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Proteintech ythdf2 rabbit proteintech
Ythdf2 Rabbit Proteintech, supplied by Proteintech, used in various techniques. Bioz Stars score: 96/100, based on 431 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Figure 2. Downregulation of <t>YTHDF2,</t> HRSP12, or an RNase P/MRP Component Inhibits m6A-Mediated mRNA Decay (A) Schematic representation of m6A reporter mRNAs. The m6A target sites are highlighted in red. (B–D) Effects of downregulation of the indicated protein or RNA on the abundance of m6A reporter mRNA. HeLa cells were transiently transfected with the indicated siRNA. One day later, the cells were cotransfected with one of the reporter plasmids and with phCMV-MUP as a reference plasmid. Two days later, total-RNA samples were prepared and analyzed by quantitative real-time RT-PCR (qRT-PCR). The levels of m6A reporter mRNAs were normalized to those of MUP mRNA. The normalized levels of m6A Mut reporter mRNAs were arbitrarily set to 100%. n = 3 for (B) and (C); n = 5 for (D); *p < 0.05; **p < 0.01. (E and F) The inﬂuence of downregulation of the indicated protein on the half-life of m6A reporter mRNA. As performed in (B)–(D), except that the cells were treated with 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB, a potent transcription inhibitor) before cell harvesting. (E) WB showing the speciﬁc downregulation. (F) mRNA half-life plotted as a function of time. Total-RNA samples were prepared at the indicated time points. The levels of m6A reporter mRNAs were normalized to those of MUP mRNA. The normalized levels at 0 h were arbitrarily set to 100%. The y axis represents the level of mRNA remaining (percentage) on the logarithmic scale. (G and H) Complementation experiments using either parental or HRSP12-KO HAP1 cells and transiently expressed FLAG-HRSP12 or its variant. Parental or HRSP12-KO HAP1 cells were transiently cotransfected with one of the m6A reporter plasmids, phCMV-MUP, and a plasmid expressing FLAG-HRSP12 or its variant. (G) WB proving a speciﬁc KO of HRSP12 and comparable expression of FLAG-HRSP12 and its variant. (H) The efﬁciency of m6A-mediated mRNA decay. n = 3; **p < 0.01. Two-tailed, equal-sample-variance Student’s t test was conducted to calculate the p values.

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Figure 2. Downregulation of YTHDF2, HRSP12, or an RNase P/MRP Component Inhibits m6A-Mediated mRNA Decay (A) Schematic representation of m6A reporter mRNAs. The m6A target sites are highlighted in red. (B–D) Effects of downregulation of the indicated protein or RNA on the abundance of m6A reporter mRNA. HeLa cells were transiently transfected with the indicated siRNA. One day later, the cells were cotransfected with one of the reporter plasmids and with phCMV-MUP as a reference plasmid. Two days later, total-RNA samples were prepared and analyzed by quantitative real-time RT-PCR (qRT-PCR). The levels of m6A reporter mRNAs were normalized to those of MUP mRNA. The normalized levels of m6A Mut reporter mRNAs were arbitrarily set to 100%. n = 3 for (B) and (C); n = 5 for (D); *p < 0.05; **p < 0.01. (E and F) The inﬂuence of downregulation of the indicated protein on the half-life of m6A reporter mRNA. As performed in (B)–(D), except that the cells were treated with 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB, a potent transcription inhibitor) before cell harvesting. (E) WB showing the speciﬁc downregulation. (F) mRNA half-life plotted as a function of time. Total-RNA samples were prepared at the indicated time points. The levels of m6A reporter mRNAs were normalized to those of MUP mRNA. The normalized levels at 0 h were arbitrarily set to 100%. The y axis represents the level of mRNA remaining (percentage) on the logarithmic scale. (G and H) Complementation experiments using either parental or HRSP12-KO HAP1 cells and transiently expressed FLAG-HRSP12 or its variant. Parental or HRSP12-KO HAP1 cells were transiently cotransfected with one of the m6A reporter plasmids, phCMV-MUP, and a plasmid expressing FLAG-HRSP12 or its variant. (G) WB proving a speciﬁc KO of HRSP12 and comparable expression of FLAG-HRSP12 and its variant. (H) The efﬁciency of m6A-mediated mRNA decay. n = 3; **p < 0.01. Two-tailed, equal-sample-variance Student’s t test was conducted to calculate the p values.

