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Addgene inc flag ha ythdc1
Flag Ha Ythdc1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/flag+ha+ythdc1/pmc12839532-31-13-14?v=Addgene+inc
Average 93 stars, based on 9 article reviews

flag ha ythdc1 - by Bioz Stars, 2026-07

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NS1 enhances m 6 A addition to NS mRNA. ( A ) Experimental procedure for the identification of host factors interacting with NS mRNA using mass spectrometry. ( B ) The MS/MS spectrum of the identified YTHDC1 peptide immunoprecipitated by NS mRNA. ( C ) Growth kinetics of WSN/WT in the absence of YTHDC1. Infection of either A549 or A549-YTHDC1 knockout cells with WSN/WT at an MOI of 0.01. Supernatants were harvested at the indicated time points and viruses titrated by plaque assay. Error bars represent the mean ± SD (n = 3). Statistical significance was assessed using Student’s t-test: **** P <0.0001; h.p.i., hours post-infection. ( D ) m 6 A modification level of NS mRNA in WSN/WT infection. SON and HPRT1 serve as positive and negative controls, respectively. A549 cells were infected with WSN/WT for 6 h. Equal amounts of RNA subjected to immunoprecipitation using either a control IgG antibody or an anti-m 6 A antibody, followed by extraction and RT-qPCR analysis of the immunoprecipitated RNA. Error bars represent the mean ± SD (n = 3). ( E ) m 6 A modification levels of IAV genome segments. A549 cells were infected with either WSN/WT or WSN/Ans1 for 6 h. The cells were then lysed, and equal amounts of RNA subjected to immunoprecipitation using either a control IgG antibody or an anti-m 6 A antibody. Immunoprecipitated RNA was extracted and subjected to RT-qPCR using specific primers for viral genes. Data is presented as the mean from three independent experiments. ( F ) m 6 A modification levels of ( Left panel ) viral and ( Right panel ) host mRNAs. HEK 293 T cells were transfected with either NS1 overexpression vector or empty vector, together with Ans1 vector for NS mRNA transcription. After 48 h, cells were lysed, and equal amounts of RNA from each experimental condition subjected to immunoprecipitation using either a control IgG antibody or an anti-m 6 A antibody. Immunoprecipitated RNA was extracted and subjected to RT-qPCR using NS and SON gene-specific primers. Error bars represent the mean ± SD (n = 3). Statistical significance was determined using Student’s t-test: * P <0.05 and **** P <0.0001. ( G ) Impact of NS1 overexpression on m 6 A levels in WSN/Ans1 infection. HEK 293 T cells were transfected with increasing doses of an NS1 overexpression vector. After 48 h, the cells were infected with WSN/Ans1 or ( H ) WSN/WT for 6 h. Equal amounts of RNA were subjected to immunoprecipitation using either a control IgG antibody or an anti-m 6 A antibody, followed by extraction and RT-qPCR analysis of the immunoprecipitated RNA. Error bars represent the mean ± SD (n = 3). ( I ) M segment splicing ratio in WSN/WT and WSN/Ans1 infections. Total RNA from A549 cells infected with WSN/WT and WSN/Ans1 viruses at an MOI of 2 was isolated at the indicated time points. Quantification of M1 and M2 mRNAs was performed by RT–qPCR. Error bars represent the mean ± SD (n = 3). Statistical significance was determined using Student’s t-test: **** P <0.0001; h.p.i., hours post-infection.

Journal: Emerging Microbes & Infections

Article Title: Influenza NS1 drives N 6 -methyladenosine (m 6 A)-mediated autoregulation of viral mRNA splicing

