Mai M Abd El-Aziz

1. MRCPath Self Help Course 10th December 2009 Define, with examples, the following:a) Exon Splicing Enhancerb) Nonsense Mediated Decayc) Cryptic Splice SiteHow would you investigate the effect of a potential splicing mutation? Mai M Abd El-Aziz

2. Exon Splicing Enhancer • Pre-mRNA sequences important for splicing

3. Exon Splicing Enhancer • Specific short oligonucleotide sequences contain wide spectrum of sequences (~6-8 nucl) but they are hard to detect because of their degeneracy • Are recognised in mRNA by serine/arginine (SR) rich proteins • Enhance pre-mRNA splicing when present in exons • Play important roles in constitutive and alternative splicing • Computational methods were used to identify 238 candidate ESEs (e.g. the RESCUE-ESE (Relative Enhancer and Silencer Classification by Unanimous Enrichment) • ESEs are clinically significant because synonymous point mutations previously thought to be silent mutations located in an ESEs can lead to exon skipping and the production of a non functioning protein.

4. Models for ESE function • SR proteins bound to an ESE stabilise the binding of U2AF-65KDa to the PPT through interaction mediated by U2AF35 • SR also binds to U1snRNP-70 and U2Af-35 The ‘U2AF-recruitment’ model for ESE function

5. Models for ESE function • The ESE independent interactions are sufficient for the stable binding of U2AF-65 to the PPT. This involves BBP which interacts with U2AF-65 and U1 snRNP associated with PRP40 • The ESE dependent interactions involves one or more SR proteins bound to an ESE including SR-family and SR related proteins. Both U1 snRNP reactions are required for stable binding of U2 snRNP to the BBPfully assembled spliceosome U1 snRNP promotes two sets of cross-intron interactions ESE- dependant and ESE-independent

6. ESE & examples of genetic diseases • Disruption of an ESE in exon 3 of MLH1 gene is the cause of HNPCC in a Quebec family. • Silent mutation within exon 7 in SMN1 gene SMA through exon 7 exclusion • Nonsense mutation in exon 27 within a purine rich element of dystrophin gene BMD

7. Nonsense Mediated Decay • A form of RNA surveillance • In vivo quick degradation of an mRNA carrying a premature termination codon at least 50 nucleotides upstream of the last splice junctions • This can avoid the potentially lethal consequences of producing a truncated polypeptide which could interfere with vital cell functions dominant-negative effect or gain-of-function effects.

8. Mechanism of NMD • After pre-mRNA splicing a multi-protein complex is deposited upstream of each exon-exon junction on nascent mRNAs. • Upf3 joins the post-splicing complex via direct or indirect interaction with RNPS1 • The post-splicing complex binds TAP to initiate mRNA nuclear transport • Some post-splicing complex proteins remain on the mRNA, others dissociate • During translation if termination occurs upstream of the last exon-exon junction, interaction between the translation release factors, eRF1 and eRF3, and the postsplicing /hUpf1-3 complex trigger mRNA decapping leading to NMD • Non-stop decay TAP: mRNA export receptor, EJC or Mark , Upf: upframeshift proteins

9. NMD & the clinical outcome • Altering the pattern of inheritance Ex. -Thalassemia 5' PTCs in the -globin gene recessive trait 3' PTCs  atypical dominant form CLCN1 ad or ar myotonia congenita ABCC6 ad or ar Pseudoxanthoma elasticum (PXE) Retinal degeneration, Robinow syndrome & brachydactyly-type B

10. NMD & the clinical outcome (cont.) • Causing distinct traits to manifest from mutations in the same gene • EX. (PCWH)peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, Waardenburg syndrome, and Hirschsprung disease • WS4Waardenburg and Hirschsprung diseases

11. NMD & the clinical outcome (cont.) • Modifying the specific clinical phenotype • NMD could be a protective mechanism EX. - Missense mut. in COL1A1 severe form of Osteogenesis Imperfecta (OI) (D-veE) - Truncating mut.  milder form of OI • NMD could have an adverse effect on C/P EX. - Truncating mut. in HEXA  severe form Tay-Sachs disease (NMD) - Rare missense mut.  less severe form since truncating mutants may retain residual activity EX. Dystrophin gene DMD

12. Cryptic Splice Site • Cryptic or latent splice sites coincidentally resemble the sequences of authentic splice sites • Cryptic splice site can be activated 1. Directly: if a mutation change the sequence so the splicing apparatus recognise it as a normal splice site 2. Indirectly: A mutation affects an authentic splice site causing splice donor or acceptor site to be faulty so the splicing apparatus scans for other possible alternatives and select a cryptic splice site • The use of intronic cryptic splice site will introduce new aa • The use of exonic cryptic splice site will lead to deletion of coding DNA

