Journal: bioRxiv

Article Title: Pooled scanning of protein variants identifies novel RNA-binding mutants

doi: 10.1101/2025.04.02.646914

Figure Lengend Snippet: (A) Experimental scheme of RBR-scan. RBR-scan plasmid libraries comprise multiple genes with point mutations fused to distinct peptide barcodes. Pools of protein variants are purified and subjected to RNA-affinity purification. The abundance of peptide barcodes in the RNA-bound vs. the input fraction is determined by LC-MS. Mutants with RNA-binding defects will show depletion of the corresponding barcode in the RNA-bound fraction relative to WT barcodes. The structure of the MS2 coat protein (PDB: 1ZDI) and its cognate stem loop RNA (orange) are shown with a key RNA-binding residue highlighted in green. (B) Domain organizations of GST-fused MS2 coat protein variants with N-terminal peptide barcodes. See methods for barcode composition rules. (C) Coomassie stain of GST-fused barcoded MS2 coat proteins (predicted MW: 43.5 kDa). (D) Western blot of individual MS2 coat protein variants after RNA affinity purification. (E) Relative peptide barcode abundances as measured by LC-MS in an equimolar pool of the four protein variants. Bars represent the mean ± SEM. Data from 2 technical replicates. (F) Same as (E) but after RNA affinity purification. Bars represent the mean barcode enrichment ratio (i.e., ratio of input to pull-down) relative to input ± SEM. Heatmap shows the log 2 (fold-change) of the enrichment ratio relative to WT. Data from 3 technical replicates. P values are from ANOVA and Holm-Sidak test.

Article Snippet: Biotinylated RNA was synthesized by in vitro transcription using a template containing three MS2 stem loop repeats (HiScribe T7 High Yield RNA Synthesis Kit, NEB E2040S) following manufacturer protocol except UTP was added at a ratio of 6.5:1 UTP:biotin-16-UTP (Sigma-Aldrich, 11388908910).

Techniques: Plasmid Preparation, Purification, Affinity Purification, Liquid Chromatography with Mass Spectroscopy, RNA Binding Assay, Residue, Staining, Western Blot

Journal: bioRxiv

Article Title: Pooled scanning of protein variants identifies novel RNA-binding mutants

doi: 10.1101/2025.04.02.646914

Figure Lengend Snippet: (A) Experimental scheme of low-throughput RBR-scan proof-of-concept experiment. BC, barcode. 3A, R49A-K57A-K61A mutant. (B) Heatmaps of mutation frequencies (% of reads) at each amino acid (x-axis) for plasmid pool of commercially synthesized MS2 coat protein variants paired to different barcodes (y-axis), as determined by nanopore sequencing. Positions of point mutations are indicated. (C) Frequency of mutant reads associated with each barcodes. Desired mutation (mut, black) is the correctly assigned mutation, WT sequences are in white, and other reads (gray) indicate mutation that do not correspond to either.

Article Snippet: Biotinylated RNA was synthesized by in vitro transcription using a template containing three MS2 stem loop repeats (HiScribe T7 High Yield RNA Synthesis Kit, NEB E2040S) following manufacturer protocol except UTP was added at a ratio of 6.5:1 UTP:biotin-16-UTP (Sigma-Aldrich, 11388908910).

Techniques: Mutagenesis, Plasmid Preparation, Synthesized, Nanopore Sequencing

Journal: bioRxiv

Article Title: Pooled scanning of protein variants identifies novel RNA-binding mutants

