Mut

1. Ex vivo analysis of splicing assays Splicing reporter Cultured cell transfection RNA isolation RNA Reverse Transcription 5’ 3’ cDNA PCR PCR products Agarose gel analysis Wt Mut Outcome of the experiment : a system to analyze the role of RNA binding proteins on alternative splicing in cultured cells. Questions that can be answered: what are the factors that regulate a splice site selection ? Where are the regulatory sequences that are responsible for a splicing event ?

2. WT LMNA A Full length mRNA Lamin A EXON 11 EXON 12 1824C>T Mut LMNA B Figure 1: The use of a cryptic 5’ splice site is at the origin of the Hutchison Gilford Progeria Syndrome A) In normal individuals, a distal 5’ splice site is used in exon 11 of LMNA gene. The encoded mRNA will be translated into prelamin A. This protein will undergo 4 steps of postranslational maturation. Mature lamin A is a component of the nuclear envelope . B) In HGPS patients, there is an activation of a cryptic splice site in exon 11 of LMNA gene. The use of this cryptic splice site leads to the deletion of 150 nucleotides, thus 50 aminoacids, from prelamin A. This truncated prelamin A will undergo an incomplete maturation and remain farnesylated, thus aggregating in the nuclear periphery. The nuclear abnormalities that are caused by the truncated protein, called progerin, are responsible for progeria. D150 mRNA Progerin EXON 11 EXON 12 Normal nuclei Progeria nuclei

3. A 0.5 to 2 µg of plasmid DNA per well (in a 6 well plate) 2 to 4 µl of dreamfect per µg of plasmid DNA B Incubate 20 min at room temperature C D Assay for splicing of your minigene reporter +/- 24hours post transfection • Figure 2: Quick protocol for splicing reporter transfection • Prepare two solutions, one containg plasmid DNA and one containing Dreamfect ReagentTM (never vortex dreamfect!) • Combine the two solutions carefully: drop wise addition, mixing by gently pippetting up and down. • Add the solution to the cells with freshly replaced medium. Rock the tissue culture plate to homogenize. • Incubate the cells at 37°C in a CO2 incubator, evaluate splicing of your reporter.

4. A B Wt Mut WT bglobin Intron Lamin intron 11 12 bglo exon 1 Lamin exon 11 12 Lamin Lamin 500 bp 400 bp 300 bp Mut bglobin Intron Lamin intron 11 12 bglo exon 1 Lamin exon 11 12 Lamin Lamin Figure 3 : Anticipated results A) When transiently transfected in Hela cells, wild type bglo-LMNA splicing reporter produces only the full length splicing isoform. On the other hand 1824C>T bglo-LMNA reporter shows a very important use of the cryptic splice site, after agarose gel analysis of RT-PCR products (B).