Journal: Molecular cell

Article Title: Endoribonucleolytic Cleavage of m 6 A-Containing RNAs by RNase P/MRP Complex.

doi: 10.1016/j.molcel.2019.02.034

Figure Lengend Snippet: Figure 2. Downregulation of YTHDF2, HRSP12, or an RNase P/MRP Component Inhibits m6A-Mediated mRNA Decay (A) Schematic representation of m6A reporter mRNAs. The m6A target sites are highlighted in red. (B–D) Effects of downregulation of the indicated protein or RNA on the abundance of m6A reporter mRNA. HeLa cells were transiently transfected with the indicated siRNA. One day later, the cells were cotransfected with one of the reporter plasmids and with phCMV-MUP as a reference plasmid. Two days later, total-RNA samples were prepared and analyzed by quantitative real-time RT-PCR (qRT-PCR). The levels of m6A reporter mRNAs were normalized to those of MUP mRNA. The normalized levels of m6A Mut reporter mRNAs were arbitrarily set to 100%. n = 3 for (B) and (C); n = 5 for (D); *p < 0.05; **p < 0.01. (E and F) The inﬂuence of downregulation of the indicated protein on the half-life of m6A reporter mRNA. As performed in (B)–(D), except that the cells were treated with 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB, a potent transcription inhibitor) before cell harvesting. (E) WB showing the speciﬁc downregulation. (F) mRNA half-life plotted as a function of time. Total-RNA samples were prepared at the indicated time points. The levels of m6A reporter mRNAs were normalized to those of MUP mRNA. The normalized levels at 0 h were arbitrarily set to 100%. The y axis represents the level of mRNA remaining (percentage) on the logarithmic scale. (G and H) Complementation experiments using either parental or HRSP12-KO HAP1 cells and transiently expressed FLAG-HRSP12 or its variant. Parental or HRSP12-KO HAP1 cells were transiently cotransfected with one of the m6A reporter plasmids, phCMV-MUP, and a plasmid expressing FLAG-HRSP12 or its variant. (G) WB proving a speciﬁc KO of HRSP12 and comparable expression of FLAG-HRSP12 and its variant. (H) The efﬁciency of m6A-mediated mRNA decay. n = 3; **p < 0.01. Two-tailed, equal-sample-variance Student’s t test was conducted to calculate the p values.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-YTHDF2 Proteintech Cat#24744-1-AP; RRID: AB_2687435 Rabbit polyclonal anti-HRSP12 Thermo Scientific Cat#PA5-31352; RRID: AB_2548826 Rabbit polyclonal anti-POP1 Proteintech Cat#12029-1-AP; RRID: AB_2166477 Rabbit polyclonal anti-RPP25 Proteintech Cat#15461-1-AP Mouse polyclonal anti-RPP20 ModiQuest Cat#MQ 1.201-100 Rabbit polyclonal anti-METTL3 Proteintech Cat#15073-1-AP; RRID: AB_2142033 Rabbit polyclonal anti-METTL14 Sigma-Aldrich Cat#HPA038002; RRID: AB_10672401 Rabbit monoclonal anti-CNOT1 Proteintech Cat#14276-1-AP; RRID: AB_10888627 Rabbit polyclonal anti-XRN1 Bethyl Laboratories Cat#A300-443A; RRID: AB_2219047 Rabbit polyclonal anti-XRN2 Bethyl Laboratories Cat#A301-103A; RRID: AB_2218876 Mouse monoclonal anti-snRNP70 Santa Cruz Biotechnology Cat#sc-390899 Rabbit polyclonal anti-m6A Synaptic System Cat#202 003; RRID: AB_2279214 Mouse monoclonal anti-b-actin Sigma-Aldrich Cat#A5441; RRID: AB_476744 Rabbit polyclonal anti-GAPDH Ab frontier Cat#LF-PA0212 Mouse monoclonal anti-FLAG Sigma-Aldrich Cat#A8592; RRID: AB_439702 Mouse monoclonal anti-FLAG Sigma-Aldrich Cat#A220 Mouse monoclonal anti MYC Calbiochem Cat#OP10L; RRID: AB_2266865 Rat monoclonal anti-HA Roche Cat#11 867 423 001 Rabbit polyclonal anti-GST Bethyl Laboratories Cat#A190-122A; RRID: AB_67419 Mouse monoclonal anti-His GE Healthcare Cat#27-4710-01; RRID: AB_771435 Bacterial and Virus Strains Escherichia coli Rosetta(DE3)pLysS Novagen Cat#70956 Escherichia coli BL21 (DE3) Novagen Cat#69450 Chemicals, Peptides, and Recombinant Proteins Cycloheximide Sigma-Aldrich Cat#01810 Dexamethasone (Dex) Sigma-Aldrich Cat#D4902 5,6-dichloro-1-b-D ribofuranosylbenzimidazole (DRB) Sigma-Aldrich Cat#D1916 GST This paper N/A GST-human YTHDF2 This paper N/A GST-legionella RavZ This paper N/A His-human HRSP12 This paper N/A Critical Commercial Assays Dynabeads Protein G for Immunoprecipitation Invitrogen Cat#10003D Dynabeads Protein A for Immunoprecipitation Invitrogen Cat#10002D SMARTer smRNA-Seq Kit for Illumina Clontech Cat#635031 Oligotex mRNA Midi Kit QIAGEN Cat#70042 NE-PER Nuclear and Cytoplasmic Extraction Reagents Thermo Scientific Cat#78835 Deposited Data mRNA-seq, CLIP-seq, and PARE-seq This paper SRP155278 Mendeley Data dataset This paper https://doi.org/10.17632/rxjghsgczt.1 Experimental Models: Cell Lines Human: HeLa Korea Cell Line Bank KCLB No. 10002; RRID: CVCL_0030 Human: HEK293T ATCC ATCC number: CRL-3216; RRID: CVCL_0063 (Continued on next page) Molecular Cell 74, 1–14.e1–e8, May 2, 2019 e1

