Journal: bioRxiv

Article Title: Novel uAUG creating variants in the 5’UTR of ENG causing Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.12.18.520932

Figure Lengend Snippet: (A) cDNA of the long (NM_001114753.3) isoform of ENG transcripts. Positions of the identified 5’UTR uAUG-creating variants from this project and published studies – as well as position of the associated uStop codon at position c.125, and of main start (c.1) and stop (c.1975) codons are indicated. 5’UTR, 5’ Untranslated Region; CDS, CoDing Sequence; WT, wild-type; uoORF, overlapping upstream Open Reading Frame; eCDS, elongated CDS. (B) Schematic presentation of the pcDNA3.1-L-ENG constructs prepared and used for the evaluation of ENG steady-state levels in HeLa cells. Arrows indicate specific primers targeting the cDNA of ENG with extra sequences containing restriction sites to allow specific cloning in the expression vector. Position of the Myc-His tag is represented by the black square on the plasmid. CMV, cytomegalovirus promoter; WT, wild-type; Var, Variant; 5’UTR, 5’ UnTranslated Region; L, Long. (C and D) Western blot results on total proteins extracted from transfected HeLa cells with 1 µg of pcDNA3.1-L-ENG constructs or from transduced HUVEC cells with 20 MOI of lentiviruses containing ENG, respectively. Two bands of different molecular weights are observed for endoglin likely corresponding to more glycosylated (upper band) and less/non glycosylated (lower band) ENG monomers . Anti-Myc and anti-ENG correspond to the used antibodies for the target protein from HeLa and HUVECs, respectively, and anti-βactin corresponds to the antibody used against the reference protein. kDa, kilodalton; M, protein ladder; WT, wild-type, C-, negative control corresponding to pcDNA3.1-empty vector. Shown results are representative of 5 independent experiments. (E) Decrease of luciferase activity observed with uAUG-creating variants in ENG . Schematic presentation of the pGL3b-(ENG)-luciferase prepared and used in this assay is shown in the upper panel. Arrows indicate specific primers targeting the promoter of ENG with extra sequences containing restriction sites to allow specific cloning in the expression vector. Luc, luciferase. In the lower panel, shown results correspond to Firefly/Renilla ratios normalized to the wild-type (WT) in 5 independent experiments. ***, p-value < 10 −3 (two-factor ANOVA followed by Tukey’s multiple comparison test of variants versus WT).

Article Snippet: Four hours after transfection, cells were stimulated overnight with 5 pg/ml of BMP9 in serum-free medium (R&D Systems) and luciferase activity was measured with the twinlite Firefly and Renilla Luciferase Reporter Gene Assay System (PerkinElmer).

Techniques: Sequencing, Construct, Clone Assay, Expressing, Plasmid Preparation, Variant Assay, Western Blot, Transfection, Negative Control, Luciferase, Activity Assay

Journal: Scientific Reports

Article Title: Translational Inhibition of α-Neurexin 2

doi: 10.1038/s41598-020-60289-8

Figure Lengend Snippet: The 5′UTR of NRXN2α represses protein expression of downstream cistron. ( A ) Schematic diagram of plasmid constructs. The horizontal line represents the 5′ UTR of NRXN2α ; the black rectangles indicate the open reading frame of firefly luciferase (Fluc) and the open rectangles indicate the open reading frame of human NRXN2α gene. ( B ) SH-SY5Y cells were co-transfected with luciferase reporters and p3PRluc. Firefly luciferase activity was measured 24 h after transfection, and Renilla luciferase activity was used to normalize for transfection efficiency. Data are shown as relative values over the samples transfected with empty control p4P plasmid. ( C ) Western Blot of luciferase protein. HEK293 cells were transiently co-transfected with luciferase reporters and EGFP expression plasmid. ( D ) Density analysis of the luciferase protein. The density was normalized to that of β-actin. ( E ) Quantification of the firefly luciferase mRNAs in SH-SY5Y cells transfected with reporter plasmids. The luciferase mRNA levels were normalized to β-actin mRNA levels. ( F ) Quantification of the NRXN2α mRNAs in HEK293 cells transfected with NRXN2α expression plasmids. The NRXN2α mRNA levels were normalized to β-actin mRNA levels. ( G ) Western Blot of neurexin2α protein expression. HEK293 cells were transiently co-transfected with NRXN2α and EGFP expression plasmid. ( H ) Density analysis of the neurexin2α protein. The density was normalized to that of β-actin. All values were expressed as means ± S.D., N = 3. ***P < 0.001 and **P < 0.01 by the Student’s t test. The full-length blots for ( C , G ) are presented in Supplementary Fig. .

Article Snippet: Renilla luciferase reporter plasmid p3PRluc was generated by amplifying coding sequence of Renilla luciferase from plasmid phRL-CMV(Promega) by PCR and replacing the coding sequence of firefly luciferase in plasmid pGL3promoter at HindIII/XbaI cutting sites.

Techniques: Expressing, Plasmid Preparation, Construct, Luciferase, Transfection, Activity Assay, Control, Western Blot

Journal: Scientific Reports

Article Title: Translational Inhibition of α-Neurexin 2

doi: 10.1038/s41598-020-60289-8

Figure Lengend Snippet: G-quadruplex and uAUGs played a synergistic role in the inhibitory effect of NRXN2α-5′UTR. SH-SY5Y cells ( A – C ), SK-N-SH and U-87MG cell ( D , E ) were co-transfected with various luciferase reporter plasmids and p3PRluc. Firefly luciferase activity was measured 24–48 h after transfection, and Renilla luciferase activity was used to normalize for transfection efficiency. Data are shown as relative values over the samples transfected with plasmids containing wild type sequences of NRXN2α -5′UTR or control plasmids. ( A ) Comparison of luciferase activity between reporter with wild type and G4M10 mutant of full-length 5′UTR. ( B ) Comparison of luciferase activity among reporter with wild type and corresponding mutants of 5′UTR-401/462 nt. ( C ) Comparison of luciferase activity among reporter with wild type, uAUG triple mutation only and combined mutations of uAUGs and G4 sequence in full-length 5′UTR. ( D ) Comparison of luciferase activity between reporter with wild type of full-length 5′UTR and control plasmids in SK-N-SH and U-87MG cell. ( E ) Comparison of luciferase activity among reporter with wild type, mutant with triple mutations of three uAUGs and mutant of both three uAUGs mutation and G-quadruplex mutation in full-length 5′UTR. ( F ) Western Blot of luciferase protein. U-87MG cells were transiently co-transfected with luciferase reporters and EGFP expression plasmid. All values above were expressed as means ± S.D., N = 3. ***P < 0.001, **P < 0.01 and *P < 0.05 by One-Way ANOVA ( B , C , E ) or t -test ( A , D ). The full-length blo t s for ( F ) are presented in Supplementary Fig. .

Article Snippet: Renilla luciferase reporter plasmid p3PRluc was generated by amplifying coding sequence of Renilla luciferase from plasmid phRL-CMV(Promega) by PCR and replacing the coding sequence of firefly luciferase in plasmid pGL3promoter at HindIII/XbaI cutting sites.

Techniques: Transfection, Luciferase, Activity Assay, Control, Comparison, Mutagenesis, Sequencing, Western Blot, Expressing, Plasmid Preparation