Journal: bioRxiv

Article Title: Kinome-wide RNAi screen uncovers role of Ballchen in maintenance of gene activation by trithorax group in Drosophila

doi: 10.1101/2020.11.26.399824

Figure Lengend Snippet: (A, B) Ballchen mutant flies ( ball 2 ) were crossed with two different alleles of Pc ( Pc 1 and Pc XL5 ), while Pc alleles ( Pc 1 and Pc XL5 ) crossed with w 1118 flies were used as control. Heterozygous Pc/+ males, Pc 1 /+ (A) and Pc XL5 /+ (B), from control crosses exhibit strong extra sex combs phenotype. In contrast, ball 2 strongly suppressed both the Pc alleles to none or few extra sex combs in ball 2 /Pc 1 (A) as well as ball 2 /Pc XL5 (B) flies. 200 male flies were analyzed for each cross and data shown represents two independent experiments. Based on strength of phenotype, male flies were categorized according to the severity of extra sex combs phenotype. These categories are: -, no extra sex combs; +, 1-2 hairs on 2 nd leg; ++, more than 3 hairs on 2 nd leg; +++, more than 3 hairs on 2 nd leg and 1-2 hairs on 3 rd leg; ++++, strong sex combs on both 2 nd and 3 rd pairs of legs. (C) ball 2 mutant flies were crossed to two different alleles of trx ( trx 1 and trx E2 ), and ball 2 /trx males in F1 were scored for loss of pigmentation in A5 abdominal segment (A5 to A4 transformation, marked with asterisk). The trx alleles ( trx 1 and trx E2 ) crossed with w 1118 flies i.e. trx 1 /+ and trx E2 /+ , show A5 to A4 transformation when compared to wild type flies. Strong enhancement of A5 to A4 transformation can be seen in ball 2 /trx double mutants when compared to trx/+ . Representative images for wild-type phenotype (top right) and A5 to A4 transformation (marked by white asterisk) are presented for trx/ball male flies (bottom right). All crosses were done in triplicates and independent t-tests were performed for analyzing each category (* p ≤ 0.05, ** p ≤ 0.01, *** p≤ 0.001 or **** p≤ 0.0001). (D) Significantly low levels of abd-A , Abd-B and pnt expression was detected through qRT-PCR in homozygous ball 2 embryos when compared with w 1118 embryos. Independent t-tests were performed for each gene analysis (* p ≤ 0.05, ** p ≤ 0.01, *** p≤ 0.001 or **** p≤ 0.0001). (E-H) Immunostaining of stage 15 embryos with anti-Abd-B is shown in w 1118 (E, F) as well as homozygous ball 2 (G, H) embryos. As compared to w 1118 (F), ball 2 embryos showed strongly diminished Abd-B (H) expression.

Article Snippet: Following antibodies were used during this study: mouse anti-Abd-B (DSHB, 1A2E9, IF: 1:40), rabbit anti-TRX (gift from R. Paro, IF: 1:20), rabbit anti-PC (Santa Cruz, D220, IF: 1:20), rabbit anti-BALL (Gift from A. Herzig, IF 1:20, ChIP: 2μl) mouse anti-GFP (Roche, 11814460001, IF:1:50), mouse anti-Tubulin (Abcam, ab44928, WB: 1:2000), mouse anti-FLAG M2 (Sigma Aldrich, WB: 1:2000, ChIP: 5μl), mouse anti-H2A-ubi (Millipore, 05-678, ChIP: 5μl).

