Recombinant Human DICER1 protein, MYC/DDK-tagged

• Cat.No.: DICER1-784H
• Product Overview: Recombinant Human DICER1, transcript variant 2, fused with MYC/DDK tag at C-terminal was expressed in HEK293.

Specification

• Species: Human
• Source: HEK293
• Tag: DDK&Myc
• Description: This gene encodes a protein possessing an RNA helicase motif containing a DEXH box in its amino terminus and an RNA motif in the carboxy terminus. The encoded protein functions as a ribonuclease and is required by the RNA interference and small temporal RNA (stRNA) pathways to produce the active small RNA component that represses gene expression. Alternative splicing results in multiple transcript variants.
• Form: 25 mM Tris.HCl, pH 7.3, 100 mM glycine, 10% glycerol.
• Molecular Mass: 218.5 kDa
• Purity: > 80% as determined by SDS-PAGE and Coomassie blue staining
• Concentration: >50 ug/mL as determined by microplate BCA method

Gene Information

• Gene Name: DICER1 dicer 1, ribonuclease type III [ Homo sapiens ]
• Official Symbol: DICER1
• Synonyms: DICER1; dicer 1, ribonuclease type III; Dicer1, Dcr 1 homolog (Drosophila); endoribonuclease Dicer; Dicer; dicer 1; double stranded RNA specific endoribonuclease; HERNA; K12H4.8 LIKE; KIAA0928; K12H4.8-LIKE; helicase MOI; helicase-moi; Dicer1, Dcr-1 homolog; helicase with RNAse motif; dicer 1, double-stranded RNA-specific endoribonuclease; DCR1; MNG1
• Gene ID: 23405
• mRNA Refseq: NM_030621
• Protein Refseq: NP_085124
• MIM: 606241
• UniProt ID: Q9UPY3
• Chromosome Location: 14q32.2
• Pathway: Gene expression, organism-specific biosystem; MicroRNA (miRNA) Biogenesis, organism-specific biosystem; Regulatory RNA pathways, organism-specific biosystem; Small Interfering RNA (siRNA) Biogenesis, organism-specific biosystem; mRNA processing, organism-specific biosystem;
• Function: ATP binding; ATP-dependent helicase activity; double-stranded RNA binding; endonuclease activity; helicase activity; hydrolase activity; metal ion binding; miRNA binding; nucleotide binding; protein binding; ribonuclease III activity; ribonuclease III activity

Citation

PACT establishes a posttranscriptional brake on mitochondrial biogenesis by promoting the maturation of miR-181c

Journal: The Journal of Biological Chemistry    PubMed ID: 35598827    Data: 2022/5/19

Authors: Asli E. Dogan, Syed M. Hamid, Ebru Erbay

Article Snippet:Anti-rabbit (5450–0011) and mouse IgG (H + L) (5220–0337) were from SeraCare.Anti-rabbit (5450–0011) and mouse IgG (H + L) (5220–0337) were from SeraCare.. Recombinant human DICER1 protein was from Creative BioMart.. Recombinant human PACT protein (NBP2-51787) was from Novus Biologicals.Recombinant human PACT protein (NBP2-51787) was from Novus Biologicals.

PACT suppresses mitobiogenesis through mature miR-181c. A , list of mitochondrial targets of miR-181. B and C , Prkra +/+ or Prkra ?/? MEFs were transfected with empty vector (Empty Vec.) or FLAG-PACT and total RNA extracts were analyzed by qRT-PCR to determine ( B ) miR-181c and U6 small nuclear RNA ( U6 ) (n = 6) and ( C ) pre-miR-181c and U6 RNA expression (n = 6). D and E , HEK293T cells were transfected with scrambled or PRKRA siRNA and total RNA extracts were analyzed by qRT-PCR for ( D ) miR-181c and U6 RNA and ( E ) pre-miR-181c and U6 RNA expression (n = 3). F , left panel , DICER cleavage assay performed using synthetic pre-miR-181c (10 μM) as substrate with recombinant DICER or PACT (0.2 μg) at 37 °C for 4 h. Samples are separated in 15% Urea-PAGE and detected with SYBR gold staining. M indicates microRNA marker. The average band intensities for the mature miR product are indicated at the top of the gel. Right panel , 5 ml of same samples (in the left panel ) were analyzed by Western blotting using specific antibodies for DICER and PACT (n = 3). G – I , HEK293T cells were transfected with scrambled or miR-181c mimic (100 nM) (n = 3); ( G ) Mitochondria enriched fraction (MF) and total cell lysates (CL) proteins were analyzed by Western blotting using specific antibodies for PGC1α, TFAM, antibody cocktail against ETC proteins, SIRT1, NRF1, and β-actin. H , total genomic DNA was analyzed by qRT-PCR to determine mtDNA ( mitochondrial major and minor arc ): nucDNA ( B2M ) ratio (n = 3). I , mitochondrial respiration was analyzed by OCR. Arrows indicate time for drug injections (n = 5; data were normalized to mitochondrial mass quantified from total OXPHOS protein levels from same samples and represented as arbitrary units (A.U)). J – L , HEK293T cells were transfected with control or miR-181c AntagomiR (100 nM). J , MF and CL protein lysates were analyzed by Western blotting using specific antibodies for PGC1α, TFAM, antibody cocktail against ETC proteins, SIRT1, NRF1, and β-actin (n = 3). K , total genomic DNA was analyzed by qRT-PCR to determine mtDNA ( mitochondrial major and minor arc ): nucDNA ( B2M ) ratio (n = 3). L , mitochondrial respiration was analyzed by OCR. Arrows indicate time for drug injections (n = 5; data were normalized to mitochondrial mass quantified from total OXPHOS protein levels from same samples and represented as arbitrary units (A.U)). M , MEF cells were transfected with scrambled or miR-181c mimic (100 nM) and complex I (mOD/min), III (Units/μg), and IV (mOD/min) activity was measured by ELISA (n = 3). N , MEF cells were transfected with control or miR-181c AntagomiR (100 nM) and complex I (mOD/min), III (Units/μg), and IV (mOD/min) activity was measured by ELISA (n = 3). O and P , Prkra +/+ or Prkra ?/? MEF cells transfected with scrambled or miR-181c mimic (100 nM). O , total genomic DNA was analyzed by qRT-PCR for mtDNA (mitochondrial Cox1 or Nd4 ): nucDNA (nuclear ApoB ) ratio (n = 3). P , mitochondrial respiration was analyzed by OCR. Arrows indicate time for drug injections (n = 5; data were normalized to mitochondrial mass that was quantified from total OXPHOS protein levels from the same samples and represented as arbitrary units (A.U)). Protein expression was calculated relative to β-actin for whole cell lysate and Ponceau S. for mitochondrial fraction and depicted at the top of each blot. Data are mean ± SD. Unpaired t test with Welch’s correction or one-way ANOVA. ? p ≤ 0.05, ?? p ≤ 0.01, ??? p ≤ 0.001, ns: not significant. CL, cell lysate; Cyto, cytoplasmic fraction; ETC, electron transfer chain; MEF, mouse embryonic fibroblast; MF, mitochondrial fraction; mtDNA, mitochondrial DNA; nucDNA, nuclear DNA; OCR, oxygen consumption rate; siRNA, silencer RNA.