doi: 10.1080/22221751.2025.2572761

Figure Lengend Snippet: NS1 enhances m 6 A addition to NS mRNA. ( A ) Experimental procedure for the identification of host factors interacting with NS mRNA using mass spectrometry. ( B ) The MS/MS spectrum of the identified YTHDC1 peptide immunoprecipitated by NS mRNA. ( C ) Growth kinetics of WSN/WT in the absence of YTHDC1. Infection of either A549 or A549-YTHDC1 knockout cells with WSN/WT at an MOI of 0.01. Supernatants were harvested at the indicated time points and viruses titrated by plaque assay. Error bars represent the mean ± SD (n = 3). Statistical significance was assessed using Student’s t-test: **** P <0.0001; h.p.i., hours post-infection. ( D ) m 6 A modification level of NS mRNA in WSN/WT infection. SON and HPRT1 serve as positive and negative controls, respectively. A549 cells were infected with WSN/WT for 6 h. Equal amounts of RNA subjected to immunoprecipitation using either a control IgG antibody or an anti-m 6 A antibody, followed by extraction and RT-qPCR analysis of the immunoprecipitated RNA. Error bars represent the mean ± SD (n = 3). ( E ) m 6 A modification levels of IAV genome segments. A549 cells were infected with either WSN/WT or WSN/Ans1 for 6 h. The cells were then lysed, and equal amounts of RNA subjected to immunoprecipitation using either a control IgG antibody or an anti-m 6 A antibody. Immunoprecipitated RNA was extracted and subjected to RT-qPCR using specific primers for viral genes. Data is presented as the mean from three independent experiments. ( F ) m 6 A modification levels of ( Left panel ) viral and ( Right panel ) host mRNAs. HEK 293 T cells were transfected with either NS1 overexpression vector or empty vector, together with Ans1 vector for NS mRNA transcription. After 48 h, cells were lysed, and equal amounts of RNA from each experimental condition subjected to immunoprecipitation using either a control IgG antibody or an anti-m 6 A antibody. Immunoprecipitated RNA was extracted and subjected to RT-qPCR using NS and SON gene-specific primers. Error bars represent the mean ± SD (n = 3). Statistical significance was determined using Student’s t-test: * P <0.05 and **** P <0.0001. ( G ) Impact of NS1 overexpression on m 6 A levels in WSN/Ans1 infection. HEK 293 T cells were transfected with increasing doses of an NS1 overexpression vector. After 48 h, the cells were infected with WSN/Ans1 or ( H ) WSN/WT for 6 h. Equal amounts of RNA were subjected to immunoprecipitation using either a control IgG antibody or an anti-m 6 A antibody, followed by extraction and RT-qPCR analysis of the immunoprecipitated RNA. Error bars represent the mean ± SD (n = 3). ( I ) M segment splicing ratio in WSN/WT and WSN/Ans1 infections. Total RNA from A549 cells infected with WSN/WT and WSN/Ans1 viruses at an MOI of 2 was isolated at the indicated time points. Quantification of M1 and M2 mRNAs was performed by RT–qPCR. Error bars represent the mean ± SD (n = 3). Statistical significance was determined using Student’s t-test: **** P <0.0001; h.p.i., hours post-infection.

Article Snippet: Expression plasmids YTHDC1 (#85167) and METTL14 (#53740) were from Addgene.

Techniques: Mass Spectrometry, Tandem Mass Spectroscopy, Immunoprecipitation, Infection, Knock-Out, Plaque Assay, Modification, Control, Extraction, Quantitative RT-PCR, Transfection, Over Expression, Plasmid Preparation, Isolation