13. Examples of cryptic splice site mutations • Activation of exonic cryptic splice site • Limb girdle muscular dystrophy (LGMD2A) patient with a silent mutation GGC>GGT leading to replacement of G624G which led to activation of cryptic donor splice site within exon 16 resulting in aberrant splicing and loss of coding sequence from exon 16 and introduction of frameshift • Activation of intronic cryptic splice site • Cystic fibrosis with CFTR mutation 3849+10KbC>T activates a cryptic splice site that lies 10 kb inside intron 19.

14. How would you investigate the effect of a potential splicing mutation? • Splicing mutation could lead to • Exon skipping • Intron retention • Frameshift mutation • Premature truncation of the protein (PTP) • Nonsense Mediated decay • In-frame deletions or insertions could lead to variability in disease expression

15. In-silico Methods • Splice-site scores and splice-site prediction http://rulai.cshl.edu/new_alt_exon_db2/HTML/score.html • RESCUE-ESE ESEfinder 2.0 (http://rulai.cshl.edu/tools/ESE) http://genes.mit.edu/burgelab/rescue-ese/ http://cubweb.biology.columbia.edu/pesx/ • Splice-site predictor programme developed by the Berkeley Drosophila Genome Project http://www.fruitfly.org/seq_tools/splice.html Prediction programs for detection of aberrations associated with variants in canonical splice sites should not be considered a replacement for in vitro studies, but rather a means to prioritize variants for in vitro analysis and confirmation of any associated splicing defects

16. Laboratory methods • Isolation of total RNA from fresh blood cells or LCLs or target disease tissue • Set-up of RT-PCR • Study the effect of ss mutations on the cDNA • Intinc expression system (Ho et al., 2008) intron-inclusive cDNA that unites the advantages of the cDNA and minigene systems. This allow simultaneous invest. Of the effect of exonic mutations on RNA splicing and protein function on PKU patients with PAH mutations • In certain SS mu predicted to result in major disruption of the proteins milder phenotype may be encountered w could be due to leaky mutations allowing for small amount of normal transcript. This can be dealt with by designing RT-PCR primers hybridising to the skipped exons. AT, MNK, ATP7A, propionic acidemia

17. Laboratory methods (cont.) • Protein truncation test (PTT) Is based on a combination of RT-PCR, transcription and translation • PCR is carried out on a cDNA via RT-PCR from RNA, or large exons in Genomic DNA. • The Essential feature of PTT is a specifically designed tailed sense primer. This contains four different regions: 1. At it’s 5’ end a T7 RNA-polymerase promoter sequence, which facilitates the in vitro production of RNA 2. A 5-7 bp spacer 3. A eukaryotic translation initiation sequence (Kozak sequence) which includes a ATG start codon, facilitating the initiation of protein synthesis 4. The 3’ region contains a gene specific sequence designed so that the sequence amplified reads in-frame from the ATG. • Large deletions, duplications and splicing mutations may be detected by agarose gel electrophoresis at this stage.

18. Laboratory methods (cont.) • Protein truncation test (PTT) (cont.) • After amplification the PCR product is added to a coupled in vitro transcription-translation system e.g. transcription/translation systems. For detection a labelled amino acid is included. The label can be either a radionucleotide such as S35, which is visualised by autoradiography or biotin for detection by chemiluminescence. • The resultant proteins are run out on a SDS-PAGE gel for sizing against normal control products and protein markers. • Studying the effect of ss mutations in vitro and in vivo • By examining the effect on the protein by Western blotting IHC • Animal models

19. References • Blencowe BJ. Exonic splicing enhancers: mechanism of action, diversity and role in human genetic diseases. Trends Biochem Sci. 2000 Mar;25(3):106-10. Review. • Fairbrother WG, Yeh RF, Sharp PA, Burge CB. Predictive identification of exonic splicing enhancers in human genes. Science. 2002; 297(5583):1007-13. • Churbanov A, Vorechovský I, Hicks C. computational prediction of splicing regulatory elements shared by Tetrapoda organisms. BMC Genomics. 2009;10:508. • Nissim-Rafina M, Kerem B. Splicing regulation as a potential genetic modifier. Trends Genet. 2002;18(3):123-7. • Lykke-Andersen J, Shu MD, Steitz JA. Communication of the position of exon-exon junctions to the mRNA surveillance machinery by the protein RNPS1. Science. 2001;293(5536):1836-9.