Techniques: Mutagenesis, Transformation Assay, Expressing, Quantitative RT-PCR, Immunostaining

Journal: bioRxiv

Article Title: Kinome-wide RNAi screen uncovers role of Ballchen in maintenance of gene activation by trithorax group in Drosophila

doi: 10.1101/2020.11.26.399824

Figure Lengend Snippet: (A) Venn diagram analysis showed 85% co-occupancy of BALL on known TRX binding sites. Heat map illustration of BALL binding sites across genome show enrichment of BALL within ± 1kb of TSSs of genes. (B) Genome browser view of BALL association compared with patterns of TRX, PC, H3K27ac and H3K4me3 association at pnt and pnr genes. (C) Genome browser view of ChIP-seq data showing binding profile of BALL and its comparison with TRX, PC, H3K27ac and H3K4me3 association across the bithorax complex (BX-C). Highly enriched BALL and TRX correlates with high levels of both H3K27ac and H3K4me3 and absence of PC at CG14906 present just downstream of Ubx. In contrast, BX-C genes i.e. Ubx , abd-A and Abd-B which are silent in S2 correlates with highly enriched PC all across BX-C. Both BALL and TRX are present at few overlapping binding sites in BX-C.

Article Snippet: Following antibodies were used during this study: mouse anti-Abd-B (DSHB, 1A2E9, IF: 1:40), rabbit anti-TRX (gift from R. Paro, IF: 1:20), rabbit anti-PC (Santa Cruz, D220, IF: 1:20), rabbit anti-BALL (Gift from A. Herzig, IF 1:20, ChIP: 2μl) mouse anti-GFP (Roche, 11814460001, IF:1:50), mouse anti-Tubulin (Abcam, ab44928, WB: 1:2000), mouse anti-FLAG M2 (Sigma Aldrich, WB: 1:2000, ChIP: 5μl), mouse anti-H2A-ubi (Millipore, 05-678, ChIP: 5μl).

Techniques: Binding Assay, ChIP-sequencing

Journal: Cell reports

Article Title: VELCRO-IP RNA-seq reveals ribosome expansion segment function in translation genome-wide

doi: 10.1016/j.celrep.2020.108629

Figure Lengend Snippet: (A) Secondary structure models of the human ( H. sapiens ) and baker’s yeast ( S. cerevisiae ) 18S rRNA region containing ES9S, highlighted in green and blue, respectively. Predicted structural changes in ES9S because of species-specific variation in sequence. Sequence divergence from the human/mouse ES9S are annotated in red. Secondary structure models of ES9S were predicted using Vienna RNAfold ( http://rna.tbi.univie.ac.at ) and visualized using VARNA ( http://varna.lri.fr ). See also . (B) Schematic of the RT-PCR analysis of the ES9S region using cDNA generated from total RNA from six species (E11.5, stage E11.5 FVB mouse embryo; chicken, Gallus gallus ; axolotl, Ambystoma mexicanum ; frog, Xenopus laevis ; zebrafish, Danio rerio ; yeast, Saccharomyces cerevisiae ) and primers specific for the 18S rRNA region containing ES9S (see ). (C) Multiple sequence alignment of the variable ES9S region in highly conserved 18S rRNA. PCR product sequencing after RT-PCR spanning the ES9S region with the outer primers in (B) for six species confirms the annotated species-specific ES9S sequence. Nucleotides divergent from human/mouse ES9S are highlighted in red. (D) Concept of revealing extended rRNA ES interactions on the ribosome with mRNAs or proteins. This enables analysis of ES9S interactions, the ES of choice in this work, via the 40S ribosomal subunit with positional resolution to identify and map ES9S binding mRNA elements underlying unexplored ES-directed translation regulation. (E) Schematic of the VELCRO-IP (variable expansion segment-ligand chimeric ribosome-IP) approach to investigate ES-mediated translation regulation through mRNA interactions. Generating FLAG-tagged humanized ribosome strains that exclusively contain human ES9S in yeast 18S rRNA and tagged WT control yeast strains in parallel enables an ES engineering system that contains rRNA and protein tags and allows the manipulation of any ES. (F) Mapping of the components of the ES engineering system onto the cryoelectron microscopy (cryo-EM) structure of the yeast 80S and 40S ribosome (PDB: 4V6I). The sites of rRNA tag insertion,the last 10 amino acids of the C terminus of Rps2/uS5, and ES9S are highlighted according to the schematic representation.

