Analysis of RSV-F mRNA processing. A) Map of exon-intron structure and poly(A) signals of the precursor mRNA encoded by pIFwt. 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 pIFwt. Cytoplasmic RNA was isolated from transfected cells and reverse transcribed by oligo-dT priming (pIFwtcDNA). 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 pIΔIFwt and pIFwt 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 pIFwt 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.).