Techniques: Transfection, Plasmid Preparation, Quantitative RT-PCR, Cell Harvesting, Variant Assay, Expressing, Two Tailed Test

Figure 4. YTHDF2-Bound Transcripts Increase in Abundance and Half-Life after Downregulation of YTHDF2, HRSP12, or a Component of RNase P/MRP (A–F) Cumulative distribution function (CDF) plots for the relative change in the abundance of transcripts. Total-RNA samples were puriﬁed and subjected to mRNA sequencing (n= 2). CDFplotsof transcripts among non-targets (3,905), CLIP–IPtargets (1,974), CLIP targets(3,250), and CLIPXIP targets (1,276) were plotted as the relative changes in abundance of transcripts within each group after downregulation of YTHDF2 (A), HRSP12 (B), POP1 (C), RPP20 (D), METTL3 (E), or CNOT1 (F). (G–I) CDF plots for the relative change in the half-life of transcripts. To determine the half-life of transcripts using HeLa cells depleted of the indicated protein, mRNA sequencing experiments were carried out as described in STAR Methods (n = 2). CDF plots of transcripts were built as relative changes in the half-life of transcripts within each group after downregulation of YTHDF2 (G), HRSP12 (H), or POP1 (I). p values were calculated by the two-tailed Mann-Whitney U test.