Article Snippet: Purified embryo mRNA was fragmented to 100-200 nt RNA fragments by magnesium-buffer based degradation using the NEBNext Magnesium RNA Fragmentation Module (NEB, E6150S).

Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, Generated, Binding Assay, Microscopy, Cryo-EM Sample Prep

Journal: Cell reports

Article Title: VELCRO-IP RNA-seq reveals ribosome expansion segment function in translation genome-wide

doi: 10.1016/j.celrep.2020.108629

Figure Lengend Snippet: Schematic representation of the VELCRO-IP approach. Yeast strains expressing chimeric (hES9S) orWT ribosomes are generated by rDNA complementation. The same strains also carry endogenously C-terminally FLAG-tagged RPS2/uS5. 40S ribosomal subunits from powderized lysates of each strain are isolated on FLAG agarose beads and washed. For VELCRO-IP qRT-PCR (proof of principle), in vitro transcripts (IVTs) (see ) are incubated with ribosome beads. Upon 3xFLAG peptide elution of 40S-RNA complexes, total RNA is eluted, and IVT RNA enrichment is determined by qRT-PCR specific for Fluc and the 18S rRNA tag. For VELCRO-IP RNA-seq (genome-wide), mRNAs from total RNA from stage E11.5 mouse embryos are purified and fragmented to 100–200 nt, and refolded RNA fragments are used as input for IP and FLAG elution of mRNA-ribosome complexes. After yeast rRNA depletion from eluted RNAs, ribosome-bound mRNA fragments are sequenced to identify hES9S-specific mouse mRNA elements.

Article Snippet: Purified embryo mRNA was fragmented to 100-200 nt RNA fragments by magnesium-buffer based degradation using the NEBNext Magnesium RNA Fragmentation Module (NEB, E6150S).

Techniques: Expressing, Generated, Isolation, Quantitative RT-PCR, In Vitro, Incubation, RNA Sequencing Assay, Genome Wide, Purification

Journal: Cell reports

Article Title: VELCRO-IP RNA-seq reveals ribosome expansion segment function in translation genome-wide

doi: 10.1016/j.celrep.2020.108629

Figure Lengend Snippet: (A) VELCRO-IP qRT-PCR: a zoomed-in view on the interactions between hES9S and Hoxa9 P4 stem-loop or other target 5′ UTRs that can be identified by VELCRO-IP. The 4-nt inactive P4 mutant M5 (P4(M5)) serves as a negative control. (B) IVTs of 475–510 nt in length contain the native spacer (–, negative control), P4-native (P4), or P4(M5)-native (P4(M5)) embedded in flanking constant regions (5′ TIE and 3′ Fluc ORF sequence) (see ). The Fluc ORF portion can be used for qPCR amplification to compare the three RNA constructs. TIE, translation inhibitory element. (C) Western blot (WB) analysis of same volumes of lysate (input), unbound fraction, and 3xFLAG peptide-eluted protein from beads to monitor ribosome enrichment of tagged (Rps2-FLAG) and untagged (Rps5) 40S and 60S (Rpl10a) components in IVT RNA samples, in combination with WT and hES9S yeast ribosomes. Cytoplasmic enzyme Pgk1 served as a negative control. The fraction loaded of input, unbound, and elution samples is expressed as a percentage of the original lysate volume. A representative experiment of n = 5 is shown. (D) Analysis of total RNA in the 3xFLAG peptide elution by qRT-PCR using the same volumes of RNA per sample for the RT. Fluc transcript enrichment was assessed by normalizing Ct values to those of the respective 18S rRNA tag to control for ribosome-IP efficiency per sample. Respective hES9S samples were compared with WT samples to assess RNA fold enrichment of IVT RNAs. Average RNA fold enrichment ± SEM, n = 5. See also - . (E) Schematic of embryo mRNA fragmentation for VELCRO-IP RNA-seq. Total RNA extraction of stage E11.5 mouse embryos yields 2%–3% of mRNA isolated on oligo(dT) beads. mRNA is fragmented with magnesium ions to a length of 100–200 nt, which overall recovers >75% of input mRNAs as fragments. (F) Fragmented mouse mRNAs from C3H10T1/2 cells in 1-μg aliquots at different time points of fragmentation (4, 5, and 6 min) were analyzed on an mRNA Pico Chip (Agilent) on a Bioanalyzer (Agilent). A zoomed-in view of the Bioanalyzer quantification (top) and virtual gel images (bottom) is shown. The marker (gray line, lane M) is overlaid for reference. See also - . (G) Fragmented mouse mRNAs from stage E11.5 embryos in 1-μg aliquots fragmented for 5 min at 94°C from two independent repeats of embryo harvest, RNA isolation, mRNA purification, and fragmentation (1 and 2). This yields fragments of 100–200 nt. RNAs were analyzed as in (F). See also .

