Journal: Nucleic Acids Research

Article Title: Cap-independent translation through the p27 5′-UTR

doi: 10.1093/nar/gkm512

Figure Lengend Snippet: Transfection of bicistronic mRNAs into human breast cancer cells. Top: Diagram of mRNA molecules that were synthesized by in vitro transcription. The arrows indicate the direction of the p27 5′-UTR sequence (reverse is the antisense orientation and forward is the sense orientation). Bottom: The in vitro synthesized mRNAs were transfected into two human breast cancer cell lines (MCF7 and MDA-MB-231) and Renilla and firefly luciferase activities were determined. For each cell line, the ratio of firefly to Renilla luciferase activity is shown for the antisense 5′-UTR (Reverse, gray bars) and the sense 5′-UTR (Forward, black bars).

Article Snippet: The dual luciferase bicistronic construct pTKLL was constructed by ligating an XbaI/BglII fragment from pRL-TK (Promega) with an AvrII/BglII fragment from pGL2CAT. pTKLL-472, carrying the human p27 5′-UTR between the Renilla and firefly luciferase coding regions, was constructed by ligating the XbaI/BglII fragment from pRL-TK (Promega) with an AvrII/BglII fragment from pGL2CAT/Luc-472 ( ).

Techniques: Transfection, Synthesized, In Vitro, Sequencing, Luciferase, Activity Assay

Journal: Nucleic Acids Research

Article Title: Cap-independent translation through the p27 5′-UTR

doi: 10.1093/nar/gkm512

Figure Lengend Snippet: Transfection of bicistronic reporter constructs into MCF7 cells. ( A ) Diagram of constructs used in the transfections and probes used for RNase protection assays. Construct names are indicated on the right. The p27 5′-UTR is a 472 nt sequence upstream of the p27 start codon. It is inserted between the coding regions for Renilla luciferase (R-Luc) and firefly luciferase (FF-Luc). The region spanned by the in vitro synthesized mRNA used as a control in RNase protection assays is shown at the bottom. ( B ) The bicistronic constructs were transfected into MCF7 human breast cancer cells. One day after transfection, the cells were harvested and the activity of both Renilla luciferase (left) and firefly luciferase (right) was determined. Black bars represent enzyme activity in the constructs carrying the TK promoter (pTKLL and pTKLL472) and gray bars represent activity in the promoterless constructs (pLL and pLL472). RLU indicates relative light units. Error bars represent SD. ( C ) RNase protection assay using Probe 1 (see panel A). Lane 1 is undigested probe. Lane 2 is probe treated with RNase. Lane 3 is probe hybridized with in vitro synthesized mRNA. Lane 4 is probe hybridized with RNA isolated from untransfected cells. In lanes 5–8, probe was hybridized with RNA from cells transfected with the constructs indicated at the top of each lane. Lane 9 is labeled marker RNAs of the size indicated on the right. The arrowhead indicates a band of ∼100 nt and is the size expected for accurate transcription initiation from the TK promoter. The asterisks mark major bands observed after transfecting cells with constructs lacking the TK promoter. ( D ) RNase protection assay using Probe 2. Lanes are the same as described for C. The arrowhead indicates a band of ∼227 nt, which is the size expected if transcription is initiated at any point upstream of the probe sequence. ( E ) RNase protection assay using Probe 3. Lanes are as describe for C. The arrowhead in lane 3 indicates a band of ∼367 nt which is the expected size for cells transfected with the constructs carrying the p27 5′-UTR. The asterisks indicate bands of ∼210 and ∼125 nt which are also observed in lanes 6 and 8. The open arrowhead in lane 5 indicates a band of ∼166 which is the expected size for cells transfected with the constructs lacking the p27 5′-UTR. ( F ) RNase protection assay using Probe 4. Lanes are as described for C. Arrowhead in lane 3 indicates a band of ∼530 nt and is the major band observed for in vitro transcribed mRNA as well as in cells transfected with constructs carrying the p27 5′-UTR. The open arrowhead in lane 5 indicates a band of the expected size for cells transfected with constructs lacking the p27 5′-UTR. ( G ) The RNase protection assay with Probe 4 was repeated using less in vitro synthesized mRNA (lane 4) in order to obtain bands of similar intensity to those observed using RNA from cells transfected with constructs containing the p27 5′-UTR (lanes 5 and 6).

