Figure 3

Analysis of RSV-F mRNA processing. A) Map of exon-intron structure and poly(A) signals of the precursor mRNA encoded by pIF^wt. Arrows indicate location of primers used for the PCR analyses. The scale indicates the distance to the transcriptional start site. AATAAA: consensus signal for polyadenylation. B) Characterisation of splicing. 293T cells were transfected with pIF^wt. Cytoplasmic RNA was isolated from transfected cells and reverse transcribed by oligo-dT priming (pIF^wtcDNA). A PCR spanning the splice sites was performed with primers: 5'UTR-s and RSV-F-ia. The size of the PCR-products was compared to the size obtained in parallel PCR using pIΔIF^wt and pIF^wt plasmid-DNA (pDNA) as templates. H₂O: negative control. C) Characterisation of poly(A) signal usage. 293T cells were transfected with the indicated plasmids. Cytoplasmic RNA was isolated from transfected cells and reverse transcribed by priming with Oligo(dT)Add-a. As a control for DNA contamination, the reverse transcription reaction was also performed without the enzyme (-). The cDNA was amplified by PCR with primers 5' UTR-s and Oligo(dT)Add-a and the size of the PCR products was determined by agarose gel electrophoresis. D) PCR products from pIF^wt transfected cells were cloned and sequenced. The 3' end of the sequence obtained in 9 of 10 clones (RSV-F mRNA exp.) is shown aligned to the RSV-F sequence of the parental plasmid (RSV-F mRNA theor.).