doi: 10.1101/2025.04.02.646914

Figure Lengend Snippet: (A) Peptide barcode design. See methods. (B) Hydrophobicity (calculated by sequence-specific retention calculator (SSRC) ) plotted against peptide barcode mass (Da) for 155 peptide barcodes designed in silico . (C) Peptide barcode analysis of barcodes attached to WT MS2 coat protein. Comparison of the log 2 (counts) from Illumina sequencing (x-axis) and log 2 (abundances) from mass spectrometry (y-axis) of 155 peptide barcodes. Horizontal lines indicate residual values. Barcodes were excluded from RBR-scan experiments (faded points) if they were outside of the residual cutoff (± 2). (D) MBP-fusion protein pools were designed to contain N-terminal peptide barcodes flanked by two PreScission Protease (PSP) sites and a 6xHis-tag. Barcodes of PSP-digested pro-peptides enriched by Ni-NTA were trypsinized to yield peptide barcodes. See methods and for barcode composition rules. (E) High throughput OE-PCR scheme. Involves three PCRs where PCRs 1 and 2 use a WT template and external primers a and d paired with internal mutant primers b and c to generate fragments AB and CD, respectively. Primer a contains the peptide barcode DNA sequence and is different for each mutant. The WT template used in fragment PCRs 1 and 2 is made with dUTP (green) so that it can be enzymatically degraded by uracil-specific excision reagent (USER) in PCR 3. PCR 3 incorporates external primers e and f instead of a and d as an additional safeguard against contamination from the WT template. All PCRs were conducted in multi-well plates to maintain high throughput. Red circle indications mutation. (F) Digestion of MS2 coat protein template with uracil-DNA glycosylase (UDG) or USER. (G) OE-PCR with mismatched AB and CD fragments. Combination of fragments AB1 with CD1 and AB2 with CD2 form PCR products at the expected size (500 bp), whereas the combination of fragments AB1 with CD2 does not. (H) Restriction digest assay for OE-PCR products from W32A with and without HindIII site (expected size of full length MS2 CP: 500 bp, expected sizes of HindIII fragments: 193 bp and 307).

Article Snippet: Biotinylated RNA was synthesized by in vitro transcription using a template containing three MS2 stem loop repeats (HiScribe T7 High Yield RNA Synthesis Kit, NEB E2040S) following manufacturer protocol except UTP was added at a ratio of 6.5:1 UTP:biotin-16-UTP (Sigma-Aldrich, 11388908910).

Techniques: Sequencing, In Silico, Comparison, Illumina Sequencing, Mass Spectrometry, High Throughput Screening Assay, Overlap Extension Polymerase Chain Reaction, Mutagenesis

Journal: bioRxiv

Article Title: Pooled scanning of protein variants identifies novel RNA-binding mutants

doi: 10.1101/2025.04.02.646914

Figure Lengend Snippet: (A) Scheme of high-throughput RBR-scan for MS2 coat protein. Peptide barcodes assigned to mutations were fused to the corresponding protein variant by a modified two-step OE-PCR . Barcoded DNA libraries were pooled and cloned into an expression vector in batch. Protein pools were expressed and purified from a single bacterial culture. Barcoded protein pools were incubated with biotinylated stem-loop RNA, followed by streptavidin-mediated affinity purification of protein–RNA complexes. RNA-binding defects were assessed by LC-MS analysis of peptide barcode abundances relative to WT. Residues selected for RBR-scan analysis are annotated on the MS2 coat protein primary sequence (UniProt P03612). Circles represent location of point mutations for RBR-scan pools. Mutants previously tested that showed no effect on RNA binding are in gray; known RNA binding mutation in blue; untested mutations in white). (B) PCR products from two RBR-scan plasmid libraries (expected size: 754 bp) and an empty vector (expected size: 278). E, empty vector. (C) Mutation frequencies (% of reads) of each amino acid (x-axis) along the MS2 coat protein sequence. Barcoded MS2 coat protein variants are shown (y-axis), and barcodes 1 and 2 denote two different peptide barcodes and codons for each amino acid within the same pool. Positions of intended mutations are indicated at the top of the heatmap, following the same color scheme as for RNA binding phenotypes. (D) Coomassie stain of MBP-fused peptide barcoded MS2 coat protein pools (predicted MW: 60.5 kDa). (E) Mass spectrometry analysis of peptide barcode abundances MS2-coat protein pools. Point mutations indicated on x-axis. Bars represent the mean log 2 (barcode abundance). Dots represent abundances from individual barcodes across replicates. Data from 4 biological replicates.

Article Snippet: Biotinylated RNA was synthesized by in vitro transcription using a template containing three MS2 stem loop repeats (HiScribe T7 High Yield RNA Synthesis Kit, NEB E2040S) following manufacturer protocol except UTP was added at a ratio of 6.5:1 UTP:biotin-16-UTP (Sigma-Aldrich, 11388908910).