Article Snippet: The dual luciferase bicistronic construct pTKLL was constructed by ligating an XbaI/BglII fragment from pRL-TK (Promega) with an AvrII/BglII fragment from pGL2CAT. pTKLL-472, carrying the human p27 5′-UTR between the Renilla and firefly luciferase coding regions, was constructed by ligating the XbaI/BglII fragment from pRL-TK (Promega) with an AvrII/BglII fragment from pGL2CAT/Luc-472 ( ).

Techniques: Transfection, Construct, Sequencing, Luciferase, In Vitro, Synthesized, Control, Activity Assay, Rnase Protection Assay, Isolation, Labeling, Marker

Journal: Nucleic Acids Research

Article Title: Cap-independent translation through the p27 5′-UTR

doi: 10.1093/nar/gkm512

Figure Lengend Snippet: Transfection of pGL4-based bicistronic vectors into MCF7 cells. ( A ) Diagram of bicistronic constructs. ( B ) The left panel shows activity of the upstream cistron (Firefly luciferase) and the right panel shows activity of the downstream cistron (Renilla luciferase). Bars labeled 472 indicate constructs with the p27 5′-UTR in the forward orientation and bars labeled 472R indicate constructs with the p27 5′-UTR in the reverse orientation. For bars labeled −Promoter cells were transfected with constructs lacking a promoter upstream of the first cistron and for bars labeled +Promoter cells were transfected with constructs carrying the SV40 promoter. The number above each bar indicates the mean of three separate transfections. The error bars represent SD.

Article Snippet: The dual luciferase bicistronic construct pTKLL was constructed by ligating an XbaI/BglII fragment from pRL-TK (Promega) with an AvrII/BglII fragment from pGL2CAT. pTKLL-472, carrying the human p27 5′-UTR between the Renilla and firefly luciferase coding regions, was constructed by ligating the XbaI/BglII fragment from pRL-TK (Promega) with an AvrII/BglII fragment from pGL2CAT/Luc-472 ( ).

Techniques: Transfection, Construct, Activity Assay, Luciferase, Labeling

Journal: Nucleic Acids Research

Article Title: Cap-independent translation through the p27 5′-UTR

doi: 10.1093/nar/gkm512

Figure Lengend Snippet: siRNA knockdown of luciferase expressed from bicistronic constructs. ( A ) The pGL4-based bicistronic reporter shown at the top of the diagram was cotransfected into MCF7 cells with a control scrambled siRNA, an siRNA targeted to firefly luciferase (siFLuc), or an siRNA targeted to Renilla luciferase (siRLuc). Firefly and Renilla luciferase activities were determined and are expressed as percent of the control with the scrambled siRNA. The number above each bar represents the relative luciferase activity (% of control). ( B ) Same as in A except that firefly luciferase is the upstream cistron and Renilla luciferase is the downstream cistron.

Article Snippet: The dual luciferase bicistronic construct pTKLL was constructed by ligating an XbaI/BglII fragment from pRL-TK (Promega) with an AvrII/BglII fragment from pGL2CAT. pTKLL-472, carrying the human p27 5′-UTR between the Renilla and firefly luciferase coding regions, was constructed by ligating the XbaI/BglII fragment from pRL-TK (Promega) with an AvrII/BglII fragment from pGL2CAT/Luc-472 ( ).

Techniques: Knockdown, Luciferase, Construct, Control, Activity Assay

Journal: Nucleic Acids Research

Article Title: Cap-independent translation through the p27 5′-UTR

doi: 10.1093/nar/gkm512

Figure Lengend Snippet: RT-PCR analysis of transcripts produced from bicistronic reporter plasmids. Top: Diagram of the transcripts that should be produced following transfection when transcription initiates from the TK promoter of the bicistronic reporter construct lacking an insert (pTKLL) and the construct carrying the p27 5′-UTR (pTKLL-472). Arrows below each diagram indicate the position of primers used for RT-PCR analysis. RT-PCR should produce a DNA fragment of ∼1635 nt if there is no splicing due to a cryptic splice acceptor site in the p27 5′-UTR. The expected size for the fragment from pTKLL transfected cells is ∼1160 nt. Bottom: The bicistronic reporters were transfected into MCF7 cells as indicated. The control reactions (C) were from untransfected cells. RNA was isolated and used for RT-PCR with the primer pair indicated in the diagram. Reactions in which all conditions were identical except that reverse transcriptase was omitted (−RT) were performed for each of the RNA templates.