Journal: Molecular cell

Article Title: Endoribonucleolytic Cleavage of m 6 A-Containing RNAs by RNase P/MRP Complex.

doi: 10.1016/j.molcel.2019.02.034

Figure Lengend Snippet: Figure 4. YTHDF2-Bound Transcripts Increase in Abundance and Half-Life after Downregulation of YTHDF2, HRSP12, or a Component of RNase P/MRP (A–F) Cumulative distribution function (CDF) plots for the relative change in the abundance of transcripts. Total-RNA samples were puriﬁed and subjected to mRNA sequencing (n= 2). CDFplotsof transcripts among non-targets (3,905), CLIP–IPtargets (1,974), CLIP targets(3,250), and CLIPXIP targets (1,276) were plotted as the relative changes in abundance of transcripts within each group after downregulation of YTHDF2 (A), HRSP12 (B), POP1 (C), RPP20 (D), METTL3 (E), or CNOT1 (F). (G–I) CDF plots for the relative change in the half-life of transcripts. To determine the half-life of transcripts using HeLa cells depleted of the indicated protein, mRNA sequencing experiments were carried out as described in STAR Methods (n = 2). CDF plots of transcripts were built as relative changes in the half-life of transcripts within each group after downregulation of YTHDF2 (G), HRSP12 (H), or POP1 (I). p values were calculated by the two-tailed Mann-Whitney U test.

Techniques: Sequencing, Two Tailed Test, MANN-WHITNEY

Journal: Molecular cell

Article Title: Endoribonucleolytic Cleavage of m 6 A-Containing RNAs by RNase P/MRP Complex.

doi: 10.1016/j.molcel.2019.02.034

Figure Lengend Snippet: Figure 5. HRSP12 Directly Binds to a Transcript and Elicits Rapid RNA Degradation via the YTHDF2-HRSP12-RNase P/MRP Pathway (A) Metagene proﬁles of the CLIP peaks of HRSP12 and YTHDF2 (Wang et al., 2014). (B) Venn diagrams for the number of genes (abundance of transcripts: FPKM R 5) containing HRSP12 peaks or YTHDF2 peaks. (C–G) CDF plots of the relative changes in abundance of transcripts among non-targets (2,213), H–Y (1,271), Y–H (1,634), and HXY targets (700) after down- regulation of YTHDF2 (C), HRSP12 (D), POP1 (E), RPP20 (F), or CNOT1 (G). (H) The logo of the consensus motif for HRSP12 binding. The consensus motif was discovered by MEME (upper, 3.4 3 1030) or HOMER (lower, p = 1.0 3 1073).

Techniques: Binding Assay

Figure 6. Transcripts Bound by Both HRSP12 and YTHDF2 Are Preferably Cleaved Downstream of the YTHDF2-Binding Site by RNase P/MRP (A) Nucleotide composition analysis of the 10-nucleotide region surrounding PARE MaxSeqs (2,257), where the cleavages were commonly dependent on YTHDF2, HRSP12, and POP1. (B) Metagene proﬁles of PARE MaxSeq, HRSP12 MaxPeak, and YTHDF2 MaxPeak. (C) Venn diagrams for the number of genes (abundance of transcripts: FPKM R 5) containing PARE MaxSeq, HRSP12 MaxPeak, or YTHDF2 MaxPeak. (D) Distance proﬁles of PARE MaxSeq, HRSP12 MaxPeak, and YTHDF2 MaxPeak in individual mRNAs (115). p values were calculated by the one-tailed Mann– Whitney U test. (E) Metagene proﬁles of the positions of PARE MaxSeq, HRSP12 MaxPeak, and YTHDF2 MaxPeak in an individual mRNA (115). p values were calculated by the one-tailed Mann–Whitney U test. (F) Length distribution of total-cell mRNAs (19,288) and common mRNAs (115). (G–I) CDF plots of relative changes in the half-life of transcripts among non-targets (1,576), HXY targets (700), and HXYXP targets (328) after downregulation of the indicated protein. p values were calculated by the two-tailed Mann–Whitney U test.