Techniques: High Throughput Screening Assay, Variant Assay, Modification, Overlap Extension Polymerase Chain Reaction, Clone Assay, Expressing, Plasmid Preparation, Purification, Incubation, Affinity Purification, RNA Binding Assay, Liquid Chromatography with Mass Spectroscopy, Sequencing, Mutagenesis, Staining, Mass Spectrometry

Journal: bioRxiv

Article Title: Pooled scanning of protein variants identifies novel RNA-binding mutants

doi: 10.1101/2025.04.02.646914

Figure Lengend Snippet: (A) MS2 coat protein OE-PCR products from two different barcode-mutation configurations. (B) Alternate barcode-mutation configuration for MS2 coat protein RBR-scan pool (see ) Mutation frequencies (% of reads) of each amino acid (x-axis) along the MS2 coat protein sequence. Barcoded MS2 coat protein variants are shown (y-axis), and barcodes 1 and 2 denote two different peptide barcodes and codons for each amino acid within the same pool. (C) Source of nanopore reads associated with each peptide barcode (left) as well as the number of reads per barcode (right). Read source categories are desired mutation (mut, black), WT (white), and other (grey). Mut, mutant.

Article Snippet: Biotinylated RNA was synthesized by in vitro transcription using a template containing three MS2 stem loop repeats (HiScribe T7 High Yield RNA Synthesis Kit, NEB E2040S) following manufacturer protocol except UTP was added at a ratio of 6.5:1 UTP:biotin-16-UTP (Sigma-Aldrich, 11388908910).

Techniques: Overlap Extension Polymerase Chain Reaction, Mutagenesis, Sequencing

Journal: bioRxiv

Article Title: Pooled scanning of protein variants identifies novel RNA-binding mutants

doi: 10.1101/2025.04.02.646914

Figure Lengend Snippet: (A) Mass spectrometry analysis of peptide barcodes after RNA pull-down. Mutations are listed on the x-axis. Known RNA-binding point mutations (red circles), WT/WT-like mutations (grey circles), and putative RNA-binding mutant (black triangle) annotated. Bars and heatmap represent the mean enrichment ratio relative to WT. Dots represent enrichment ratios of individual barcodes across replicates. Data from 4 biological replicates. (B) Western blot of individual MS2 coat protein mutants after RNA pull-down with MS2 stem loop RNA or control RNA. (C) EMSAs with titrations of recombinant MS2 coat proteins and 50 nmol Alexa488-MS2 stem loop RNA in the presence of 1 g/L yeast tRNA. Complexes were separated on native gels, and RNA was imaged by Cy2 excitation. Data is representative of ≥2 experiments. (D) Densitometric quantification of the proportion of RNA bound in (C). (E–F) Fluorescence polarization assays of individual MS2 coat protein mutants with MS2 stem loop RNA (E) or control RNA (F). Data from 2 technical replicates.

Article Snippet: Biotinylated RNA was synthesized by in vitro transcription using a template containing three MS2 stem loop repeats (HiScribe T7 High Yield RNA Synthesis Kit, NEB E2040S) following manufacturer protocol except UTP was added at a ratio of 6.5:1 UTP:biotin-16-UTP (Sigma-Aldrich, 11388908910).

Techniques: Mass Spectrometry, RNA Binding Assay, Mutagenesis, Western Blot, Control, Recombinant, Fluorescence

Journal: bioRxiv

Article Title: Pooled scanning of protein variants identifies novel RNA-binding mutants

doi: 10.1101/2025.04.02.646914

Figure Lengend Snippet: (A) Coomassie stain of individual MBP-fused MS2 coat proteins (predicted molecular weight: 60.5 kDa). (B) EMSAs with titrations of recombinant MS2 coat proteins and 50 nmol Alexa488-MS2 stem loop RNA in the presence of 1 g/L yeast tRNA. Complexes were separated on native gels, and RNA was imaged by Cy2 excitation. Data is representative of ≥2 experiments. Replicate for .

Article Snippet: Biotinylated RNA was synthesized by in vitro transcription using a template containing three MS2 stem loop repeats (HiScribe T7 High Yield RNA Synthesis Kit, NEB E2040S) following manufacturer protocol except UTP was added at a ratio of 6.5:1 UTP:biotin-16-UTP (Sigma-Aldrich, 11388908910).

