highyield t7 mrna synthesis kit  (Jena Bioscience)

Journal: Nucleic Acids Research

Article Title: Small nucleolar RNAs promote the restoration of muscle differentiation defects in cells from myotonic dystrophy type 1

doi: 10.1093/nar/gkaf232

Figure Lengend Snippet: Functional studies of SNORA40 and SNORA70 in muscle cells. Immunofluorescence in normal MT with (ASO CTRL) or without (ASOs SNORA40 and SNORA70) the presence of ( A ) SNORA40 and ( E ) SNORA70 ( n ≥ 3). Relative fusion index measured at 5 days post-differentiation after the loss of ( B ) SNORA40 and ( F ) SNORA70 in normal muscle cells. More than 800 nuclei per replicate were counted to calculate the fusion index ( n ≥ 3). Relative expression of the CCND3 and MYOG mRNA differentiation markers in normal muscle cells depleted in ( C ) SNORA40 and ( G ) SNORA70 ( n ≥ 3). Relative expression of the MYF5 and CCNB1 mRNA proliferation markers in normal muscle cells depleted in ( D ) SNORA40 and ( H ) SNORA70 ( n ≥ 3). Immunofluorescence in DM1 MT with plasmids expressing either a random sequence (Rd), ( I ) SNORA40 (SNORA40), or ( M ) SNORA70 ( n ≥ 3). Relative to the fusion index measured at 5 days post-differentiation after the gain of ( J ) SNORA40 and ( N ) SNORA70 in DM1 muscle cells. More than 800 nuclei per replicate were counted to calculate the fusion index ( n ≥ 3). Relative expression of the CCND3 and MYOG mRNA differentiation markers in DM1 muscle cells expressing ( K ) SNORA40 and ( O ) SNORA70 ( n ≥ 3). Relative expression of the MYF5 and CCNB1 mRNA proliferation markers in DM1 muscle cells expressing ( L ) SNORA40 and ( P ) SNORA70 ( n ≥ 3). ASO, gapmers; CTRL, control; Alpha-tub, alpha-tubulin; FI, fusion index; U6, RNU6; CCND3, Cyclin D3; MYOG, Myogenin; MYF5, Myogenic factor 5; CCNB1, Cyclin B1; d, day of differentiation. Scale bar = 10 μm. Error bars represent the standard error of the mean (SEM). * indicates P -value< .05. Significant differences were assessed by comparing the two conditions: ASO CTRL versus ASO SNORA40 at each corresponding time point. Statistical analyses were performed using a one-way ANOVA Fisher-means comparison test for the Fl results and a two-way ANOVA Tukey’s multiple comparisons test for the qPCR results.

Article Snippet: Full-length CCND3 ivt + Ψ was in vitro transcribed using the HighYield T7 mRNA Synthesis Kit (Ψ-UTP, Jena Bioscience, Germany) before IP.

Techniques: Functional Assay, Immunofluorescence, Expressing, Sequencing, Control, Comparison

Journal: Nucleic Acids Research

Article Title: Small nucleolar RNAs promote the restoration of muscle differentiation defects in cells from myotonic dystrophy type 1

doi: 10.1093/nar/gkaf232

Figure Lengend Snippet: Interaction of CCND3 with SNORA70 and SNORA40. ( A and B ) Physical interaction of CCND3 with SNORA40 and SNORA70, respectively, as predicted with snoGPS software. Scores are indicated in bold. ( C ) The accumulation of CCND3 in the nucleoli. Isolation of nucleoli by sucrose cushion centrifugation was performed as described in . The total RNA from each fraction was extracted and the localization of CCND3 mRNA was assessed by RT-PCR ( n = 2). To confirm the correct isolation of the nucleoli, MALAT1 RNA and SNORD16 were used as nuclear (negative control) and nucleolar (positive control) markers, respectively. cDNA (50 ng of equivalent RNA) was used for each PCR reaction. ( D and E ) Direct interaction of CCND3 with SNORA40 and SNORA70, respectively. U6 and ACA36B were used as negative controls ( n = 2). ( F ) Immunoprecipitation (1 μg of RNA) of Psi in mRNA-enriched populations followed by RT-PCR. Five percent of the input was used. In vitro transcribed CCND3 RNA with and without pseudouridines were used as positive and negative controls, respectively ( n = 3). Ψ, Psi; T, total fraction; C, cytoplasmic fraction; N, nuclear fraction; Np, nucleoplasmic fraction; Nuc, nucleolar fraction; Blk, mock PCR; RNA PD, RNA pull-down; IP, Immunoprecipitation; Ig, immunoglobulin; 18S, rRNA 18S; SH3BGRL3, SH3 domain binding glutamic acid-rich-like protein 3.