Article Snippet: Purified embryo mRNA was fragmented to 100-200 nt RNA fragments by magnesium-buffer based degradation using the NEBNext Magnesium RNA Fragmentation Module (NEB, E6150S).

Techniques: Quantitative RT-PCR, Mutagenesis, Negative Control, Sequencing, Amplification, Construct, Western Blot, RNA Sequencing Assay, RNA Extraction, Isolation, Marker, Purification

Journal: Cell reports

Article Title: VELCRO-IP RNA-seq reveals ribosome expansion segment function in translation genome-wide

doi: 10.1016/j.celrep.2020.108629

Figure Lengend Snippet: (A) For VELCRO-IP RNA-seq, mRNA was isolated from stage E11.5 mouse embryos, fragmented, and used as input. Eluted and yeast rRNA-depleted RNA obtains ribosome-bound mouse mRNA fragments for library preparation and Illumina sequencing, including the mRNA fragment input for reference. The distribution of mRNA fragment lengths for all sequenced libraries is plotted with a median fragment length of 246 nt. All reads were mapped to the mouse and yeast transcriptomes, and only reads exclusively mapping to mouse mRNAs were further analyzed. (B) Eluted and yeast rRNA-depleted mouse RNA from three independent replicates of WT and hES9S VELCRO-IP experiments were analyzed on an mRNA Pico Chip (Agilent) on a Bioanalyzer (Agilent) as in . See . (C) WB analysis as in to monitor efficient IP of 40S ribosomes after VELCRO-IP. A representative experiment of n = 3 is shown. (D) Kernel density of the distribution of t-statistics for the test of differential enrichment of mRNA fragments bound to hES9S versus WT ribosomes is plotted in black. Empirical estimates of the decomposition of the test statistics distribution to null and non-null tests are plotted in gray and red, respectively. The dotted line indicates local FDR of 0.05. (E) Comparison of individual VELCRO-IP RNA-seq samples (three replicate samples per hES9S and WT). Scatterplots of normalized log read counts, colored by expression level. Pearson correlation coefficients are shown in the top-right boxes. See . (F) RNA-seq results of independent replicates (n = 3) for each WT and hES9S sample. Normalized log read counts are presented for WT and hES9S-enriched mouse mRNA fragments. Fragments (FDR < 0.05) are colored according to the mRNA region to which they map (see legend): 5′ UTR or overlapping 5′ UTR/ORF (red), 3′ UTR (green), and ORF (blue). Mouse genes are labeled for which enriched fragments in the 5′ UTR and/or 5′ region of the ORF were identified and for which 5′ UTR validation experiments were performed. Five control 5′ UTRs are marked that are equally bound to both WT and hES9S 40S subunits and served as negative controls. See and . (G) Analysis of regions mapping to 5′ UTR, ORF, or 3′ UTR in hES9S-enriched samples compared with their presence in WT or hES9S samples, each n = 3, expressed as the percentage of total read windows identified. The indicated p value is calculated by a chi-square test. (H) Gene Ontology (GO) analysis for the biological process of 87 5′ UTR regions (FDR < 0.05, n = 3) enriched by hES9S. Displayed are the expected and observed frequency of genes for the significant terms (FDR < 0.05) (expressed mRNA regions were used as the background population; see for details of the thresholds used). See for GO terms of ORF, 3′ UTR, and full mRNA (all regions), as well as .