Techniques: Staining, Molecular Weight, Recombinant

Journal: bioRxiv

Article Title: Pooled scanning of protein variants identifies novel RNA-binding mutants

doi: 10.1101/2025.04.02.646914

Figure Lengend Snippet: (A) Mutation frequencies (% of reads) of each amino acid (x-axis) along the SRSF2 sequence. Barcoded MS2 coat protein variants are shown (y-axis), where WT barcodes as well as barcode 1 from barcode-mutation pair set #1 (BC1.1), BC1.2, BC2.1, and BC2.2 are indicated. Barcodes within a set (i.e., barcode 1 and barcode 2) are associated with different codons for the same amino acid-coding mutation. Sets refer to different barcode-mutation pairings. Positions of intended mutations are indicated at the top of the heatmap (white circles, unknown phenotype; blue circles, known RNA-binding mutant). (B) PCR products from two RBR-scan plasmid libraries (expected size: 1027 bp) and an empty vector (expected size: 278). E, empty vector. (C) Coomassie stain of MBP-fused peptide barcoded SRSF2 pools (predicted MW: 72 kDa). (D) Mass spectrometry analysis of peptide barcode abundances SRSF2 pools. Point mutations indicated on x-axis. Bars represent the mean log 2 (barcode abundance). Dots represent abundances from individual barcodes across replicates. Data from 8 biological replicates. (E) Coomassie stain of individual MBP-fused SRSF2 variants. (F) EMSAs with titrations of recombinant SRSF2 RRMs and 50 nmol Alexa488-MELK RNA in the presence of 1g/L yeast tRNA. Complexes were separated on native gels, and RNA was imaged by Cy2 excitation. Data is representative of ≥2 experiments. Replicate for .

Article Snippet: Biotinylated RNA was synthesized by in vitro transcription using a template containing three MS2 stem loop repeats (HiScribe T7 High Yield RNA Synthesis Kit, NEB E2040S) following manufacturer protocol except UTP was added at a ratio of 6.5:1 UTP:biotin-16-UTP (Sigma-Aldrich, 11388908910).

Techniques: Mutagenesis, Sequencing, RNA Binding Assay, Plasmid Preparation, Staining, Mass Spectrometry, Recombinant

Journal: bioRxiv

Article Title: Computations performed by shadow enhancers and enhancer duplications vary across the Drosophila embryo

doi: 10.1101/396457

Figure Lengend Snippet: (A). Krüppel ( Kr ) locus showing the two blastoderm enhancers, CD1 (orange) and CD2 (blue). The Krüppel reporter used here drives expression of an MS2 cassette followed by a lacZ reporter gene and α-tubulin 3’UTR, under the control of the endogenous Krüppel promoter and 5’UTR. (B). The blastoderm enhancers of Krüppel drive overlapping expression patterns in the central region of the embryo during nuclear cycle 14 (nc14). Representative maximum projection images showing nascent transcription (MCP-GFP; green) and nuclei (histone-RFP; red) in mid-nc14 (anterior left, dorsal up). (C). Reporter constructs used to investigate the behavior of enhancer duplications. * indicates an enhancer is at its non-endogenous location relative to the promoter. Dotted lines represent synthetic “neutral” sequences, computationally designed to be depleted for binding sites of transcription factors active in patterning the blastoderm embryo. These sequences were used to maintain the endogenous spacing of the enhancers and promoter in the reporter constructs. (D). Live imaging of nascent transcription using MS2 system. A sequence encoding 24x MS2 repeats was incorporated into the 5’ of the reporter gene. When it is transcribed, the sequence forms RNA stem loops that are bound by a constitutively expressed MCP-GFP fusion protein. As a train of polymerases transcribe through the reporter, GFP builds up at the locus and nascent transcripts become visible. Once a transcript dissociates from the gene and diffuses away it is no longer discernible. (E). Krüppel shadow enhancers drive differing levels and dynamics of expression over time in nuclear cycle 14 (CD1, orange; CD2, blue; CD1-CD2, black). Plot shows mean fluorescence per active nucleus in a single anterior-posterior bin of 2.5% embryo length (EL) covering between 50-52.5 % EL (see also Figure S1). Error bars are standard error of the mean across multiple embryos. Each time point is separated by 20 seconds. CD1, n = 5 embryos; CD2, n = 6; CD1-CD2, n = 6.

Article Snippet: The reporter consisted of the D.melanogaster Krüppel core promoter and its surrounding sequence from the 3’ end of the CD2 enhancer to the beginning of the second exon of Krüppel ; a 1.5 kb cassette encoding 24 MS2 stem loops (from Addgene 31865, pCR4-24XMS2SL-stable plasmid); 3 kb of the lacZ gene; and the alpha-tubulin 3’ UTR.