Article Snippet: The dual luciferase bicistronic construct pTKLL was constructed by ligating an XbaI/BglII fragment from pRL-TK (Promega) with an AvrII/BglII fragment from pGL2CAT. pTKLL-472, carrying the human p27 5′-UTR between the Renilla and firefly luciferase coding regions, was constructed by ligating the XbaI/BglII fragment from pRL-TK (Promega) with an AvrII/BglII fragment from pGL2CAT/Luc-472 ( ).

Techniques: Reverse Transcription Polymerase Chain Reaction, Produced, Transfection, Construct, Control, Isolation, Reverse Transcription

Journal: Nucleic Acids Research

Article Title: Cap-independent translation through the p27 5′-UTR

doi: 10.1093/nar/gkm512

Figure Lengend Snippet: Effect of an IRE on expression of bicistronic reporters carrying the p27 5′-UTR. Top: Diagram of potential mRNA products resulting from transcription of a bicistronic reporter in which an IRE has been inserted just downstream of the transcriptional start site within the TK promoter. The top diagram shows the expected transcript if there is no cryptic splice site within the 5′-UTR. In this case, only the upstream cistron (Renilla luciferase) would be affected by iron chelation. The bottom diagram shows the expected transcript if the 5′-UTR has a cryptic splice acceptor site. The IRE would be linked to a monocistronic mRNA encoding only firefly luciferase and iron chelation would inhibit expression of this enzyme. Bottom: NIH3T3 cells were transfected with bicistronic reporters pTKLL-472 and pTKLL-472-IRE. These constructs are identical except that pTKLL-472-IRE has an IRE inserted 35 nt downstream of the TK transcriptional start site. The transfected cells were treated with (+, gray bars) or without (−, black bars) the iron chelator DFO. The cells were harvested and Renilla (R) and firefly (F) luciferase activities were determined. For each transfection, the value of luciferase activity in the absence of DFO was set to 1.

Article Snippet: The dual luciferase bicistronic construct pTKLL was constructed by ligating an XbaI/BglII fragment from pRL-TK (Promega) with an AvrII/BglII fragment from pGL2CAT. pTKLL-472, carrying the human p27 5′-UTR between the Renilla and firefly luciferase coding regions, was constructed by ligating the XbaI/BglII fragment from pRL-TK (Promega) with an AvrII/BglII fragment from pGL2CAT/Luc-472 ( ).

Techniques: Expressing, Luciferase, Transfection, Construct, Activity Assay

Journal: bioRxiv

Article Title: Early to Middle Pleistocene transition shapes the evolution of human-specific mutations associated with height and basal metabolic rate

doi: 10.1101/2024.06.23.600148

Figure Lengend Snippet: a : Expression of ACSF3 in the liver of great apes. The x-axis shows both the mean expression (vertical lines) and the expressions of each individual (dots; N=2 for each species) . The phylogeny is shown on the left ( Methods ). b-c : GWAS associations (top panel), fine-mapping (middle panel) and eQTL associations with ACSF3 (bottom panel) of the 11 fine-mapped SNPs within one credible set. For GWAS associations with height ( b ) and BMR ( c ), SNPs within the genomic region (chr16: 89500494-89557054, extended 10 kbp upstream and downstream of the 11 fine-mapped SNPs; GRCh37) are also shown. For fine-mapping, the posterior inclusion probability (PIP) of each SNP is shown. For eQTL associations, the -log 10 ( P ) in whole blood in GTEx is shown. For all the panels, the purple rhombus represents the index SNP (rs34590044) and the color represents the LD (r ) between other SNPs and index SNP. For the 11 SNPs within the credible set, upper triangles represent increased effects and lower triangles represent decreased effects of AMH-specific alleles. d) : Expression levels of ACSF3 when the region containing rs34590044 was inhibited by CRISPRi in HepG2 cells. NC: control. N=3. Error bars are 1 s.e.m. e : Protein levels of ACSF3 when the region containing rs34590044 was inhibited by CRISPRi in HepG2 cells. NC: control. N=3. Error bars are 1 s.e.m. f : Activity of the luciferase reporter of EV rs34590044-A or -C in HepG2 cells. Two-tailed t-test was performed between EV and rs34590044-A or rs34590044-A and rs34590044-C. N=3. EV: empty vector. Error bars are 1 s.e.m. g : Association between rs34590044 and the expression of ACSF3 in whole blood in GTEx. The x-axis shows the genotypes of rs34590044 and the number of individuals and the y-axis shows the normalized expression of ACSF3 . The raw P value is shown on the top. The figure is obtained from GTEx ( URLs ).