YTHDC1 disrupts the interaction between SRSF3 and NS mRNA. ( A ) NS segment splicing ratio in A549-YTHDC1 knockout cells. A549 or A549-YTHDC1 knockout cells were infected with WSN/WT at an MOI of 2. Total RNA was extracted at the indicated time points. ( Left panel ) Levels of NS1 and NEP mRNAs were measured by RT–qPCR. Error bars represent the mean ± SD (n = 3). Statistical significance was determined using Student’s t-test: **** P <0.0001; h.p.i., hours post-infection. ( Right panel ) Efficiency of YTHDC1 knockout was validated by western blot. ( B ) NS segment splicing ratios in A549-SRSF3 knockdown cells: A549 or A549-siSRSF3 knockdown cells were infected with WSN/WT at an MOI of 2. Total RNA was extracted at specific time points. ( Left panel ) Levels of NS1 and NEP mRNAs, measured by RT–qPCR. Error bars represent the mean ± SD (n = 3). Statistical significance was determined using Student’s t-test: **** P <0.0001; h.p.i., hours post-infection. ( Right panel ) Efficiency of SRSF3 knockdown was validated by western blot. ( C ) Effect of YTHDC1 and m 6 A modification on binding of SRSF3 to NS mRNA. A549 and A549-YTHDC1 knockout cells were infected with either wild-type or mutant WSN viruses at an MOI of 2. At six hours post-infection, cells were lysed, and equal amounts of RNA were immunoprecipitated using either a control IgG antibody or an anti-SRSF3 antibody. The immunoprecipitated RNA was extracted and analyzed by RT-qPCR. Error bars represent the mean ± SD (n = 3). Statistical significance was determined using Student’s t-test: ** P <0.01, *** P <0.001 and **** P <0.0001. ( D ) ( Left panel ) Influence of YTHDC1 overexpression on SRSF3-NS mRNA interaction. HEK 293 T cells were transfected with increasing doses of YTHDC1 overexpression vector. At 48 h, cells were infected with WSN/WT at an MOI of 2 for 6 h. Equal amounts of RNA were immunoprecipitated with control IgG or anti-SRSF3 antibodies, then extracted and analyzed by RT-qPCR analysis. ( Right panel ) YTHDC1 binding to NS mRNA in cells infected with WSN/WT and WSN/A385C viruses. A549 cells were infected with each virus for 6 h, followed by YTHDC1-RIP to assess binding levels. Error bars represent the mean ± SD (n = 3). ( E ) Identification of SRSF3 binding sites on NS mRNA using modified SRSF3-RIP-qPCR (as described in A and B). A549 cells were infected with WSN/WT at an MOI of 2 for 6 h. Equal amounts of RNA were immunoprecipitated using either control IgG or anti-SRSF3 antibodies, followed by extraction and RT-qPCR analysis. (Left panel) Results from the first round of SRSF3-RIP with primers spanning the entire NS segment. (Right panel) Results from the second round of SRSF3-RIP using primers targeting the ED region of NS1. All data were normalized to the IgG control with the corresponding input RNA. Error bars represent the mean ± SD (n = 3). ( F ) Construction of NS deletion mutants for SRSF3 binding site identification. ( G ) SRSF3 binding to NS deletion mutants in the absence of YTHDC1. A549-YTHDC1 knockout cells were transfected with plasmids containing either full-length NS or NS deletion mutants (#1 Mut and #2 Mut) for 48 h, followed by modified SRSF3-RIP-qPCR. Error bars represent the mean ± SD (n = 3). Statistical significance was determined using Student’s t-test: **** P <0.0001. ( H ) Hypothetical model illustrating the competitive binding of YTHDC1 and SRSF3 on NS mRNA.

Journal: Emerging Microbes & Infections

Article Title: Influenza NS1 drives N 6 -methyladenosine (m 6 A)-mediated autoregulation of viral mRNA splicing