Techniques: Expressing, Construct, Binding Assay, Imaging, Sequencing, Fluorescence

Journal: bioRxiv

Article Title: Computations performed by shadow enhancers and enhancer duplications vary across the Drosophila embryo

doi: 10.1101/396457

Figure Lengend Snippet: (A). Comparing mean expression over time in nuclear cycle 14 (nc14) driven by the CD1 enhancer at its endogenous position (CD1, orange) and at the promoter (CD1*, red). Shown is mean fluorescence per active nucleus (a.u.) within bins in the anterior, middle and posterior of the expression pattern. Each bin is 2.5% embryo length (EL) in width. Error bars show the standard error of the mean and data points are 20 secs apart. CD1, n = 3 embryos; CD1* n = 6. (B). Fraction of total nuclei in the indicated anterior-posterior bin that are actively driving transcription, over time in nc14. Nuclei are counted as active if there is a detectable spot of MS2 fluorescence. Total number of nuclei in a given bin in nc14 is ∼30. Error bars show the standard error of the mean across multiple embryos. (C). Evolution of the expression pattern driven by CD1 (left) and CD1* (right) in the first 16 mins of nc14. Line traces show the mean fluorescence in active nuclei across the pattern and show time points ∼100 secs apart. (D). Initial transcription rate across the patterns driven by CD1 and CD1*, estimated by finding the maximum derivative of the initial rise in fluorescence for each transcriptionally active nucleus; data points show mean across all active nuclei across a number of embryos for each anterior-posterior bin, +/-standard error. (E). Mean fluorescence per active nucleus (a.u.) driven by the CD2 enhancer at its endogenous position (blue) and upstream of the promoter (CD2*, indigo) across nc14. CD2, n = 6 embryos; CD2* n = 4. (F). Fraction of active nuclei driven by CD2 and CD2* over time in nc14. (G). Evolution of the expression pattern driven by CD2 (left) and CD2* (right) over the first 35 mins of nc14. The window of time shown is longer than in the equivalent plots in (C) because the CD2 enhancer takes longer to start driving expression and to reach its peak level. (H). Initial transcription rate across the expression patterns driven by CD2 and CD2* (see D).

Article Snippet: The reporter consisted of the D.melanogaster Krüppel core promoter and its surrounding sequence from the 3’ end of the CD2 enhancer to the beginning of the second exon of Krüppel ; a 1.5 kb cassette encoding 24 MS2 stem loops (from Addgene 31865, pCR4-24XMS2SL-stable plasmid); 3 kb of the lacZ gene; and the alpha-tubulin 3’ UTR.

Techniques: Expressing, Fluorescence

Journal: Molecular Therapy. Nucleic Acids

Article Title: One-Step piggyBac Transposon-Based CRISPR/Cas9 Activation of Multiple Genes

doi: 10.1016/j.omtn.2017.06.007

Figure Lengend Snippet: Identification of the Best sgRNAs in Gene Activation by 293FT-SAM (A) PB vectors used for creating 293FT-SAM. (B) Generation of 293FT-SAM cell line by co-transfecting 293FT with PB-SAM and PBase expression vectors. qPCR results showed that SAM components MS2-p65-HSF1 (MSPH) and dCas9-VP64 were stably expressed for at least 90 days. (C–E) qPCR results of gene expression activation of individual TFs (ASCL1, NEUROG1, NEUROG2, OLIG2, SOX10, LHX3, NKX2-2, MNX1, MYT1, FOXG1, LHX2, SOX8, OLIG1, SOX2, ISL1, SOX11, SOX9, POU3F2, NFIA, NFIB, and NFIX) and lncRNAs (RMST, HAR1B, and HAR1A) after transfecting 293FT-SAM cell line with corresponding sgRNA vectors. A total of three sgRNAs per gene was tested separately to evaluate their activation efficiency. The rightmost bars for each gene represent the extent of gene activation when all three sgRNA vectors for that gene were co-transfected. (F) A pie chart showing the range of fold change of gene expression after each sgRNA vector was transfected into the 293FT-SAM line. For each gene, three sgRNAs were tested separately. A total of 72 sgRNAs was tested. Basically, 43 sgRNAs were able to augment target gene expression by >2-fold. (G) For the 24 genes targeted, 19 genes were activated to be expressed at >2-fold. The qPCR results were normalized to GAPDH mRNA level. P2A, self-cleaving peptide P2A sequence; Hygro, hygromycin resistance cassette; Blast, blasticidin resistance cassette; NLS, nuclear localization signal. In (B)–(E), data are presented as mean ± SEM (n = 3).