Article Snippet: The enhancer luciferase reporter constructs with the control luciferase construct Renilla were co-transfected into cells using PEI (Polysciences).

Techniques: Expressing, Control, Activity Assay, Luciferase, Two Tailed Test, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Structural basis of specific DNA binding by the transcription factor ZBTB24

doi: 10.1093/nar/gkz557

Figure Lengend Snippet: ChIP-Seq analysis identifies a 12-bp consensus DNA sequence recognized by ZBTB24. ( A ) Mouse ZBTB24 protein is schematically shown, including the BTB domain, AT-hook, and zinc finger (ZF) domain with eight tandem ZFs. ( B ) Pie chart showing the distribution of HA-ZBTB24 ChIP-Seq peaks in the genome. Upstream, -50 kb to -5 kb from TSS; promoter, −5 kb to +0.5 kb from TSS; TES, −0.5 kb to +5 kb from TES; downstream, +5 kb to +50 kb from TES; distal, other regions. ( C ) ZBTB24-binding motif identified from ChIP-Seq data. ( D ) CentriMo analysis showing the distribution of the motif to the peak summits. ( E ) Distribution of ZBTB24 signal over ±2 kb around the centers of ZBTB24 peaks with (red) and without (blue) the ZBTB24 motif. The signal of input was subtracted from the signal of ZBTB24. HA#3 and HA#4 are the two HA-ZBTB24 stable clones used for ChIP-Seq. ( F ) Genome browser screenshots showing a prominent ZBTB24-binding peak in the Cdca7 promoter.

Article Snippet: The Cdca7 luciferase ( Cdca7-Luc ) reporter construct was generated by cloning a ∼1.6-kb Cdca7 promoter region into the pGL3-Basic vector (Promega).

Techniques: ChIP-sequencing, Sequencing, Binding Assay, Clone Assay

Journal: Nucleic Acids Research

Article Title: Structural basis of specific DNA binding by the transcription factor ZBTB24

doi: 10.1093/nar/gkz557

Figure Lengend Snippet: The C382Y and C407G mutations abolish the abilities of mouse ZBTB24 to bind specific DNA and induce CDCA7 expression. ( A and B ) C382 and C407 are respectively one of the four Zn ligands in ZF4 and ZF5. C382Y and C407G correspond to ICF missense mutations. ( C ) A Coomassie brilliant blue-stained gel showing purified GST-ZBTB24 fragments comprising of ZF1–8 with no mutation (WT) and with the C382Y and C407G mutations. ( D ) EMSA using GST fusion proteins described in panel (C) and a specific DNA probe containing the ZBTB24-binding motif (described in Figure ). The protein concentrations used are indicated. ( E ) Rescue experiments. HA-tagged WT or mutant ZBTB24 was stably expressed in Zbtb24 -deficient mESCs and their ability to restore CDCA7 level was examined. Shown are western blots with the indicated antibodies. WT (J1), Cdca7 −/− and Zbtb24 −/− mESCs were used as controls. ( F ) Luciferase reporter assays. HA-tagged WT or mutant ZBTB24 or the empty HA vector was co-transfected with Cdca7 promoter-driven luciferase reporter constructs in Zbtb24 -deficient mESCs, and the cell lysates were used for luciferase activity assays 24 h post-transfection. Cdca7-Luc , reporter with intact ZBTB24-binding motif; Cdca7Δ-Luc , reporter with the ZBTB24-binding motif deleted. Luciferase activity results, relative to samples transfected with the empty HA vector, from three independent experiments (mean ± SD) are shown; ** P < 0.01.

Article Snippet: The Cdca7 luciferase ( Cdca7-Luc ) reporter construct was generated by cloning a ∼1.6-kb Cdca7 promoter region into the pGL3-Basic vector (Promega).

Techniques: Expressing, Staining, Purification, Mutagenesis, Binding Assay, Stable Transfection, Western Blot, Luciferase, Plasmid Preparation, Transfection, Construct, Activity Assay