doi: 10.1080/22221751.2025.2572761

Figure Lengend Snippet: YTHDC1 disrupts the interaction between SRSF3 and NS mRNA. ( A ) NS segment splicing ratio in A549-YTHDC1 knockout cells. A549 or A549-YTHDC1 knockout cells were infected with WSN/WT at an MOI of 2. Total RNA was extracted at the indicated time points. ( Left panel ) Levels of NS1 and NEP mRNAs were measured by RT–qPCR. Error bars represent the mean ± SD (n = 3). Statistical significance was determined using Student’s t-test: **** P <0.0001; h.p.i., hours post-infection. ( Right panel ) Efficiency of YTHDC1 knockout was validated by western blot. ( B ) NS segment splicing ratios in A549-SRSF3 knockdown cells: A549 or A549-siSRSF3 knockdown cells were infected with WSN/WT at an MOI of 2. Total RNA was extracted at specific time points. ( Left panel ) Levels of NS1 and NEP mRNAs, measured by RT–qPCR. Error bars represent the mean ± SD (n = 3). Statistical significance was determined using Student’s t-test: **** P <0.0001; h.p.i., hours post-infection. ( Right panel ) Efficiency of SRSF3 knockdown was validated by western blot. ( C ) Effect of YTHDC1 and m 6 A modification on binding of SRSF3 to NS mRNA. A549 and A549-YTHDC1 knockout cells were infected with either wild-type or mutant WSN viruses at an MOI of 2. At six hours post-infection, cells were lysed, and equal amounts of RNA were immunoprecipitated using either a control IgG antibody or an anti-SRSF3 antibody. The immunoprecipitated RNA was extracted and analyzed by RT-qPCR. Error bars represent the mean ± SD (n = 3). Statistical significance was determined using Student’s t-test: ** P <0.01, *** P <0.001 and **** P <0.0001. ( D ) ( Left panel ) Influence of YTHDC1 overexpression on SRSF3-NS mRNA interaction. HEK 293 T cells were transfected with increasing doses of YTHDC1 overexpression vector. At 48 h, cells were infected with WSN/WT at an MOI of 2 for 6 h. Equal amounts of RNA were immunoprecipitated with control IgG or anti-SRSF3 antibodies, then extracted and analyzed by RT-qPCR analysis. ( Right panel ) YTHDC1 binding to NS mRNA in cells infected with WSN/WT and WSN/A385C viruses. A549 cells were infected with each virus for 6 h, followed by YTHDC1-RIP to assess binding levels. Error bars represent the mean ± SD (n = 3). ( E ) Identification of SRSF3 binding sites on NS mRNA using modified SRSF3-RIP-qPCR (as described in A and B). A549 cells were infected with WSN/WT at an MOI of 2 for 6 h. Equal amounts of RNA were immunoprecipitated using either control IgG or anti-SRSF3 antibodies, followed by extraction and RT-qPCR analysis. (Left panel) Results from the first round of SRSF3-RIP with primers spanning the entire NS segment. (Right panel) Results from the second round of SRSF3-RIP using primers targeting the ED region of NS1. All data were normalized to the IgG control with the corresponding input RNA. Error bars represent the mean ± SD (n = 3). ( F ) Construction of NS deletion mutants for SRSF3 binding site identification. ( G ) SRSF3 binding to NS deletion mutants in the absence of YTHDC1. A549-YTHDC1 knockout cells were transfected with plasmids containing either full-length NS or NS deletion mutants (#1 Mut and #2 Mut) for 48 h, followed by modified SRSF3-RIP-qPCR. Error bars represent the mean ± SD (n = 3). Statistical significance was determined using Student’s t-test: **** P <0.0001. ( H ) Hypothetical model illustrating the competitive binding of YTHDC1 and SRSF3 on NS mRNA.

Article Snippet: Expression plasmids YTHDC1 (#85167) and METTL14 (#53740) were from Addgene.

Techniques: Knock-Out, Infection, Quantitative RT-PCR, Western Blot, Knockdown, Modification, Binding Assay, Mutagenesis, Immunoprecipitation, Control, Over Expression, Transfection, Plasmid Preparation, Virus, Extraction

Working model describing NS1-mediated regulation of its own mRNA splicing via m 6 A modification . Upon entry of NS mRNA into the nucleus, NS1 interacts with METTL3 and METTL14 to facilitate the m 6 A addition on NS mRNA. The m 6 A modification is then recognized by the m 6 A reader YTHDC1, which blocks the association between NS mRNA and SRSF3, thereby suppressing splicing.

Journal: Emerging Microbes & Infections

Article Title: Influenza NS1 drives N 6 -methyladenosine (m 6 A)-mediated autoregulation of viral mRNA splicing

doi: 10.1080/22221751.2025.2572761

Figure Lengend Snippet: Working model describing NS1-mediated regulation of its own mRNA splicing via m 6 A modification . Upon entry of NS mRNA into the nucleus, NS1 interacts with METTL3 and METTL14 to facilitate the m 6 A addition on NS mRNA. The m 6 A modification is then recognized by the m 6 A reader YTHDC1, which blocks the association between NS mRNA and SRSF3, thereby suppressing splicing.