Article Snippet: The sgRNA (tracrRNA and crRNA) with MS2 stem loop fragment ( A) (Addgene, 61427) was digested and cloned into the PB vector via Gibson assembly.

Techniques: Activation Assay, Expressing, Stable Transfection, Gene Expression, Transfection, Plasmid Preparation, Targeted Gene Expression, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: One-Step piggyBac Transposon-Based CRISPR/Cas9 Activation of Multiple Genes

doi: 10.1016/j.omtn.2017.06.007

Figure Lengend Snippet: A Schematic Representation of Constructing PB-CRISPRa All-in-One Vectors with Multisite Gateway Cloning Strategy The PB-SAM vector was amplified to become PB-SAM R1-R2 DEST vector containing attR1 and attR2 sites, a ccdB cassette, and a chloramphenicol resistance cassette (CmR). PB-sgRNA (MS2) vectors containing different sgRNA inserts were amplified to be attached to appropriate attB sites. All-in-one vectors were assembled by four-way LR reactions. P2A, self-cleaving peptide P2A sequence; Hygro, hygromycin resistance cassette; Blast, blasticidin resistance cassette; Amp R , ampicillin resistance cassette; Kan R , kanamycin resistance cassette; NLS, nuclear localization signal; ITR, inverted terminal repeat sequence.

Article Snippet: The sgRNA (tracrRNA and crRNA) with MS2 stem loop fragment ( A) (Addgene, 61427) was digested and cloned into the PB vector via Gibson assembly.

Techniques: Cloning, Plasmid Preparation, Amplification, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: CD46 splice variant enhances translation of specific mRNAs linked to an aggressive tumor cell phenotype in bladder cancer

doi: 10.1016/j.omtn.2021.02.019

Figure Lengend Snippet: CD46-CYT2 regulates IRES-dependent translation via hnRNPA1 (A) EJ-1 cells were transiently transfected with StrepII-GST-CYT1/2 and/or FLAG-hnRNPA1. The cell lysates were precipitated with StrepII-Tactin and immunoblotted with an anti-FLAG antibody. (B) Co-immunoprecipitation experiments were conducted with an CD46 antibody in CD46-KO cells stably expressing CD46-CYT1 or CD46-CYT2, respectively. (C) Lysates from 293T cells transfected with FLAG-hnRNPA1 were incubated with either purified protein GST-CYT1 or GST-CYT2. Bound FLAG-hnRNPA1 proteins were immunoblotted by anti-FLAG. (D) EJ-1 cells expressing sh-LacZ or sh-hnRNPA1 were transfected with indicated IRES-dependent reporters, respectively. The firefly and Renilla luciferase activities were measured. (E and F) EJ-1 cells expressing sh-LacZ or sh-hnRNPA1 were transfected with CD46-CYT2 or pHAGE-negative control (NC) (control/empty vector) and the indicated IRES-dependent reporter plasmids. The firefly and Renilla luciferase activities were measured. (G) Relative luciferase activity of wild-type (WT) or CD46-KO EJ-1 cells transfected with Lenti-NC (control) or FLAG-hnRNPA1 with the indicated tethering reporter. (H) CD46-KO cells stably expressing CD46-CYT2, MS2-GST together with sh-LacZ, or sh-hnRNPA1 were transfected with the 3′ MS2 stem-loop-tagged IRES sequence of HIF1a or c-Myc. After 48 h of culture, cells were lysed and incubated overnight with glutathione Sepharose beads. Precipitates were subjected to western blotting with anti-hnRNPA1 or anti-CD46 antibodies. Levels of hnRNPA1 and CD46 are normalized against input and expressed as fold change relative to base expression determined using control sh-LacZ. (I) Cell lysates from WT or CD46-KO cells were incubated with IgG or anti-hnRNPA1 antibody and immunoprecipitated with protein A/G-conjugated beads. Bound RNAs were then eluted, purified, and subjected to qPCR for CCND1 and c-Myc mRNAs.

Article Snippet: For tethering assays, a luciferase reporter containing four copies of the MS2 binding site was generated by cloning the MS2 stem loop region of single guide RNA (sgRNA) (MS2) plasmids (Addgene, #61424) into the NheI and EcoRI cut PCDH-Rluc-MCS-Fluc.