Article Snippet: Purified embryo mRNA was fragmented to 100-200 nt RNA fragments by magnesium-buffer based degradation using the NEBNext Magnesium RNA Fragmentation Module (NEB, E6150S).

Techniques: RNA Sequencing Assay, Isolation, Sequencing, Expressing, Labeling

Journal: Cell reports

Article Title: VELCRO-IP RNA-seq reveals ribosome expansion segment function in translation genome-wide

doi: 10.1016/j.celrep.2020.108629

Figure Lengend Snippet: (A) Based on the analysis in and , full 5′ UTRs (as annotated in ENSEMBL) were experimentally validated. Schematic of the 4xS1m pulldown to probe the interactions of control and candidate 5′ UTR-4xS1m in vitro -transcribed RNAs with WT and hES9S yeast ribosomes. (B) 4xS1m pulldown of candidate 5′ UTR-4xS1m RNA with WT and hES9S yeast ribosomes for three control 5′ UTRs as negative controls and four candidate 5′ UTRs were tested alongside Hoxa9 P4 as a positive control. After the formation of ribosome-RNA RNPs in vitro , beads are split in half for total RNA and protein. Ribosome-RNA RNP enrichment in vitro is monitored by qRT-PCR for tagged 18S and 25S rRNA and other RNA classes normalized to the input (RNA on beads) and by WB. Fold enrichment of RNAs was determined by qRT-PCR using the same volumes of eluted RNA and normalizing Ct values of each sample to their respective RNA input (WT or hES9S). Yeast actin ( act1 ) mRNA and yeast UsnRNA1 serve as negative controls. WB analysis was performed for 40S and 60S subunit RPs of the same volumes of protein released from beads by RNase A. The fraction loaded of input and elution samples is expressed as a percentage of the original lysate volume. The P4-4xS1m/WT sample was used to normalize for RNA fold enrichment (set to 1). Average RNA fold enrichment, SEM, n = 3; ns, not significant; long exp., long exposure. See . (C) Bicistronic mRNA reporter genes containing no insert in the intergenic region (pRF, vector) and candidate or control 5′ UTRs were transiently transfected into mouse C3H10T1/2 cells. Cells were split in half for protein lysates for luciferase activity measurement and total RNA extraction for qRT-PCR analysis. Relative luciferase activity is expressed as a Fluc(IRES)/Rluc(cap-initiation) ratio normalized to respective Fluc/Rluc mRNA levels and expressed as average activity ± SEM, n = 3–8. pRF serves as negative control, the encephalomyocarditis virus (EMCV) and hepatitis C virus (HCV) IRESs serve as IRES controls, EMCV IRES activity was used as a cutoff, and the full-length (FL) Hoxa9 IRES-like element and P4-native served as Hoxa9 IRES-like references.

Article Snippet: Purified embryo mRNA was fragmented to 100-200 nt RNA fragments by magnesium-buffer based degradation using the NEBNext Magnesium RNA Fragmentation Module (NEB, E6150S).

Techniques: In Vitro, Positive Control, Quantitative RT-PCR, Plasmid Preparation, Transfection, Luciferase, Activity Assay, RNA Extraction, Negative Control

Journal: Cell reports

Article Title: VELCRO-IP RNA-seq reveals ribosome expansion segment function in translation genome-wide

doi: 10.1016/j.celrep.2020.108629

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Purified embryo mRNA was fragmented to 100-200 nt RNA fragments by magnesium-buffer based degradation using the NEBNext Magnesium RNA Fragmentation Module (NEB, E6150S).

