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Article Title: Ribosomal frameshifting in the CCR5 mRNA is regulated by miRNAs and the NMD pathway
Figure Legend Snippet: Dual-luciferase reporters, frameshifting data from other cell types, and frameshift peptide LC-MS/MS sequencing data a , Schematic of dual-luciferase constructs used to test the CCR5 frameshifting signal. Transcription is driven from the SV40 early enhancer/promoter and transcription termination and polyadenylation utilizes the SV40 late poly(A) signal. The in-frame control is p2luci, encoding a firefly/ Renilla luciferase fusion protein. In the out of frame reporter (OOF), firefly luciferase lies in the –1 reading frame with respect to the Renilla open reading frame. In the HIV –1 PRF reporter, the –1 PRF signal of HIV-1 was cloned in between the two luciferase reporters, and the firefly ORF is in the –1 frame with respect to Renilla . The CCR5 –1 PRF reporter is the same, except that it contains the CCR5 –1 PRF signal. In the CCR5 slip site mutant (SSM), the UUUAAAA slippery heptamer of the CCR5 –1 PRF signal was mutated to GCGCGCG. The PTC reporter is based on the CCR5 –1 PRF reporter in which a premature termination codon was inserted following the Renilla open reading frame. b , Efficient –1 PRF promoted by the CCR5 sequence in CHO and Vero cells. Error bars denote standard error. c , d , LC-MS/MS analysis of the CCR5/β-Gal –1 PRF fusion protein. c , Primary amino acid sequence of the predicted fusion protein. Matched peptides are shown in bold red. Leader sequence is highlighted in cyan. CCR5 0-frame sequence is yellow, and CCR5 –1 frame peptide is highlighted in green. Non-highlighted sequence is β-galactosidase. d , Partial list of matching peptides identified by MS/MS analysis. Highlighted sequences show MSMS spectra of TSRIPVVHAVFALKSQ (2–17) and of TSRIPVVHAVFALKSQDGHLWGG (2–24) with N-terminal acetylation.
Techniques Used: Luciferase, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing, Construct, Clone Assay, Mutagenesis
Figure Legend Snippet: Specific stimulation of CCR5-mediated –1 PRF by miR-1224 a , HeLa cells were transfected with 0–30 nmol of miR-1224 miRNA expressing constructs and with HIV-1 or CCR5 –1 PRF reporters. b , PRF assays of HeLa cells mock-transfected (0), or transfected with scrambled miRNA (Scr), a miR-1224 antagomir (anti-1224), miR-1224, miR1224 + anti-miR-1224, or miR-1224 plus an siRNA directed against argonaute 1. c , Ablation of the miRNA processing machinery affects –1 PRF promoted by the HIV-1 and CCR5 frameshift signals. –1 PRF assays were performed using cells transfected with siRNAs targeting Argonaute 1 ( AGO1 ), Argonaute 2 ( AGO2 ), DGCR8 , exportin 5 ( XPO5 ) or scrambled sequences (Scr). Error bars denote standard error. * P
Techniques Used: Transfection, Expressing, Construct
Figure Legend Snippet: Close relationship of CCR5 sequences among simians a , Phylogenetic trees. The larger tree uses Danio rerio CCR5 as an outgroup while the smaller inset tree uses two Lemur species. Bootstrap support is included at each node; branch lengths are provided where appropriate. The low bootstrap values between some clades are indicative of the very small number of phylogenetically informative bases and high propensity of invariant bases. b , Sequence alignment of CCR5 coding sequences. The portion of the CCR5 coding sequence alignment containing the –1 PRF signal is shown from the seaview sequence editor. In total, these 45 species were trimmed to 7,000 bases each and comprise 827 complete sites, of which 241 are invariant and 586 were informative for phylogenetic analyses. Danio rerio (at bottom) provides an outgroup; when considering the conservation of the region surrounding the –1 PRF signal at position 408, it was removed. This results in 170 sites, of which 69 are invariant, and 58 informative. With the exception of the Lemur, the –1 PRF region is strikingly conserved among primates.
