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Gene Structure and Function Jo Field Thursday 10th December 2009

Gene Structure and Function Jo 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

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Gene Structure and Function Jo Field Thursday 10th December 2009

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  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

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