Techniques: Recombinant, SYBR Green Assay, Protease Inhibitor, Avidin-Biotin Assay, Semi-Dry Trans-Blot, Western Blot, Modification, Reporter Assay, Purification, Gel Extraction, Clone Assay, In Vitro, Synthesized, Generated, Software

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Transcriptional control of the B3GALT5 gene by a retroviral promoter and methylation of distant regulatory elements.

doi: 10.1096/fj.13-236273

Figure Lengend Snippet: Figure 1. Detection of transcription factors HNF1/ and Cdx1/2 in human colon cancers and cell lines expressing various amounts of B3GALT5 LTR transcript. A) RNA extracted from cancers and cell lines was reverse transcribed, and normalized amounts of the resultant first-strand cDNAs were mixed with the indicated amounts of competitor (truncated) cDNAs and subjected to PCR (25 and 35 cycles for -actin and B3GALT5 LTR, respectively), using primers specific to -actin and B3GALT5 LTR transcript, respectively. Note the different amounts of B3GALT5 LTR competitor cDNA used in the case of colon cancers, cell lines (present figure), and matched pairs of colon biopsies (Supplemental Fig. S1A). Twenty percent of each amplification reaction was electrophoresed in a 1% agarose gel and visualized by ethidium bromide staining. The target doublet corresponded to the alternative splicing that had been reported (10). Representative gels are shown. Samples were not all run on the same gel, as indicated by the vertical white spaces between the gels. B) Densitometric scanning of gel images was performed to quantitate the amounts of B3GALT5 LTR transcript, which were calculated from a standard curve and normalized to the amounts calculated for -actin. Results are means sd of 3 determinations. Note the different scales used for colon cancer and the cell lines. C) Nuclear extracts (5–10 g of protein) were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane that was blotted with anti-HNF1 antibody (recognizing both HNF1 and HNF1), or anti-Cdx1, anti-Cdx2, or anti-histone H3 antibodies, followed by an HRP-labeled secondary antibody and chemiluminescence detection. Longer exposures were necessary to detect a visible spot for Cdx1 and Cdx2. For comparative quantitation, see Supplemental Fig. S1B.

Article Snippet: Aliquots of nuclear extracts (5–10 g of protein) were separated by 10% SDS-PAGE; transferred to a nitrocellulose membrane (Trans-Blot SD Semi-Dry Transfer Cell; Bio-Rad Laboratories S.r.l., Milan, Italy); and blotted with rabbit polyclonal anti-HNF1 / (sc-8986, 1:200; Santa Cruz Biotechnology Italia, Milan, Italy), rabbit polyclonal anti-H3 (D2B12 4620, 1:500; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-Cdx1 (PAB4713, 1:200; Abnova, Taipei, Taiwan), or mouse monoclonal antiCdx2 (M01, 1:200; Abnova) antibodies, according to our published protocol (18).

Techniques: Expressing, Reverse Transcription, Agarose Gel Electrophoresis, Staining, Alternative Splicing, SDS Page, Membrane, Labeling, Quantitation Assay

Journal: Cell reports

Article Title: Toll-like receptor 4 and CD11b expressed on microglia coordinate eradication of Candida albicans cerebral mycosis

doi: 10.1016/j.celrep.2023.113240

Figure Lengend Snippet:

Article Snippet: APP was semi-quantified using Western blot (4%–12% Nupage Bis-Tris gel, Thermo Fisher Scientific, Waltham MA) and quantitative PCR. (ab32136, 1:5000 Abcam, Cambridge MA; Mm01344172, Invitrogen, Carlsbad, CA).

Techniques: Recombinant, Lysis, Magnetic Beads, Software, Enzyme-linked Immunosorbent Assay