Techniques Used: Sequencing
Figure Legend Snippet: Specific stimulation of CCR5-mediated –1 PRF by miR-1224 a , Sequence of the CCR5 –1 PRF signal is shown. The UUUAAAA slippery site is italicized, stems 1 and 2 of the mRNA pseudoknot are coloured blue and red, respectively, and unpaired bases are black. Sequences of miR-1224, miR-711, and miR-1413, and their potential hybridization patterns with CCR5 sequence are indicated. b , Stimulation of –1 PRF by miR-1224 in HeLa cells. Cells were transfected with 5 nM of the indicated miRNA expressing constructs, or mock transfected. After 24 h incubation, cells were transfected with the indicated –1 PRF dual-luciferase reporters, and frameshift assays were performed 24–36 h. later. c , miR-141 stimulates CCR5 mediated –1 PRF in CHO cells. CHO and Vero cells were transfected with 5 nM of the indicated miRNA expressing constructs, with scrambled miRNA sequences, or mock transfected. After 24 h incubation, cells were transfected with the indicated –1 PRF dual-luciferase reporters, and frameshift assays were performed 24–36 h later. Error bars denote standard error. * P
Techniques Used: Sequencing, Hybridization, Transfection, Expressing, Construct, Incubation, Luciferase
Figure Legend Snippet: Mapping the CCR5/miR-1224 interaction: miR-1224 stimulates CCR5 –1 PRF by stabilizing Stem 2 through a proposed triple helical interaction a , Putative miR-1224 binding sites were changed to their complements (green). b , Gel-shift assays using miR1224 and M1, M2 and M3 variants of the CCR5 signal (described in ) using native conditions. n = 6 for each sample (three times each of two technical replicates). c , Models diagramming experimental results. The CCR5 pseudoknot is cartooned with wild-type sequences shown as black lines, mutant sequences shown as green lines, and miR-1224 in red. Proposed triple helical interactions between the CCR5 –1 PRF promoting pseudoknot and miR-1224 are shown in the Native and M2 models. Extended Data Fig. 5
Techniques Used: Binding Assay, Electrophoretic Mobility Shift Assay, Mutagenesis
Figure Legend Snippet: Models of the CCR5 –1 PRF stimulating mRNA pseudoknot a , Best-fit two-dimensional model based on chemical modification analyses. b , Three-dimensional model, two views. Slippery site is green, stem 1 is dark blue, unpaired bases and the loop within stem 1 are light blue, paired bases in stem 2 are red, and unpaired bases in stem 2 are yellow.
Techniques Used: Modification
Figure Legend Snippet: mRNA stability studies a , Rabbit β-globin reporter containing a doxycycline repressible promoter and SV40 derived polyA signal is shown. The native CCR5 –1 PRF signal was cloned into exon 1. Controls included insertion of the CCR5 SSM –1 PRF signal, PTCs in all three reading frames at this position, or insertion of the 27 nucleotide TNF-α-derived A-U rich element (ARE) immediately following the rabbit β-globin open reading frame. b , Time course measurements of rabbit β-globin reporter abundances transcriptionally arrested with doxycycline. c , Time course measurements of native CCR5 mRNA reporter abundances from HeLa-TZM BL cells transcriptionally arrested with actinomycin D. Cells were transfected with SMG1 miRNA or scrambled miRNA control. d , Effects of various RNAs on CCR5 mRNA steady-state abundance. HeLa-TZM BL cells expressing CCR5 were transfected as follows: scrambled siRNA control (scr), human SMG1 siRNA (Smg), miR-1224 (1224), a mIR-1224 antagomir (anti), and combinations thereof. b – d , n = 9 (three times on three independent biological replicates). Error bars denote standard error. * P
Techniques Used: Derivative Assay, Clone Assay, Transfection, Expressing
Figure Legend Snippet: Mapping and modelling the interactions of miR-1224 with the CCR5 –1 PRF signal in vitro and in live cells a , Dilutions of CCR5 –1 PRF signal (R5) transcript were mixed with [ 32 P]-labelled miR-1224 RNA (miR), and incubated at 30 °C (Native), or denatured at 90 °C and slowly cooled (Refolded). Samples separated through native PAGE were quantified and data plotted onto single site binding isotherms. K D values and standard deviations are indicated. b , In vivo pull-down of native CCR5 mRNA in live cells. Biotinylated miR-1224 precursor or a scrambled biotinylated control (Scr) were transfected into HeLa TZM BL cells expressing CCR5. Fold enrichment of affinity purified mRNAs were analysed by quantitative PCR with reverse transcription (qRT–PCR) using CCR5 - or GAPDH -specific primer sets. c , HeLa cells were co-transfected with dual-luciferase plasmids containing either the CCR5 or HIV-1 –1 PRF signal sequences and affinity-purified mRNAs were analysed as in b. d , EMSA assays were performed using miR1224 and M1, M2 and M3 variants of the CCR5 signal using native conditions. Single site binding isotherms generated from these data are plotted. K D values are indicated. For a and d , n = 6 for each sample (three times each of two technical replicates). For b and c , n = 9 for each sample (three times each for three biological replicates. Error bars denote standard deviation. * P
Techniques Used: In Vitro, Incubation, Clear Native PAGE, Binding Assay, In Vivo, Transfection, Expressing, Affinity Purification, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Luciferase, Generated, Standard Deviation
Figure Legend Snippet: Gel shifts and SHAPE analysis of CCR5 + miR-1224 a , b , In vitro binding of mIR-1224 with the CCR5 –1 PRF signal. Serial dilutions from 30 nM to 0.25 nM of a CCR5 –1 PRF signal (R5) containing transcript were mixed with equal volumes of 1.0 nM [ 32 P]-labelled synthetic miR-1224 RNA (miR), and incubated for 30 °C for 30 min ( a , Native), or at 90 °C for 5 s, cooled quickly to 60° and then slowly to 37° ( b , Refolded). Samples were separated through 10% native PAGE, dried, and intensities of retarded bands were quantified using phosphorimager and BioRad Quantity One software. c , d , miR-1224 does not bind to the HIV-1 –1 PRF signal in vitro . c , ‘Native’ annealing conditions. d , ‘Refolded’ annealing conditions. For a – d , n = 6 for each sample (three times each of two technical replicates). e , SHAPE analysis of the CCR5 –1 PRF signal in the absence (lane 7) or presence (lane 8) of miR-1224.
