Journal: Nucleic Acids Research

Article Title: Post-transcriptional control of KRAS : functional roles of 5′UTR RNA G-quadruplexes, long noncoding RNA, and hnRNPA1

doi: 10.1093/nar/gkaf886

Figure Lengend Snippet: Formation of rG4 structures in the 5′UTR of KRAS mRNA. ( A ) Ribonucleotide sequence of the 5′UTR of KRAS mRNA (the sequence does not contain the intron and refers to the mature post-splicing form). The cartoon shows the three nonoverlapping rG4 structures formed within the first 80 nt of the 5′UTR of KRAS mRNA (s-80). ( B ) Sequences of the rG4 motifs present in the first 80 nt of KRAS 5′UTR and 1H Nuclear magnetic resonance (NMR) imino proton spectra of 200 μM utr-1, utr-z, and utr-c rG4 at 25°C and 46°C in 100 nM KCl, 10 mM KPi buffer. ( C ) Circular dichroism (CD) spectra at 25°C and 85°C of 3 μM s-80 (80-mer oligoribonucleotide) in 50 mM Tris–HCl, pH 7.4, 100 mM KCl. ( D ) Structure of the lentiviral plasmids carrying the KRAS-5′UTR, with the three rG4 motifs, upstream of Scarlet. Construct pLV-KRAS-5′UTRmut Scarlet contains the three rG4 motif mutated, whereas the other constructs carry mutational deletions of one or two rG4 motifs, as reported in the figure panel. ( E ) RNA immunoprecipitation (RIP) with G4-specific antibody BG4 in 293T cells stably trasduced with the lentiviral plasmids. The plot shows the rG4 enrichment over input. GAPDH was used as a control. Data are shown as mean ± standard deviation (SD; n = 3, independent experiments) (Dunn’s multiple comparison test).

Article Snippet: Phoenix Ampho (RRID:CVCL_H716), 293T (RRID:CVCL_0045), MIA PaCa-2 (RRID:CVCL_0045), and Panc-1 (RRID:CVCL_0480) were obtained from ATCC (Virginia, USA).

Techniques: Sequencing, Nuclear Magnetic Resonance, Circular Dichroism, Construct, RNA Immunoprecipitation, Stable Transfection, Control, Standard Deviation, Comparison

Journal: Nucleic Acids Research

Article Title: Post-transcriptional control of KRAS : functional roles of 5′UTR RNA G-quadruplexes, long noncoding RNA, and hnRNPA1

doi: 10.1093/nar/gkaf886

Figure Lengend Snippet: RIP, ChIP, ChIP-seq and effect of 5′UTR on transcription and protein levels. ( A ) The luciferase plasmids pKRASpro and pKRASpro-utr were developed to study the effects of the 5′UTR on transcription. In pKRASpro , luciferase expression is driven by the KRAS promoter (−377/−1 compared to TSS), whereas in pKRASpro-utr , luciferase expression is driven by the KRAS promoter (−377/−1) and the 5′UTR of KRAS (+1/+180) containing the rG4 motifs utr-1, utr-z, and utr-c. The plasmid p3XMEF2-luc , in which luciferase is driven by is driven by three tandem repeats of the MEF2 binding site [CTA(TATT) 4 TAG], positioned upstream of a minimal c-fos promoter, served as a control. ( B , C ) RIP ( B ) and ChIP ( C ) assays with the G4-specific antibody BG4 showing the enrichment of rG4 and G4 DNA motifs compared to input in 293T cells transfected with pKRASpro-utr or p3XMEF2-luc (control), as indicated. The concentration and time of PDS treatment are 1 μM, 24 h. Data are expressed as mean ± SD, n = 3 (independent experiments), a t -test was performed for the indicated comparisons. ( D ) ChIP-seq shows genomic coverage of DNA sequences after BG4 purification mapped within the KRAS and MYC genomic loci. ( E , F ) Luciferase (left) and mRNA levels (right) in 293T cells transfected with pKRASpro or pKRASpro-utr and treated or not with the indicated μM concentrations of PDS for 16 h. Data are expressed as mean ± SD, n = 4, independent experiments. Dunn’s multiple comparison test was performed.

Article Snippet: Phoenix Ampho (RRID:CVCL_H716), 293T (RRID:CVCL_0045), MIA PaCa-2 (RRID:CVCL_0045), and Panc-1 (RRID:CVCL_0480) were obtained from ATCC (Virginia, USA).

