Gene Structure and Function Jo Field Thursday 10th December 2009

1. Gene Structure and FunctionJo Field Thursday 10th December 2009 • How does splicing contribute to the spectrum of mutations? • Make sure that you explain the terms: • donor and acceptor splice site • exon skipping • cryptic splice site • consensus sequence • exon splice enhancer • Make sure you understand, and explain to colleagues, the Shapiro and Senapathy scores

2. How does splicing contribute to the spectrum of mutations? • Plan • Brief introduction to splicing process • Basic process, consensus sequences, splice donor/acceptor & branch sites - Splice site scores (Shapiro & Senapathy) • ESEs etc. • Alternative splicing • Effect of mutations on splicing (give some examples) • intron retention, exon skipping, activation of cryptic splice sites • mutations in ESEs • mutations affecting splice regulatory proteins (trans-acting mutations, mRNA gain-of function) • Conclusion • therapeutic options - regulate splicing • future prospects to identify splicing mutations

3. How does splicing contribute to the spectrum of mutations? • Introduction • Splicing = mRNA processing, involving intron removal and exon joining • Importance of splicing consensus sequences • consensus sequences - distinctive sequences found in the majority of splice junction sequences, conserved • most introns start GT- and end -AG • Splice donor site(5’ end of intron) contains invariant GT & other conserved bases • Splice acceptor site (3’ end of intron) contains AG and polypyrimidine tract • Branch site (<40 nt upstream of 3’ end of intron) contains invariant A

4. How does splicing contribute to the spectrum of mutations? • Introduction - 2 • G at 5’ most end of intron involved in nucleophilic attack on A of branch site to form branched lariat structure • Branched intronic structure excised and exons joined • Mediated by spliceosome - complex of small nuclear RNAs and >150 proteins • snRNAs (as snRNPs) involved in RNA-RNA base pairing with splice consensus sequences • recognise splice consensus sequences and bring appropriate DNA regions together for splicing

5. How does splicing contribute to the spectrum of mutations? • Introduction - 3 • Strength of splice sites can be measured by scoring systems such as the Shapiro and Senapathy (S&S, SS) score • helps predict possible splice sites in novel DNA regions • can assess possible effect of mutations in splice site consensus sequences • Other online/software-based splice site prediction tools • e.g. Berkeley Drosophila Genome Project splice site prediction by Neural Network

6. How does splicing contribute to the spectrum of mutations? • Shapiro & Senapathy Score • Shapiro and Senapathy tabulated nucleotide frequencies in consensus splice sites from a wide range of eukaryotes • calculated a percentage for each nucleotide position • e.g. for splice donor site, last nucleotide of exon = 11% A, 3% C, 78% G & 8% T, while first nucleotide of intron = 100% G • For a particular splice site sequence, add up the frequencies for each nucleotide for each position, and express as a percentage of the maximum “best” splice site score • For instance, for the 5’ (splice donor) site, • S&S score = t - tmin where t = total of nt frequencies tmax - tmin for each position • tmax = highest possible total and tmin = lowest possible total

7. How does splicing contribute to the spectrum of mutations? • Shapiro & Senapathy Score - 2 • The Shapiro and Senapathy score for the splice acceptor site is calculated in a similar way • average of the score for the four nucleotides at the intron-exon boundary (-3 to +1) and the 8 best nucleotides from the polypyrimidine tract • as most splice acceptor sites have at least two purines in these positions • but does not allow an AG together in the polypyrimidine tract region

8. How does splicing contribute to the spectrum of mutations? • Introduction - cont • Other sequence elements can affect splicing efficiency • Positively acting Exonic/intronic splicing enhancers (ESE/ISE) • Negatively acting Exonic/intronic splicing suppressor/silencer (ESS/ISS) • Extensive, complex & diverse • direct splicing machinery to appropriate sites • inhibit use of cryptic splice sites • Some ESEs promote splicing by binding to SR protein family (serine/arginine-rich splicing factors) • Some ESSs/ISSs repress splicing by binding to heterogeneous nuclear ribonucleoproteins (hnRNPs) • However, many elements have not had corresponding trans-acting mediators identified

9. How does splicing contribute to the spectrum of mutations? • Introduction - cont • Briefly mention alternative splicing • several different transcript isoforms can be produced from a single gene • regulated by elements involved in the control of constitutive splicing (e.g. SR proteins) • as well as alternative splicing regulators • Alternative splicing increases biological complexity encoded by a gene and can be involved in tissue-specific/developmental stage-specific gene expression