Article Snippet: Expression plasmids YTHDC1 (#85167) and METTL14 (#53740) were from Addgene.

Techniques: Modification

Fig. 2. YTHDC1 Knockdown Prolongs the Duration of the Early Stage of Mitosis

Journal: Biological & pharmaceutical bulletin

Article Title: N 6 -Methyladenosine Reader Protein YTHDC1 Supports Proper Mitotic Progression Partly through Regulation of TPX2-Aurora A Signaling.

doi: 10.1248/bpb.b24-00542

Figure Lengend Snippet: Fig. 2. YTHDC1 Knockdown Prolongs the Duration of the Early Stage of Mitosis

Article Snippet: Doxycycline (Dox)-inducible cell lines expressing FLAG and HA-tagged wild-type or mutant YTHDC1 (A549/YTHDC1-WT or -W377A/W428A) were established as previously described.11) Plasmids pcDNA3-FLAG-HA-hYTHDC1 was a gift from Samie Jaffrey (Addgene plasmid # 85167; http://n2t.net/ addgene:85167; RRID:Addgene_85167).12) FLAG-HA epitopetagged YTHDC1 was subcloned into pENTR4-no-ccDB (686-1) [a gift from Eric Campeau & Paul Kaufman (Addgene plasmid # 17424; http://n2t.net/addgene:17424; RRID: Addgene_17424)].13) The small interfering RNA (siRNA)-resistant YTHDC1 plasmid was created by introducing a silent mutation into the siYTHDC1#1 target site by PCR using the following primers: (sense) 5′-CTGGTCTCCAAGCCACTGAGCTCATCTGTTAGC-3′ and (antisense) 5′-CTGCCTCGAATGGACAGAAGGCTTTTGTCG-3′.

Techniques: Knockdown

Fig. 3. The Spindle Assembly Checkpoint Is Involved in the YTHDC1 Knockdown-Induced Delay in Mitotic Progression

Journal: Biological & pharmaceutical bulletin

Article Title: N 6 -Methyladenosine Reader Protein YTHDC1 Supports Proper Mitotic Progression Partly through Regulation of TPX2-Aurora A Signaling.

doi: 10.1248/bpb.b24-00542

Figure Lengend Snippet: Fig. 3. The Spindle Assembly Checkpoint Is Involved in the YTHDC1 Knockdown-Induced Delay in Mitotic Progression

Techniques: Knockdown

Fig. 4. YTHDC1 Supports Mitotic Progression Partly through Regulation of Centrosomal Abundance of Aurora A

Journal: Biological & pharmaceutical bulletin

Article Title: N 6 -Methyladenosine Reader Protein YTHDC1 Supports Proper Mitotic Progression Partly through Regulation of TPX2-Aurora A Signaling.

doi: 10.1248/bpb.b24-00542

Figure Lengend Snippet: Fig. 4. YTHDC1 Supports Mitotic Progression Partly through Regulation of Centrosomal Abundance of Aurora A

Techniques:

Fig. 5. YTHDC1 Represses the Centrosomal Abundance of Aurora A through the Regulation of TPX2 Expression

Journal: Biological & pharmaceutical bulletin

Article Title: N 6 -Methyladenosine Reader Protein YTHDC1 Supports Proper Mitotic Progression Partly through Regulation of TPX2-Aurora A Signaling.

doi: 10.1248/bpb.b24-00542

Figure Lengend Snippet: Fig. 5. YTHDC1 Represses the Centrosomal Abundance of Aurora A through the Regulation of TPX2 Expression

Techniques: Expressing

Figure 1. YTHDC1 and YTHDC2 interact with ZFC3H1 and U1-70K. (A) The main components of the MTREC complex (outlined by a dotted line) and associated factors in S. pombe (left), and their human homologs, including the PAXT complex (outlined by a dotted line) and YTH proteins (right). (B) HEK293 cells were either transfected with FLAG-tagged YTHDC1 or mock transfected. After 18–24 h, cell lysates were collected and subjected to immunoprecipitation using FLAG M2 beads. Precipitates were then treated with either buffer or a combination of RNase A and RNase T1 and reisolated to remove RNA-dependent interacting proteins. Samples were separated by SDS–PAGE and immunoblotted for the indicated proteins. Note