Techniques: Transfection, Immunoprecipitation, Stable Transfection, Expressing, Incubation, Purification, Luciferase, Negative Control, Control, Plasmid Preparation, Activity Assay, Sequencing, Western Blot

Journal: Molecular Therapy. Nucleic Acids

Article Title: CD46 splice variant enhances translation of specific mRNAs linked to an aggressive tumor cell phenotype in bladder cancer

doi: 10.1016/j.omtn.2021.02.019

Figure Lengend Snippet: The CYT1 and CYT2 domain of CD46 regulates protein translation (A) Schematics of the tethering reporter assay. (B) The tethering of CYT1 or CYT2 to the EMCV and EV-71A IRES reporters led to a decrease in translation in comparison with the control. The relative luciferase activity is shown of EJ-1 cells transfected with MS2-myc (control), MS2-myc-CYT1, or MS2-myc-CYT2 with the indicated tethering reporter plasmids. (C) The tethering of CYT1 or CYT2 to CCND1, HIF1a, or c-Myc IRES led to an increase in translation. The relative luciferase activity is shown of EJ-1 cells transfected with MS2-myc (control), MS2-myc-CYT1, or MS2-myc-CYT2 with the indicated tethering reporter. (D) CD46-CYT2 promotes the IRES-dependent translation of HIF1a and c-Myc. Top: schematic representation of bicistronic reporter constructs with different IRESs. Bottom: EJ-1 cells were transfected with the indicated plasmids. The firefly and Renilla luciferase activities were measured, and the ratios of firefly luciferase activity over Renilla luciferase activity were calculated. (E) EJ-1 and 5637 cells transfected with sh-CD46-CYT2 or sh-LacZ were cultured in Met-free DMEM for 1 h in the presence of AHA to capture newly synthesized proteins and immunoblotted using the indicated antibodies. Levels of HIF1a and c-Myc are normalized against GAPDH and expressed as fold change relative to base expression determined using control shRNA. All data represent mean ± SD and were analyzed by an unpaired two-tailed Student’s t test (n = 3). ∗p < 0.05 versus control; ∗∗p < 0.01, ∗∗∗p < 0.001. n.s., not significant.

Article Snippet: For tethering assays, a luciferase reporter containing four copies of the MS2 binding site was generated by cloning the MS2 stem loop region of single guide RNA (sgRNA) (MS2) plasmids (Addgene, #61424) into the NheI and EcoRI cut PCDH-Rluc-MCS-Fluc.

Techniques: Reporter Assay, Comparison, Control, Luciferase, Activity Assay, Transfection, Construct, Cell Culture, Synthesized, Expressing, shRNA, Two Tailed Test

Journal: Cell reports

Article Title: Signal Integration by Shadow Enhancers and Enhancer Duplications Varies across the Drosophila Embryo

doi: 10.1016/j.celrep.2019.01.115

Figure Lengend Snippet: (A) Krüppel (Kr) locus showing the two blastoderm enhancers, CD1 (orange) and CD2 (blue). The Krüppel reporter used here drives expression of an MS2 cassette followed by a lacZ reporter gene and α-tubulin 3′ UTR, under the control of the endogenous Krüppel promoter and 5′ UTR.

Article Snippet: The reporter consisted of the D. melanogaster Krüppel core promoter and its surrounding sequence from the 3 ’ end of the CD2 enhancer to the beginning of the second exon of Krüppel ; a 1.5 kb cassette encoding 24 MS2 stem loops (from Addgene 31865, pCR4–24XMS2SL-stable plasmid); 3 kb of the lacZ gene; and the alpha-tubulin 3 ’ UTR.

Techniques: Expressing, Control

Journal: Cell reports

Article Title: Signal Integration by Shadow Enhancers and Enhancer Duplications Varies across the Drosophila Embryo

doi: 10.1016/j.celrep.2019.01.115

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The reporter consisted of the D. melanogaster Krüppel core promoter and its surrounding sequence from the 3 ’ end of the CD2 enhancer to the beginning of the second exon of Krüppel ; a 1.5 kb cassette encoding 24 MS2 stem loops (from Addgene 31865, pCR4–24XMS2SL-stable plasmid); 3 kb of the lacZ gene; and the alpha-tubulin 3 ’ UTR.

Techniques: Virus, Imaging, Transgenic Assay, Generated, Recombinant, Plasmid Preparation, Software, Membrane, In Vitro