Techniques: RNA Immunoprecipitation - Chromatin Immunoprecipitation, ChIP-sequencing, Luciferase, Expressing, Plasmid Preparation, Binding Assay, Control, Transfection, Concentration Assay, Purification, Comparison

Journal: Nucleic Acids Research

Article Title: Post-transcriptional control of KRAS : functional roles of 5′UTR RNA G-quadruplexes, long noncoding RNA, and hnRNPA1

doi: 10.1093/nar/gkaf886

Figure Lengend Snippet: Influence on KRAS gene expression of G4 and rG4 in the promoter and 5′UTR mRNA, respectively. ( A ) Schematic representation of plasmid pKRASpro_mut containing upstream of luciferase the KRAS promoter with mutations in the 32R motif that destabilise G4 formation. ( B ) Relative luciferase levels in 293T cells transfected with 1 μg of the indicated plasmids and 100 ng of pRenilla, as an internal reference. Data are expressed as mean ± SD, n = 4, independent experiments. Dunn’s multiple comparison test. ( C ) Putative representation of G4-mid as a recruitment element for TFs, which then bind to the neighbouring 32R motif in the linear duplex conformation. ( D ) Levels of mScarlet and HPRT mRNAs measured in 293T cells stably transfected with the indicated lentiviral plasmids carrying either the wild-type KRAS-5′UTR or mutant KRAS-5′UTRmut in which utr-1, utr-c, and utr-z motifs are mutated or KRAS-5′UTR with deletion mutations carrying only one rG4 domain as shown in the scheme. Data are expressed as mean ± SD, n = 3, independent experiments. Dunn’s multiple comparison test. ( E ) Cas9 was used to remove utr-1, utr-z, and part of utr-c from KRAS-5′UTR in 293T cells. ( F ) KRAS mRNA levels relative to HPRT in wild-type and mutant (MUT) 293T cells, n = 3, independent experiments, t -test was performed. ( G ) Immunoblot analysis of KRAS protein levels in wild-type and mutant 293T cells (2 biological replicates for each clone). Right panel shows the quantification of the KRAS protein; a t -test was performed.

Article Snippet: Phoenix Ampho (RRID:CVCL_H716), 293T (RRID:CVCL_0045), MIA PaCa-2 (RRID:CVCL_0045), and Panc-1 (RRID:CVCL_0480) were obtained from ATCC (Virginia, USA).

Techniques: Gene Expression, Plasmid Preparation, Luciferase, Transfection, Comparison, Stable Transfection, Mutagenesis, Western Blot

Journal: Nucleic Acids Research

Article Title: Post-transcriptional control of KRAS : functional roles of 5′UTR RNA G-quadruplexes, long noncoding RNA, and hnRNPA1

doi: 10.1093/nar/gkaf886

Figure Lengend Snippet: Stability of KRAS mRNA and effect of lncRNA. ( A ) Scheme of the experiment for monitoring the effect of the 5′UTR of KRAS on mRNA stability. ( B – D ) Wild-type (WT) and CRISPR/Cas9 edited 293T cells (MUT cells, in which the rG4 motifs in 5′UTR were deleted) were treated or not with 1 μM PDS for 12 h and then with 10 μg/ml Chx or with 20 nM ActD ( C , D ). The cells were harvested at the indicated time points after treatment. Experimental mRNAt/mRNA 0 plots were best fitted to a standard first-order decay equation. n > 3, independent experiments, t -test was performed. ( E ) Pulse-chase labelling of newly synthesized mRNA with 4sU in wild-type and MUT 293T cells. The cells were treated with 4sU for 4 h and subject to thiol-specific biotinylation with Biotin-HPDP. Newly synthesized RNA was pulldown with streptavidin beads, recovered and subjected to RT-qPCR. ( F ) WT and MUT 293T cells were treated with 4sU for 4 h and mRNA determined as a function of time by RT-qPCR. Data are expressed as mean ± SD, n = 3, independent experiments, t -test.

Article Snippet: Phoenix Ampho (RRID:CVCL_H716), 293T (RRID:CVCL_0045), MIA PaCa-2 (RRID:CVCL_0045), and Panc-1 (RRID:CVCL_0480) were obtained from ATCC (Virginia, USA).

