8 and R-loops ( e ) and between R-loops and UV-induced cyclobutane pyrimidine dimers (CPDs) ( f ). Cells were irradiated with UV-C (purple) at 10 J/m 2 (a) or 20 J/m 2 (b, e, f) or left untreated (gray). Red fluorescence indicates PLA or immunostaining signals; nuclei were counterstained with DAPI (blue). In (d) and (e), green fluorescence marks R-loops labeled by the mRNH probe. Horizontal black bars indicate the median of each group. Asterisks indicate significant differences relative to untreated cells or between genotypes (* p < 0.05, *** p < 0.001). White scale bars, 10 μm. ( g ) Schematic model of the proposed DGCR8-mediated mechanism of R-loop regulation.

Article Snippet: Dgcr8 -knockout MEFs were purchased from Novus Biologicals and cultured according to the manufacturer’s instructions.

Techniques: Immunostaining, Transduction, Mutagenesis, Plasmid Preparation, Microscopy, Irradiation, Labeling, Fluorescence

Journal: Cell Death & Disease

Article Title: Protein disulfide isomerase blocks the interaction of LC3II-PHB2 and promotes mTOR signaling to regulate autophagy and radio/chemo-sensitivity

doi: 10.1038/s41419-022-05302-w

Figure Lengend Snippet: A – D Immunoblot analysis showing the levels of ERS pathway-related proteins GRP78, PERK, ATF6 ( A , C ) or AKT/mTOR pathway-related proteins ( B , D ) in PDI knockdown or overexpression HCT116 or MEF cells treated with γ-ray (4 Gy) or cisplatin (20 μM) irradiation for 24 h. E , F Immunoblot analysis showing the levels of ERS pathway-related proteins and AKT/mTOR pathway-related proteins in PDI knockdown or overexpression MEF cells treated with γ-ray irradiation ( E ) or cisplatin ( F ) for 24 h. G , H The existence of PDI in the co-precipitated complexes was confirmed by western blotting, IgG was employed as the negative control. Western blotting shows the association of PDI with GRP78 after Co-IP in HCT116 cells were treated with γ-ray (4 Gy) irradiation ( G ) or cisplatin (20 μM) ( H ) for 24 h. I , J The existence of PDI in the co-precipitated complexes was confirmed by western blotting, IgG was employed as the negative control. Western blotting shows the association of PDI with GRP78 after Co-IP in MEF cells were treated with γ-ray (4 Gy) irradiation ( I ) or cisplatin (20 μM) ( J ) for 24 h. K , L HCT116 cells were pretreated with or without 5 mM MK2206 ( K ) or 10 µM 4-PBA ( L ) for 2 h, then irradiated with 4 Gy γ-ray for 24 h. LC3II accumulation was measured using western blotting.

Article Snippet: The human cell lines HCT116, HEK293T, A549, HIEC, and MEF cells were kindly provided by Dr Chi Li (University of Louisville, Kentucky, USA).

Techniques: Western Blot, Knockdown, Over Expression, Irradiation, Negative Control, Co-Immunoprecipitation Assay

Journal: Cell Death & Disease

Article Title: Protein disulfide isomerase blocks the interaction of LC3II-PHB2 and promotes mTOR signaling to regulate autophagy and radio/chemo-sensitivity

doi: 10.1038/s41419-022-05302-w

Figure Lengend Snippet: A – D The interaction between PDI and PHB2 was detected by immunoprecipitation after the treatment with γ-ray (4 Gy) irradiation ( A , B ) or 20 μM cisplatin ( C , D ) for 24 h in HCT116 and MEF cells. E , F Immunofluorescence microscope analysis of co-localization of PDI (red) and PHB2 (green) in PDI knockdown or PDI overexpression HCT116 cells after 24 h of treatment with IR (4 Gy) ( E ) or cisplatin (20 μM) ( F ). Blue DAPI staining was used to stain the cell nucleus. Scale bar = 25 μm. G The interaction between PDI and PHB2 was detected by Flag-pull down in vitro. Purified Flag-PHB2 or Myc-PDI immobilized on the beads was incubated with purified Myc-PDI or Flag-PHB2. Input and bead-bound proteins were analyzed by immunoblotting with anti-PDI or anti-PHB2 antibodies. H The changes in the MMP of HCT116 cells before and after γ-ray (4 Gy) irradiation for 24 h were detected by the JC-1 probe. I Statistical map of MMP. J The expression of ROS in HCT116 cells after γ-ray (4 Gy) irradiation for 24 h. K , L Expression of mitophagy-related proteins in mitochondria treated with γ-ray (4 Gy) irradiation ( K ) after 24 h in HCT116 cells or treated with cisplatin (20 μM) after 24 h in MEF cells ( L ). Data were pooled from three independent experiments and the results are represented as mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The human cell lines HCT116, HEK293T, A549, HIEC, and MEF cells were kindly provided by Dr Chi Li (University of Louisville, Kentucky, USA).

Techniques: Immunoprecipitation, Irradiation, Immunofluorescence, Microscopy, Knockdown, Over Expression, Staining, In Vitro, Purification, Incubation, Western Blot, Expressing