2Genetic information is transferred from genes to the proteins they encode via a “messenger” RNA intermediateDNAGENEmessenger RNA(mRNA)proteintranscriptiontranslation
3Most genes have their protein-coding information interrupted by non-coding sequences called “introns”. The coding sequences are then called “exons”DNAGENEintronexon 1exon 2transcriptionprecursor-mRNA(pre-mRNA)intron
4The intron is also present in the RNA copy of the gene and must be removed by a process called “RNA splicing”proteintranslationmRNARNA splicingpre-mRNAintron
5Splicing a pre-mRNA involves two reactions intron branchpointpre-mRNAAintermediatesStep 1Aspliced mRNAStep 2
6Splicing occurs in a “spliceosome” an RNA-protein complex (simplified) (~100 proteins + 5 small RNAs)pre-mRNAspliced mRNASplicing works similarly in different organisms, for example in yeast, flies, worms, plants and animals.
7RNA is produced in the nucleus of the cell RNA is produced in the nucleus of the cell. The mRNA has to be transported to the cytoplasm to produce proteinsRibosomes are RNA-protein machines that make proteins, translating the coding information in the mRNA
9Alternative splicing In humans, many genes contain multiple introns 34512intron 2intron 3intron 4intron 1Usually all introns must be removed before the mRNA can be translated to produce protein
10However, multiple introns may be spliced differently in different circumstances, for example in different tissues.1235Heart muscle mRNA35421pre-mRNA1435Uterine muscle mRNAThus one gene can encode more than one protein. The proteins are similar but not identical and may have distinct properties. This is important in complex organisms
11Different signals in the pre-mRNA and different proteins cause spliceosomes to form in particular positions to give alternative splicingWe are studying how mRNAs and proteins interact in order to understand how these machines work in general and, in particular, how RNA splicing is regulated as it affects which proteins are produced in each cell and tissue in the body.
12Fas Fas ligand 7 6 5 6 5 7 Fas pre-mRNA APOPTOSIS Fas ligand Alternative splicing can generate mRNAs encoding proteins withdifferent, even opposite functionsFasFas ligand(membrane-associated)765657Fas pre-mRNA(+)APOPTOSIS(programmedcell death)(-)Fas ligandSoluble Fas(membrane)75
13Alternative splicing can generate tens of thousands of mRNAs from a single primary transcriptCombinatorial selection of one exon at each of four variable regions generates more than38,000 different mRNAs and proteins in the Drosophila cell adhesion molecule Dscam1248332pre-mRNAmRNAproteinThe protein variants are important for wiring of the nervous system and for immune response
14Examples of the potential consequences of mutations on splicing 35412ABCMutations occuron the DNA(in a gene)no mutationnormal mRNAmutation Atruncated mRNAmutation Bexon 3 skippedmutation Clonger exon 43541212541235412normal proteinactivetruncated proteininactiveprotein of different size (smaller or longer)inactive or aberrant function
15Pathologies resulting from aberrant splicing can be grouped in two major categories Mutations affecting proteins that are involved in splicingExamples: Spinal Muscular AtrophyRetinitis PigmentosaMyotonic Dystrophy Mutations affecting a specific messenger RNA and disturbing its normal splicing patternExamples: ß-ThalassemiaDuchenne Muscular DystrophyCystic FibrosisFrasier SyndromeFrontotemporal Dementia and Parkinsonism
16Therefore, understanding the mechanism of RNA splicing in normal cells and how it is regulated in different tissues and at different stages of development of an organism is essential in order to develop strategies to correct aberrant splicing in human pathologies