Techniques: CRISPR, Pulse Chase, Synthesized, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Post-transcriptional control of KRAS : functional roles of 5′UTR RNA G-quadruplexes, long noncoding RNA, and hnRNPA1

doi: 10.1093/nar/gkaf886

Figure Lengend Snippet: LncRNA HSALNT0012722 promotes the downregulation of KRAS . ( A ) RNAcentral v24 was interrogated with the complementary sequence of s-80 to identify lncRNAs containing this motif with the potential to form a duplex with the 5′UTR of KRAS . A total of 40 lncRNAs were fished out with a significance threshold of E > 9.6e 2 . A total of 18/40 were selected because they are complementary at the utr-1, utr-c, and utr-z level, as indicated. Four of these (highlighted in the table) were selected because they contain a region that is complementary to utr-c and utr-z (LINC01750.9, HSALNT0012723, and HSALNT0012722) or utr-1 (HSALNT0117439). A schematic representation of the mechanism of action of the lncRNA in regulating optimal levels of KRAS mRNA is provided. ( B ) Fluorescent mScarlet was inserted into plasmid pLV under the control of KRAS 5′UTR (pLV-KRAS-5′UTR Scarlet) or control 5′UTR (Addgene plasmid #184637) ( pLV-5′UTRcon ). The core sequences of lncRNA HSALNT0012722 and HSALNT0117439 complementary to utr-z/utr-c and utr-1, respectively, were cloned into pcDNA3 downstream of the CMV promoter to obtain pcHSALNT0012722 and pcHSALNT0117439 . ( C ) The mScarlet vectors were integrated into 293T cells. A total of 1 × 10 6 293 founder cells were transfected with 5 μg of the indicated pcDNA3 plasmids containing the core lncRNA sequence. The % mScarlet positive 293T cells was determined by flow cytometry 48 h after transfection with pcDNA3 , pcHSALNT0012722 or pcHSALNT0117439 . ( D , E ) Histograms showing the percentage of mScarlet positive cells ( D ) and mScarlet mRNA levels ( E ) in 293T cells stably transfected with pLV-KRAS-5′UTR Scarlet or pLV-5′UTRcon mScarlet . Data are expressed as mean ± SD, n = 8 in panel (D), n = 3 in panel (E), independent experiments. Dunn’s multiple comparison test was performed. ( F ) A RIP assay with BG4 was performed in Panc-1 cells to assess rG4 formation in the reporter by precipitating the RNA bound by BG4 and IgG. To subtract the possible formation of G4 in the mScarlet CDS, the same experiment was performed in cells that had integrated the control 5′UTR. Data are expressed as mean ± SD, n = 3, independent experiments. ( G ) A total of 3 × 10 5 Panc-1 founder cells generated as described in Fig. were co-transfected with 2 μg pcDNA3 plasmids expressing the indicated lncRNAs and 500 ng pEGFP C1 with 6 μl Lipofectamine 2000. After 48 h, the GFP + cells were scored for mScarlet positivity by flow cytometry. Data are expressed as mean of fluorescence (MOF) in the fraction of GFP + cells. Data are expressed as mean ± SD, n = 4, independent experiments. Dunn’s multiple comparison test was performed.

Article Snippet: Phoenix Ampho (RRID:CVCL_H716), 293T (RRID:CVCL_0045), MIA PaCa-2 (RRID:CVCL_0045), and Panc-1 (RRID:CVCL_0480) were obtained from ATCC (Virginia, USA).

Techniques: Sequencing, Plasmid Preparation, Control, Clone Assay, Transfection, Flow Cytometry, Stable Transfection, Comparison, Generated, Expressing, Fluorescence

Journal: Nucleic Acids Research

Article Title: Post-transcriptional control of KRAS : functional roles of 5′UTR RNA G-quadruplexes, long noncoding RNA, and hnRNPA1