Journal: Molecular cell

Article Title: Endoribonucleolytic Cleavage of m 6 A-Containing RNAs by RNase P/MRP Complex.

doi: 10.1016/j.molcel.2019.02.034

Figure Lengend Snippet: Figure 6. Transcripts Bound by Both HRSP12 and YTHDF2 Are Preferably Cleaved Downstream of the YTHDF2-Binding Site by RNase P/MRP (A) Nucleotide composition analysis of the 10-nucleotide region surrounding PARE MaxSeqs (2,257), where the cleavages were commonly dependent on YTHDF2, HRSP12, and POP1. (B) Metagene proﬁles of PARE MaxSeq, HRSP12 MaxPeak, and YTHDF2 MaxPeak. (C) Venn diagrams for the number of genes (abundance of transcripts: FPKM R 5) containing PARE MaxSeq, HRSP12 MaxPeak, or YTHDF2 MaxPeak. (D) Distance proﬁles of PARE MaxSeq, HRSP12 MaxPeak, and YTHDF2 MaxPeak in individual mRNAs (115). p values were calculated by the one-tailed Mann– Whitney U test. (E) Metagene proﬁles of the positions of PARE MaxSeq, HRSP12 MaxPeak, and YTHDF2 MaxPeak in an individual mRNA (115). p values were calculated by the one-tailed Mann–Whitney U test. (F) Length distribution of total-cell mRNAs (19,288) and common mRNAs (115). (G–I) CDF plots of relative changes in the half-life of transcripts among non-targets (1,576), HXY targets (700), and HXYXP targets (328) after downregulation of the indicated protein. p values were calculated by the two-tailed Mann–Whitney U test.

Techniques: Binding Assay, One-tailed Test, MANN-WHITNEY, Two Tailed Test

Figure 7. Cooperative Binding of HRSP12 and YTHDF2 to m6A RNA Contributes to Endoribonucleolytic Cleavage of a Subset of m6A-Con- taining Circular RNAs (A–D) In vivo CLIPs of HRSP12 or YTHDF2. HeLa cells depleted of YTHDF2 (A), HRSP12 (B), or POP1 (C and D) were cotransfected with one of the SON reporter plasmids and with phCMV-MUP as a reference plasmid. The extracts of the cells were subjected to in vivo CLIPs using either a-HRSP12 antibody (A and C) or a-YTHDF2 antibody (B and D). The relative levels of Mut reporter mRNAs in CLIP using rIgG were arbitrarily set to 100%. n = 3; *p < 0.05; n.s.: not signiﬁcant. (E) In vitro CLIP using a biotinylated probe harboring either the HRSP12-binding motif (GGUUC) or its mutant variant (GCAAC) and harboring either nonmethylated (A) or m6A site. A biotinylated random 41-mer RNA probe served as a control. n = 3.

Journal: Molecular cell

Article Title: Endoribonucleolytic Cleavage of m 6 A-Containing RNAs by RNase P/MRP Complex.

doi: 10.1016/j.molcel.2019.02.034

Figure Lengend Snippet: Figure 7. Cooperative Binding of HRSP12 and YTHDF2 to m6A RNA Contributes to Endoribonucleolytic Cleavage of a Subset of m6A-Con- taining Circular RNAs (A–D) In vivo CLIPs of HRSP12 or YTHDF2. HeLa cells depleted of YTHDF2 (A), HRSP12 (B), or POP1 (C and D) were cotransfected with one of the SON reporter plasmids and with phCMV-MUP as a reference plasmid. The extracts of the cells were subjected to in vivo CLIPs using either a-HRSP12 antibody (A and C) or a-YTHDF2 antibody (B and D). The relative levels of Mut reporter mRNAs in CLIP using rIgG were arbitrarily set to 100%. n = 3; *p < 0.05; n.s.: not signiﬁcant. (E) In vitro CLIP using a biotinylated probe harboring either the HRSP12-binding motif (GGUUC) or its mutant variant (GCAAC) and harboring either nonmethylated (A) or m6A site. A biotinylated random 41-mer RNA probe served as a control. n = 3.

Techniques: Binding Assay, In Vivo, Plasmid Preparation, In Vitro, Mutagenesis, Variant Assay, Control