10. How does splicing contribute to the spectrum of mutations? • Mutations affecting splicing • Splicing is a complex process - significant proportion of pathogenic mutations affect splicing (15-50%) • Mutations altering GT/AG dinucleotides abolish normal splicing - loss of function • Mutations in adjacent consensus sequences can have significant effect • can lead to intron retention, if no other splice sites available • incorrectly processed mRNAs can be retained in nucleus and remain untranslated • alternatively, can lead to exon skipping - use of alternative legitimate splice site, mutated exon not included in processed transcript • possible outcomes - in-frame or out-of-frame deletion/introduction of premature termination codon (PTC) • frameshifts resulting in PTC can be affected by nonsense mediated decay (NMD) if PTC > 50 nt upstream of last splice junction

11. How does splicing contribute to the spectrum of mutations? • Mutations affecting splicing - 2 • Mutations can directly or indirectly activate cryptic splice sites • sequences not normally involved in splicing • show sequence similarity to consensus splice site sequences • can result in inclusion of intronic sequence/exclusion of exonic sequence and can lead to frameshifts • Mutations in ESE/ISEs & ESS/ISS can have pathogenic effects • can resemble silent mutations (synonymous/intronic) • can be difficult to predict • can alter ratio of different splice variants

12. How does splicing contribute to the spectrum of mutations? • Mutations affecting splicing - Examples • Polymorphic TG and T repeat regions in CFTR intron 8 • 5T allele increases amount of exon 9 skipping • e.g. in variant/atypical CF • Mutation activating cryptic splice site • 3849 + 10 kb C>T in CFTR intron 19 activates cryptic splice donor site • leads to variable inclusion of an 84 nucleotide ‘exon’ from intron 19 • Mutation affecting exonic splice enhancer • C6T in SMN2 exon 7 (compared to SMN1 gene) • disrupts ESE, leading to exon 7 skipping in SMN2 - unstable transcript • SMN2 cannot compensate for loss of SMN1 in SMA • Splicing mutations may be difficult to identify using standard techniques that focus on coding regions • deep intronic mutations may be missed • use RT-PCR or higher capacity sequencing

13. How does splicing contribute to the spectrum of mutations? • Mutations affecting splicing - Trans-acting • Mutations of proteins involved in splicing regulation rather than consensus splice sites themselves • Example - HBII-52 small nucleolar RNA (snoRNA) - in SNURF-SNRPNlocus (affected in PWS) • Evidence that HBII-52 snoRNA regulates splicing of the serotonin receptor 2C • Loss of HBII-52 snoRNA in PWS leads to aberrant splicing of serotonin receptor 2C – more widespread disruption of splicing in PWS? • Also RNA gain-of-function mutations (e.g. CTG expansion in myotonic dystrophy) can disrupt normal splicing by sequestration of proteins involved in splicing regulation • eg MBNL1, muscleblind-like 1 • disrupts normal balance of antagonistic regulatory factors

14. How does splicing contribute to the spectrum of mutations? • Conclusion • Knowledge of a particular mutation’s effect on splicing can be important therapeutically • e.g. if can use targeted oligonucleotides to prevent aberrant splicing • promote exon skipping if appropriate • to increase expression of alternatively-spliced isoforms • Mutations affecting splicing are probably under-diagnosed • Splicing mutations likely to appear more frequently in future • using mutation detection techniques such as higher-throughput sequencing, allowing sequence analysis of large introns • splicing minigene assays, splicing microarrays • as knowledge of splicing regulatory elements e.g. ESE/ISEs increases • with increased characterisation of trans-acting factors regulating splicing

15. How does splicing contribute to the spectrum of mutations? • Feedback • Time limitations • ? Use of diagrams could be helpful? • References • Strachan and Read (2004). Human Molecular Genetics (Third Edition). Garland Science, London and New York (2004). • Shapiro and Senapathy (1987). RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res. 15, 7155-7174 • Wang and Cooper (2007). Splicing in disease: disruption of the splicing code and the decoding machinery. Nature Rev. Genet. 8, 749-761. • Cartegni et al (2002). Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nature Rev. Genet. 3, 285-298. • Fairbrother et al (2002). Predictive Identification of Exonic Splicing Enhancers in Human Genes. Science 297, 1007-1013 • Cartegni and Krainer (2002). Disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1. Nature Genet. 30, 377-384