Journal: Life science alliance

Article Title: N -6-methyladenosine (m6A) promotes the nuclear retention of mRNAs with intact 5' splice site motifs.

doi: 10.26508/lsa.202403142

Figure Lengend Snippet: Figure 1. YTHDC1 and YTHDC2 interact with ZFC3H1 and U1-70K. (A) The main components of the MTREC complex (outlined by a dotted line) and associated factors in S. pombe (left), and their human homologs, including the PAXT complex (outlined by a dotted line) and YTH proteins (right). (B) HEK293 cells were either transfected with FLAG-tagged YTHDC1 or mock transfected. After 18–24 h, cell lysates were collected and subjected to immunoprecipitation using FLAG M2 beads. Precipitates were then treated with either buffer or a combination of RNase A and RNase T1 and reisolated to remove RNA-dependent interacting proteins. Samples were separated by SDS–PAGE and immunoblotted for the indicated proteins. Note

Article Snippet: The FLAG-HA-tagged YTHDC1 plasmid was purchased from Addgene (plasmid #85167).

Techniques: Transfection, Immunoprecipitation, SDS Page

$Figure 3. YTHDC1/YTHDC2 and ALKBH5 are required for the nuclear retention of 59SS motif containing mRNAs. (A) U2OS cells were treated with different lentivirus shRNAs against YTHDC1, YTHDC2, ALKBH5, ZFC3H1, or scrambled control. Note that “ZFC3H1-2” denotes shRNA #2 against ZFC3H1 as described previously (Lee et al, 2022). Lysates were collected after 96 h, separated by SDS–PAGE and immunoprobed for YTHDC1, YTHDC2, ALKBH5, ZFC3H1, tubulin, or mAb414, which recognizes FG-nucleoporin proteins. To effectively deplete YTHDC1 and YTHDC2, cells were treated with lentivirus containing two shRNAs against YTHDC1 and another two against YTHDC2. (B, C) Control- or YTHDC1/2-, ALKBH5-, ZFC3H1-depleted U2OS cells were transfected with the intronless ftz reporter plasmid (±59SS) and 59SS motif containing reporter that lacks m6A methylation sites (no-m6A-ftz-Δi-59SS). 18–24 h later the cells were ﬁxed and the mRNA was visualized by FISH. Representative images are shown in (B), scale bar = 20 μm, and quantiﬁcation is shown in (C). Each bar represents the average and standard error of three independent experiments, each experiment consisting of at least 30–60 cells. t test was performed, *P < 0.05, **P < 0.01, ***P < 0.001. (D) Schematic of PCF11-IPA RNA reporter. The position of the FISH probe used to visualize the IPA transcript is marked in gray and the position of the 39 cleavage site in the intron is as indicated. (E, F) Similar to (B, C), except that Control-, YTHDC1/YTHDC2-, or ALKBH5-depleted cells were transfected with the PCF11-IPA RNA reporter. (G, H) Total RNA was isolated from whole cell lysates (G) or nuclear and cytoplasmic fractions (H) from control- or YTHDC1/2-, ZFC3H1-depleted U2OS cells. PCF11-IPA was assessed by qRT-PCR using oligo- dT to generate cDNA and intronic-speciﬁc primers for the amplicon. Levels were normalized to the control depletion. Note that no signal was detected in reactions lacking reverse transcriptase (not shown). Each bar represents the average and standard error of three (G) or two (H) independent experiments. Source data are available for this ﬁgure.$

Journal: Life science alliance

Article Title: N -6-methyladenosine (m6A) promotes the nuclear retention of mRNAs with intact 5' splice site motifs.