Article Snippet: Full-length CCND3 ivt + Ψ was in vitro transcribed using the HighYield T7 mRNA Synthesis Kit (Ψ-UTP, Jena Bioscience, Germany) before IP.

Techniques: Software, Isolation, Centrifugation, Reverse Transcription Polymerase Chain Reaction, Negative Control, Positive Control, Immunoprecipitation, In Vitro, Binding Assay

Journal: EMBO Reports

Article Title: miRNA-target complementarity in cnidarians resembles its counterpart in plants

doi: 10.1038/s44319-024-00350-z

Figure Lengend Snippet: Reagents and tools table

Article Snippet: In vitro transcription was conducted with HighYield T7 Cap 1 AG (3‘-OMe) mRNA Synthesis Kit (m5CTP) (Jena Bioscience, Germany) using 800 ng of amplified template followed by Turbo DNase treatment (Thermo Fisher Scientific) by incubation with 1 µl of DNase for 30 min at 37 °C twice sequentially.

Techniques: Sequencing, shRNA, SYBR Green Assay, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Magnetic Beads, Multiplex Assay, Sensitive Assay, Software, Imaging, Spectrophotometry, Real-time Polymerase Chain Reaction

Journal: Advanced Science

Article Title: PCBP1/2 and TDP43 Function as NAT10 Adaptors to Mediate mRNA ac 4 C Formation in Mammalian Cells

doi: 10.1002/advs.202400133

Figure Lengend Snippet: Identification and confirmation of TDP43 and PCBP1/2 as NAT10 adaptors. A) Scheme of N‐4‐acetylcytidine formation in mammalian RNAs. THUMPD1 and box C/D snoRNAs facilitate NAT10 in tRNA and 18S rRNA ac 4 C production, respectively. In mRNA ac 4 C formation, the presumed adaptor remains unknown. B) Comparison of NAT10 interactomes in HEK293T cells and mouse testis. HEK293T cells expressing N‐terminus FLAG‐tagged NAT10 and wild‐type (WT) mouse testis were subjected to affinity purification with anti‐FLAG and anti‐NAT10 antibody, respectively, and mass spectrometry analysis. RNA‐binding proteins (RBPs) including PCBP1, PCBP2, and TDP43 presented affinity toward NAT10. [ ¹⁵ ] C) Co‐immunoprecipitation (co‐IP) results showing the interactions between FLAG‐tagged NAT10 and HA‐tagged TDP43, PCBP1, and PCBP2, which were resistant to RNase A digestion. D) Endogenous immunoprecipitation (endo‐IP) results showing spontaneous coordination of TDP43, NAT10, and PCBP1/2 in 293T cells. Anti‐TDP43 antibody was applied in endo‐IP. Arrows indicated the beads band. E‐F) Diagram of N‐terminal truncated PCBP1/2 constructs and validation of the interaction between NAT10 and WT or mutant PCBPs. Co‐IP results indicated that the hnRNP K homology (KH) 1 domain in PCBP1/2 was responsible for their conjugation with NAT10.

Article Snippet: The mouse β‐globin DNA template (corresponding sequences in Table , Supporting Information) was in vitro transcribed using a High Yield T7 CAP 1 AG+ac 4 CTP mRNA Synthesis Kit (Jena Bioscience) according to the manufacturer's instructions.

Techniques: Comparison, Expressing, Affinity Purification, Mass Spectrometry, RNA Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Construct, Mutagenesis, Conjugation Assay

Journal: Advanced Science

Article Title: PCBP1/2 and TDP43 Function as NAT10 Adaptors to Mediate mRNA ac 4 C Formation in Mammalian Cells

doi: 10.1002/advs.202400133

Figure Lengend Snippet: Depletion of PCBP1/2 and TDP43 resulted in decreased mRNA ac 4 C abundance in HEK293T cells. A) Comparison of the mRNA expression levels of PCBP1/2, TDP43 , and NAT10 between the control group (si NC ) and upon PCBP1/2 or TDP43 knockdown ( siPCBP1/2 and siTDP43 , respectively) by RT‐qPCR. Gene expression levels were normalized to GAPDH . Mean ± SEM, n = 3. P ‐values were calculated using the two‐tailed Student's t ‐test between the knockdown groups and si NC group. ***P < 0.001. n.s ., not significant. B) Comparison of the protein expression levels of PCBP1/2, TDP43, and NAT10 between the control group (si NC ) and PCBP1/2 or TDP43 knockdown groups. DDB1 was blotted as a loading control. C‐E) LC‐MS/MS detection of ac 4 C abundance (ac 4 C/C) in total RNA, poly(A) RNA, and non‐poly(A) RNA in the control group and in PCBP1/2 , TDP43 , or NAT10 knockdown groups. The ac 4 C abundance was normalized to the si NC group in each RNA type. Mean ± SEM. *P < 0.05, **P < 0.01. F‐H) LC‐MS/MS detection of m 6 A, m 5 C, and f 5 C abundance (m 6 A/A, m 5 C/C, and f 5 C/C, respectively) in poly(A) RNA in the control group and upon PCBP1/2 , TDP43 , or NAT10 knockdown. The indicated modification abundance was normalized to the control group. Mean ± SEM.

