Journal: Cell reports

Article Title: Association of RanGAP to Nuclear Pore Complex Component, RanBP2/Nup358, is Required for Pupal Development in Drosophila

doi: 10.1016/j.celrep.2021.110151

Figure Lengend Snippet: A. Schematic comparison of the domain architecture of RanGAPs from S. pombe (SpRna1p), Drosophila melanogaster (DmRanGAP), Homo sapiens (HsRanGAP1), and Arabidopsis thaliana (AtRanGAP1). Catalytic domain, green; acidic domain, pink; and nuclear envelope targeting domain, yellow. B. Western blot analysis of whole cell lysate from DLD1 parental, RanGAP1WT-Flag and RanGAP1KR-Flag cells using RanGAP1 antibody. A SUMOylated form and an unmodified form of RanGAP1 were shown in DLD1 parental and RanGAP1WT-Flag cells, while only unmodified RanGAP1 can be observed in RanGAP1KR-Flag cells. C. Live imaging of DLD1 cells harboring mCherry tagged RanGAP1WT and RanGAP1KR. RanGAP1WT-mCherry localizes primarily at the nuclear envelop whereas RanGAP1KR-mCherry is dispersed in cytoplasm. D. Cell growth analysis of HCT116 with RanGAP1WT-Flag and RanGAP1KR-Flag. (N=3, p= 0.7769). E. Analysis of time from nuclear envelope break down (NEBD) to anaphase. No statistically significant difference in mitotic timing was observed between HCT116 cells harboring RanGAP1WT-Flag versus RanGAP1KR-Flag (N=34 and N=48, respectively, p=0.0521). F. Protein transport assay in DLD1 cells harboring RanGAP1WT-Flag versus RanGAP1KR-Flag using a 46 kDa mCherry-tagged model substrate carrying classic NLS and NES domains (N= 4 and 5 for RanGAP1WT- and RanGAP1KR- expressing cells, respectively) (Niopek et al., 2016). G. First order kinetic fitting curves for nuclear export (upper panel) and import (lower panel) with apparent import/export rates (k), derived from (F).

Article Snippet: Human colorectal carcinoma tissue culture cells HCT116 were cultured in McCoy’s 5A (ATCC) supplemented with heat-inactivated 10% FBS (Atlanta Biologicals) and antibiotics (100 IU/ml penicillin and 100 μg/ml streptomycin) in 5% CO2 atmosphere at 37°C.

Techniques: Comparison, Western Blot, Imaging, Transport Assay, Expressing, Derivative Assay

Journal: Cell reports

Article Title: Association of RanGAP to Nuclear Pore Complex Component, RanBP2/Nup358, is Required for Pupal Development in Drosophila

doi: 10.1016/j.celrep.2021.110151

Figure Lengend Snippet: Key Resources Table

Article Snippet: Human colorectal carcinoma tissue culture cells HCT116 were cultured in McCoy’s 5A (ATCC) supplemented with heat-inactivated 10% FBS (Atlanta Biologicals) and antibiotics (100 IU/ml penicillin and 100 μg/ml streptomycin) in 5% CO2 atmosphere at 37°C.

Techniques: Virus, Recombinant, Cloning, Oligo Synthesis, Plasmid Preparation, Software, Microscopy

Journal: Cell reports

Article Title: Association of RanGAP to Nuclear Pore Complex Component, RanBP2/Nup358, is Required for Pupal Development in Drosophila

doi: 10.1016/j.celrep.2021.110151

Figure Lengend Snippet: Key Resources Table

Article Snippet: All plasmid constructions were generated with Platinum SuperFi DNA Polymerase (Invitrogen) with primers/synthesized oligos listed in Supplemental Table 1 .