Techniques Used: In Vitro, Binding Assay, Incubation, Clear Native PAGE, Software
Figure Legend Snippet: Structural analysis of the CCR5 –1 PRF signal a , Computationally predicted and best fit 2-dimensional mRNA structures with chemical modification data. Stems 1, 2, and the loop are indicated as S1, S2 and L. The five different segments of stem 2 are labelled a–e. Nucleotide bases showing low levels of reactivity with DMS, CMCT, and kethoxal, and ribose sugars whose 2′-OH groups with NMIA were similarly non-reactive are noted as open circles. Moderately reactive sugars and bases are denoted by grey filled circles. Strongly modified (strongly reactive) sugars and bases are represented as black filled circles. Circles proximal to the bases denote reactivity of the bases, while circles distal to the bases denote reactivity of the ribose sugars. From left to right: mRNA pseudoknot best fit to data; mRNA pseudoknot predicted by Pknots; mRNA pseudoknot predicted by NUPAK; tandem stem-loops predicted by mFold. Red bases represent chemical modification patterns inconsistent with computational predictions. b , c , Chemical modification experiments. Autoradiograms of reverse transcriptase primer extensions performed on RNA transcribed by T7 RNA Pol from template PCR amplified from CCR5 –1 PRF signal containing plasmid. Bands correspond to strong readthrough control (RT) stops 1 nucleotide 5′ of bases modified by chemical reagents. b , The CCR5 mRNA was either left unmodified (un), or modified with 3 increasing concentrations of dimethyl sulphate (DMS, reacts with A and C), 1-cyclohexyl-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMCT, reacts with U), or 1,1-dihydroxy-3-ethoxy-2-butanone (kethoxal, reacts with G) respectively. c , Primer extension reactions were performed on unmodified samples and samples incubated with 30, 65, and 110 nM NMIA (N-methyl isatoic anhydride). These are labelled 1, 2 and 3, respectively, beneath each sample, and ‘un’ denotes untreated RNA. d , The all atom r.m.s.d. plot of the 80 ns-long molecular dynamics simulation states against the reference structure (the starting state of the simulation) at the end of a 2 ns-long equilibration. All measures exclude most mobile, single-stranded, first 7 nucleotides. The full structure r.m.s.d. values are shown in black, SL1 data are plotted in cyan, and the SL2 data are plotted in red. Mean r.m.s.d. values with standard deviations are given in the box. Overall, after approximately 21 ns the structure stabilizes. e , Two views (opposite sides) of an average minimized structure based on the last 12 ns of the simulation (indicated with red arrow in d ) where all the r.m.s.d. measures converge and are most stable.
Techniques Used: Modification, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Incubation
Figure Legend Snippet: The CCR5 sequence promotes efficient frameshifting a , Measurement of –1 PRF in HeLa cells. –1 PRF efficiency was monitored in HeLa cells using dual luciferase reporters. Error bars denote an approximation of standard errors. *** P
Techniques Used: Sequencing, Luciferase
Article Title: Ribosomal frameshifting in the CCR5 mRNA is regulated by miRNAs and the NMD pathway
Article Snippet: .. The PRF signal from Homo sapiens CCR5 was amplified from pCMV-XL4 (pJD819) containing the