Article Snippet: The mouse β‐globin DNA template (corresponding sequences in Table , Supporting Information) was in vitro transcribed using a High Yield T7 CAP 1 AG+ac 4 CTP mRNA Synthesis Kit (Jena Bioscience) according to the manufacturer's instructions.

Techniques: Comparison, Expressing, Control, Knockdown, Quantitative RT-PCR, Two Tailed Test, Liquid Chromatography with Mass Spectroscopy, Modification

Journal: Advanced Science

Article Title: PCBP1/2 and TDP43 Function as NAT10 Adaptors to Mediate mRNA ac 4 C Formation in Mammalian Cells

doi: 10.1002/advs.202400133

Figure Lengend Snippet: PCBP1/2 and TDP43 facilitated the binding of NAT10 to ac 4 C‐preferred mRNAs. A) Western blots of immunoprecipitated PCBP1, PCBP2, and TDP43 in endogenous RNA‐immunoprecipitation (RIP). Rabbit isotype IgG was applied as the control. B‐D) RT‐qPCR results showing that mRNAs that served as non‐preferred ( GAPDH and EEF1A1) , highly preferred ( RRBP1 , RBBP6 , and UPF3B ), and moderately preferred ( FUS , ZFP36L2 and LAMP1 ) ac 4 C substrates were tethered by PCBP1/2 and TDP43 in varying degrees. Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. n.s . not significant. E‐F) RT‐qPCR results showing that acetylation‐preferred mRNA 5′‐UTRs were diversely tethered by PCBP1/2 and TDP43. Mean ± SEM. G) Western blots of immunoprecipitated NAT10, TDP43, PCBP1, and PCBP2 in RIP by anti‐NAT10 antibody in the control ( siNC ) and the adaptor‐depleted groups ( siPCBP1/2 and siTDP43 , respectively). The interaction between TDP43 and NAT10 was maintained when PCBP1/2 was depleted. Vice versa. H) Schematic illustration of NAT10/PCBP/TDP43 complex in mRNA acetylation. Under normal conditions, NAT10/PCBP1/TDP43 formed a stable heterogeneous tetramer, with PCBPs binding to NAT10 through their KH1 domain. By tethering mRNAs, PCBP1/2 and TDP43 recruited NAT10 to preferred mRNAs and NAT10 transferred acetyl groups to cytidines at the indicated sites. Loss of PCBP1/2 or TDP43 incurred instability in the complex, resulting in unspecific binding to non‐acetylation mRNA substrate or even failure in mRNA binding, consequently leading to aberrant ac 4 C formation. I‐K) RT‐qPCR results showing the varied connection between NAT10 and mRNAs of the indicated acetylation‐preferred groups upon PCBP1/2 and TDP43 knockdown. Mean ± SEM. L‐M) RT‐qPCR results showing the diverted connection between NAT10 and mRNA 5′‐UTRs of the UPF3B (L) and LAMP1 (M) upon PCBP1/2 and TDP43 knockdown. Mean ± SEM.

Article Snippet: The mouse β‐globin DNA template (corresponding sequences in Table , Supporting Information) was in vitro transcribed using a High Yield T7 CAP 1 AG+ac 4 CTP mRNA Synthesis Kit (Jena Bioscience) according to the manufacturer's instructions.

Techniques: Binding Assay, Western Blot, Immunoprecipitation, RNA Immunoprecipitation, Control, Quantitative RT-PCR, Knockdown

Journal: Advanced Science

Article Title: PCBP1/2 and TDP43 Function as NAT10 Adaptors to Mediate mRNA ac 4 C Formation in Mammalian Cells