doi: 10.26508/lsa.202403142

Figure Lengend Snippet: Figure 3. YTHDC1/YTHDC2 and ALKBH5 are required for the nuclear retention of 59SS motif containing mRNAs. (A) U2OS cells were treated with different lentivirus shRNAs against YTHDC1, YTHDC2, ALKBH5, ZFC3H1, or scrambled control. Note that “ZFC3H1-2” denotes shRNA #2 against ZFC3H1 as described previously (Lee et al, 2022). Lysates were collected after 96 h, separated by SDS–PAGE and immunoprobed for YTHDC1, YTHDC2, ALKBH5, ZFC3H1, tubulin, or mAb414, which recognizes FG-nucleoporin proteins. To effectively deplete YTHDC1 and YTHDC2, cells were treated with lentivirus containing two shRNAs against YTHDC1 and another two against YTHDC2. (B, C) Control- or YTHDC1/2-, ALKBH5-, ZFC3H1-depleted U2OS cells were transfected with the intronless ftz reporter plasmid (±59SS) and 59SS motif containing reporter that lacks m6A methylation sites (no-m6A-ftz-Δi-59SS). 18–24 h later the cells were ﬁxed and the mRNA was visualized by FISH. Representative images are shown in (B), scale bar = 20 μm, and quantiﬁcation is shown in (C). Each bar represents the average and standard error of three independent experiments, each experiment consisting of at least 30–60 cells. t test was performed, *P < 0.05, **P < 0.01, ***P < 0.001. (D) Schematic of PCF11-IPA RNA reporter. The position of the FISH probe used to visualize the IPA transcript is marked in gray and the position of the 39 cleavage site in the intron is as indicated. (E, F) Similar to (B, C), except that Control-, YTHDC1/YTHDC2-, or ALKBH5-depleted cells were transfected with the PCF11-IPA RNA reporter. (G, H) Total RNA was isolated from whole cell lysates (G) or nuclear and cytoplasmic fractions (H) from control- or YTHDC1/2-, ZFC3H1-depleted U2OS cells. PCF11-IPA was assessed by qRT-PCR using oligo- dT to generate cDNA and intronic-speciﬁc primers for the amplicon. Levels were normalized to the control depletion. Note that no signal was detected in reactions lacking reverse transcriptase (not shown). Each bar represents the average and standard error of three (G) or two (H) independent experiments. Source data are available for this ﬁgure.

Article Snippet: The FLAG-HA-tagged YTHDC1 plasmid was purchased from Addgene (plasmid #85167).

Techniques: Control, shRNA, SDS Page, Transfection, Plasmid Preparation, Methylation, Isolation, Quantitative RT-PCR, Amplification, Reverse Transcription

Figure 5. 59SS motif containing mRNAs are transferred from nuclear speckles to YTHDC1-containing foci in an m6A-dependent manner. (A, B) Plasmid DNA containing the ftz-Δi-59SS or no-m6A-ftz-Δi-59SS reporter genes was microinjected into U2OS nuclei. One hour post-injection, cells were ﬁxed and stained for ftz mRNA by FISH and SC35 by immunoﬂuorescence. Representative cells are shown in (A), each row represents a single ﬁeld of view. The merged image shows

Journal: Life science alliance

Article Title: N -6-methyladenosine (m6A) promotes the nuclear retention of mRNAs with intact 5' splice site motifs.

doi: 10.26508/lsa.202403142

Figure Lengend Snippet: Figure 5. 59SS motif containing mRNAs are transferred from nuclear speckles to YTHDC1-containing foci in an m6A-dependent manner. (A, B) Plasmid DNA containing the ftz-Δi-59SS or no-m6A-ftz-Δi-59SS reporter genes was microinjected into U2OS nuclei. One hour post-injection, cells were ﬁxed and stained for ftz mRNA by FISH and SC35 by immunoﬂuorescence. Representative cells are shown in (A), each row represents a single ﬁeld of view. The merged image shows

Article Snippet: The FLAG-HA-tagged YTHDC1 plasmid was purchased from Addgene (plasmid #85167).

Techniques: Plasmid Preparation, Injection, Staining

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