Article Title: Ribosomal frameshifting in the CCR5 mRNA is regulated by miRNAs and the NMD pathway
Figure Lengend 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.
Article Snippet: The PRF signal from Homo sapiens CCR5 was amplified from pCMV-XL4 (pJD819) containing the CCR5 open reading frame (Origene) using oligonucleotides with BamHI and SalI restriction sites.
Techniques: Modification, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Incubation