doi: 10.1002/advs.202400133

Figure Lengend Snippet: Affinity between PCBP1 and mRNA affected ac 4 C site preference. A) IGV browser shots of transcript reads of RRBP1 exon 14 – 16 mapping in acRIP‐seq mapping to the human reference genome. Nucleotides 3299–3313 within RRBP1 exon15 (CGCCAGCUCCCGCGG) were predicted to harbor ac4C according to PACES, further designated as RRBP1 site . B) RT‐qPCR results confirming RRBP1 exon15 mRNA acetylation in the control group ( siNC ) but lost upon PCBP1/2 , TDP43 , or NAT10 depletion. Mean ± SEM. *P < 0.05, ***P < 0.001. C) RT‐qPCR results showing PCBP1 affinity in interaction with RRBP1 exon15 mRNA in endogenous RIP. Mean ± SEM. n.s . not significant. D) RT‐qPCR results showing the affinity of NAT10 toward RRBP1 exon15 mRNA decreased or lost upon TDP43 and PCBP1/2 knockdown, respectively. Mean ± SEM. E) Illustration of RNA probe designed for RNA pull‐down. RRBP1 site was cloned from HEK293T cDNA and in vitro transcribed with biotinylated‐uridine incorporation directly or after neutral mutation in which most cytidines within RRBP1 site were substituted by other nucleosides (C3299U, C3302G, C3305U, C3307G, C3308U, C3309A, and C3311G, indicated as RRBP1 site1–WT and RRBP1 site1–mut , respectively). F) RNA pull‐down results indicated PCBP1/2 presented specific affinity toward WT RRBP1 site . G) RNA pull‐down results indicated diverted affinity between RRBP1 exon15 and NAT10/PCBP/TDP43 complex subunits in the control group and upon PCBP1/2 , TDP43 , and NAT10 knockdown. H) Schematic of RNA pull‐down analysis with cytidine and acetylated cytidine incorporated probes. WT and mutated RRBP1 exon 15 were in vitro transcribed with cytidines and acetylated cytidines as indicated. Biotinylated uridines were also incorporated for further enrichment. The RNA probes were mixed with the indicated cell lysates and subjected to affinity pull‐down. I) RNA pull‐down results showing the affinity between the PCBP/TDP43/NAT10 complex and the indicated RNA probes. PCBP1 specifically interacted with RRBP1 exon15 , discarding the incorporation of ac 4 C, while PCBP2 merely contacted with ac 4 C(–) RNAs. TDP43 presented no difference in binding with ac 4 C‐containing or ac 4 C‐lacking mRNAs. The gray values of the bands were quantified by ImageJ and normalized to the control group pulled down through probes containing WT sequences. #N/A indicated that no band detected.

Article Snippet: The mouse β‐globin DNA template (corresponding sequences in Table , Supporting Information) was in vitro transcribed using a High Yield T7 CAP 1 AG+ac 4 CTP mRNA Synthesis Kit (Jena Bioscience) according to the manufacturer's instructions.

Techniques: Quantitative RT-PCR, Control, Knockdown, Clone Assay, In Vitro, Mutagenesis, Binding Assay

Journal: Advanced Science

Article Title: PCBP1/2 and TDP43 Function as NAT10 Adaptors to Mediate mRNA ac 4 C Formation in Mammalian Cells

doi: 10.1002/advs.202400133

Figure Lengend Snippet: NAT10/PCBP/TDP43 complex functioned in mRNA acetylation in mouse testes. A) Endo‐IP results showing the spontaneous coordination of TDP43, NAT10, and PCBP1/2 in mouse testes. Anti‐TDP43 antibody was applied in endo‐IP. Arrows indicated the beads band. B) Western blot results showing TDP43 expression in spermatocytes isolated from WT mouse testes using flow cytometry sorting (FACS) (LZ, leptotene and zygotene, PD, pachytene and diplotene, MII, metaphase II and RS, round spermatids). α‐tubulin was blotted as a loading control. C) Acetylated mRNAs in mouse testes were defined through enrichment abundance between the acRIP and the inputs and IgG‐enriched groups. FC, fold change. D) Heatmap indicating enrichment levels of WT mouse testes ac 4 C targets. The color key from red to blue indicates relative enrichment extents from high to low. E) Enriched sequence motif analysis of ac4C peak clusters identified by acRIP‐seq in mouse testis. A CCHCAGSHC (H = C/U/A, S = C/G, P = 1.5E‐7) motif was detected by MEME analysis. F) IGV browser views of highly acetylated ( Enho 3′‐UTR), moderately acetylated ( Hoxd9 ), and not‐acetylated ( Eef1a1 ) transcripts mapping to the mouse reference genome (mm10). Reads in the acRIP, IgG, and input groups are presented. The intron/exon (line/box) genomic structure is shown in dark blue. G) RT‐qPCR results showing diverted acetylation abundance of highly acetylated (3′‐UTR of Enho ), moderately acetylated ( Hoxd9 ), and low acetylated mRNAs ( Eef1a1 ). Mean ± SEM. *P < 0.05, ** *P < 0.001. H) RT‐qPCR results showing TDP43 affinity toward preferred ac 4 C‐targeted mRNAs in mouse testes. Mean ± SEM. * *P < 0.01.

Article Snippet: The mouse β‐globin DNA template (corresponding sequences in Table , Supporting Information) was in vitro transcribed using a High Yield T7 CAP 1 AG+ac 4 CTP mRNA Synthesis Kit (Jena Bioscience) according to the manufacturer's instructions.

Techniques: Western Blot, Expressing, Isolation, Flow Cytometry, Control, Sequencing, Quantitative RT-PCR