doi: 10.1093/nar/gkaf886

Figure Lengend Snippet: LINC01750 lncRNAs bind to KRAS 5′UTR and affect KRAS mRNA levels ( A ) Schematic representation of the locus of LINC01750 transcripts. H3K27ac acetylation in Panc-1 cells compared to input, the positions of the four sgRNAs used to modulate lncRNA expression, and the region complementary to the 5′UTR of KRAS [indicated as complementary RNA (cRNA)] are shown. ( B ) Levels of KRAS mRNA and LINC01750 transcripts in 293T cells in which LINC01750 expression was enhanced by the dCas9-VP64 system (SAM + sgRNA) compared to control (SAM + sgRNACT). ( C ) LINC01750 transcripts were repressed in 293T cells by the dCas9-KRAB system (KRAB + sgRNA). As a control, the cells were transduced with KRAB + sgRNACT. ( D ) Levels of KRAS mRNA and LINC01750 transcripts in PDAC cells (MIA PaCa-2) in which lncRNA expression was suppressed by the dCas9-KRAB system (KRAB + sgRNA2) compared to control (KRAB + sgRNACT). ( E ) Western blot showing the level of KRAS protein in PDAC (MIA PaCa2) cells transduced with either KRAB + sgRNA2 or KRAB + sgRNACT. The histogram shows the KRAS/Tubulin ratio in the treated cells. ( F – H ) Scheme of the Biotin-streptavidin assay using RNA extracted from 293T (transduced with dCas9-VP64 to overexpress LINCO17250) (Fig. ) and PDAC (Panc-1) cells (Fig. ). Biotinylated 41-mer RNA with the utr-c and utr-z motifs was used as bait, while a 41-mer mutated at the rG4 motifs served as a control. Significant enrichment of LINC01750 transcripts was only achieved with the wild-type bait. No enrichment of HPRT and DAB1 transcripts was obtained (control). ( I ) PAGE showing RNase III-dependent degradation of KRAS 5′UTR hybridised to LncRNA. Twenty micromolar Cy5.5-labelled oligoribonucleotide containing the utr-z + utr-c motifs (41-mer 5′UTR) was melted at 95°C for 5 min and annealed for 3 h at RT in 50 mM Tris (pH7.4) in the presence or absence of 100 mM KCl. Where indicated, a 39-mer synthetic HSALNT0012722 was added in a 2:1 ratio to the 41-mer 5′UTR to allow formation of dsRNA. dsRNA was digested with 3U RNase III for 15 min at 30°C. The experiment was repeated twice with similar results.

Article Snippet: Phoenix Ampho (RRID:CVCL_H716), 293T (RRID:CVCL_0045), MIA PaCa-2 (RRID:CVCL_0045), and Panc-1 (RRID:CVCL_0480) were obtained from ATCC (Virginia, USA).

Techniques: Expressing, Control, Transduction, Western Blot

Journal: Nucleic Acids Research

Article Title: Post-transcriptional control of KRAS : functional roles of 5′UTR RNA G-quadruplexes, long noncoding RNA, and hnRNPA1

doi: 10.1093/nar/gkaf886

Figure Lengend Snippet: 5′UTR rG4 structures are bound by hnRNPA1. ( A ) RIP scheme to show that hnRNPA1 is associated with KRAS 5′UTR containing the rG4 motifs. ( B ) RIP performed with 293T cells using hnRNPA1 Ab and IgG (control) shows that hnRNPA1 is associated with the 5′UTR of KRAS mRNA containing the rG4 motifs. As a further control, we show no enrichment of GAPDH. ( C ) Same experiment as in panel (B), but using 293T cells in which pLV-KRAS-5′UTR mScarlet or pLV-KRAS-5′UTRcon mScarlet were integrated into the genome. The RIP shows that hnRNPA1 Ab but not IgG (control) pulldown RNA sequences enriched of KRAS 5′UTR. n = 3, independent experiments, t -test was performed. ( D ) RIP with 293T cells stably transduced with the lentiviral vectors described in Fig. . The hnRNPA1 enrichment in cells containing KRAS -5′UTR, KRAS -5′UTRmut, KRAS -5′UTR(D1DC); KRAS -5′UTR(DZDC) and KRAS -5′UTR(D1DZ) is shown. n = 3, independent experiments, Dunn’s multiple comparison test was performed. ( E ) Electrophoretic mobility shift assay (EMSA) showing binding of hnRNPA1 to 41-mer oligoribonucleotide containing the utr-c and utr-z motifs (41-mer 5′UTR), but not to the 41-mer Mut with mutated rG4 motifs. Increasing ratios of hnRNPA1:41-mer 5′UTR were used (1:10, 1:1, 10:1). ( F ) Typical ITC for the interaction of utr-c with UP1 (the proteolytic fragment of hnRNPA1 that retains rG4 binding and unfolding activity). ( G ) Best-fitting binding curves obtained for the binding of UP1 to utr-1, utr-z, and utr-c rG4s in 10 mM KPi buffer, 50 mM KCl.

Article Snippet: Phoenix Ampho (RRID:CVCL_H716), 293T (RRID:CVCL_0045), MIA PaCa-2 (RRID:CVCL_0045), and Panc-1 (RRID:CVCL_0480) were obtained from ATCC (Virginia, USA).

Techniques: Control, Stable Transfection, Transduction, Comparison, Electrophoretic Mobility Shift Assay, Binding Assay, Activity Assay