Techniques: Virus, Recombinant, Cloning, Oligo Synthesis, Plasmid Preparation, Software, Microscopy

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet: Schematic representation of pTRM4-TAA expression plasmid. The wild-type TRM4 gene is mutated at F602 position (TTT converted to TAA) to generate a premature termination codon (PTC) (indicated). The plasmid-borne TRM4 gene is fused with a multicomponent C-terminal tag to facilitate the detection and purification of full-length Trm4 product. The tag contains a 3C protease cleavage site that can be used to release the purified protein from the tag. The expression of TRM4 gene is under the control of Gal promoter, which is galactose-inducible. URA3 is an auxotroph selective marker in yeast. This is a high copy 2 µ plasmid.

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Expressing, Plasmid Preparation, Purification, Marker

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet: The strategy for cloning artificial snR81 using four primers overlapping PCR (also see Fig. 2). The construction is based on naturally occurring yeast snR81 box H/ACA RNA. The 5′-most primer is sense-strand sequence (forward), while the other primers have antisense sequences (reverse). The guide sequences (guide1, guide1′, guide2, and guide2′) are changed to base pair with substrate mRNAs, while the rest of the snR81 sequence remain unchanged. The amplified artificial snR81 is digested with BamHI and HindIII and subsequently cloned into pSEC plasmid under the control of the Gal promoter. LEU2 is an auxotroph selective marker in yeast.

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Clone Assay, Sequencing, Amplification, Plasmid Preparation, Marker

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet: Schematic representation of site-specific RNase H cleavage of TRM4 mRNA directed by 2′-O-methyl RNA–DNA chimera. The target sequence of TRM4 mRNA is shown and the PTC (UAA) is indicated. The antisense TRM4 2′-O-methyl RNA–DNA chimera is also shown. d represents deoxy, and m stands for 2′-O-methyl. The arrow indicates the RNase H cleavage site.

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Sequencing

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet: TLC analysis of uridine-to-Ψ conversion at the PTC within the TRM4 mRNA transcript. The PTC-containing TRM4 mRNA, coexpressed with a PTC-specific guide RNA, is purified by oligonucleotide affinity chromatography. The RNA is cleaved by RNase H (directed by a specific 2′-O-methyl RNA–DNA chimera) at the site 5′ of the PTC (UAA). The resulting 3′-half fragment is 5′ radiolabeled with 32P through dephosphorylation and rephosphorylation (see text). The labeled RNA is digested with nuclease P1 to completion. The digested sample is mixed with an equal amount of 5′-32P-adenosine-monophosphate, 5′–32P-cytidine-monophosphate, and 5′–32P-guanosine-monophosphate, and analyzed by two-dimensional TLC. The first and second dimensions are indicated. The origin and the positions of each 5′-phosphorylated nucleotide are also indicated.

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Purification, Affinity Chromatography, De-Phosphorylation Assay, Labeling

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet: Ψ-Mediated nonsense suppression detected by Western blotting. Equal amounts of total protein are loaded (lanes 1–4), and anti-Protein A and anti-GAPDH (loading control) are used for blotting. Readthrough of PTC is visualized (lane 4), where a cognate (PTC-specific) guide RNA designed to target the PTC for pseudouridylation is coexpressed. In contrast, no significant suppression is observed when no guide RNA (lane 1) or a guide RNA-containing random guide sequences is coexpressed (lane 3). Lane 2 is a positive control where the wild-type TRM4 mRNA (with no PTC) is expressed.

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Western Blot, Positive Control

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet:

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Plasmid Preparation

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet:

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques:

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet:

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques:

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet:

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques:

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet:

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Plasmid Preparation

Journal: Methods in enzymology

Article Title: Pseudouridine in mRNA: Incorporation, Detection, and Recoding

doi: 10.1016/bs.mie.2015.03.009

Figure Lengend Snippet:

Article Snippet: Biotin-streptavidin binding buffer: 0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2 Biotinylated- TRM4 antisense oligo (IDT): biotin-5′-CCACTCTTGTTGGTTCACCAGTGGC-3′ Streptavidin agarose bead (Pierce) Elution buffer: 0.1 M glycine–HCl pH 7.0

Techniques: Plasmid Preparation