BIOL 3P50 Molecular Genetics Past Lecture - March 25th, 2024 PDF

BIOL 3P50 Molecular Genetics March 25th, 2024 CHAPTER 14 RNA Splicing - part 2 Typical eukaryotic gene Original pre-mRNA transcript contains sequences that will be removed from mature mRNA = Introns Exons include all sequences included in the mature mRNA Coding and noncoding (e.g. UTRs, exons of non...

BIOL 3P50 Molecular Genetics March 25th, 2024 CHAPTER 14 RNA Splicing - part 2 Typical eukaryotic gene Original pre-mRNA transcript contains sequences that will be removed from mature mRNA = Introns Exons include all sequences included in the mature mRNA Coding and noncoding (e.g. UTRs, exons of noncoding RNAs) Process of removing introns = splicing Must occur with great precision Drosophila Dscam Cell surface proteins of immunoglobulin superfamily Act in neural patterning in the brain, and in innate immunity 24 exons, 4 with alternative forms 38,016 possible protein isoforms Mutually exclusive splicing of exon 6 Cannot be explained by steric hindrance, major/minor sites, or nonsense mediated decay Intron following exon 5 (before exon 6.1) contains docking site Can interact with different selector sequences found in front of each exon 6 variant Only one can bind with docking site at a time Interaction brings exon 6 variant close to exon 5 Other exons become coated with a splicing repressor protein Prevents their inclusion in the mRNA Selected exon is protected by RNA:RNA interactions Splicing Activators & Repressors Splicing enhancers or silencers are sequences that affect splicing at nearby sites Bound by splicing activators & repressors In alternative splicing, these may direct machinery to different splice sites under different conditions Mechanisms of silencer action e.g. HIV tat exon 3 Binding of SC35 activator to exon splicing enhancer (ESE) promotes inclusion of exon 3 Binding of A1 repressor to silencer promotes cooperative binding to adjacent sequences, preventing SC35 binding e.g. PTB splicing repressor Binds to sequences that flank an exon Interacts with U1 and inhibits its function, preventing exon inclusion Drosophila sex determination Sex determined by ratio of X chromosomes to autosomes Affects expression of Sxl gene in early embryo due to balance of transcriptional activators & repressors SisA and SisB activators from X chromosome Females have 2X Dpn repressor expressed from autosome Females and males have 1X Only females express Sxl from Pe promoter in early embryo Sxl protein Drosophila sex determination Sxl expression switches to Pm in later development Constitutive promoter, expressed in males and females Sxl protein is a splicing repressor & directs its own splicing In females, prevents inclusion of exon with stop codon Males have no Sxl due to lack of expression in early embryo mRNA includes exon with stop codon – no Sxl protein splice site blocked stop codon Drosophila sex determination Sxl also controls splicing of tra gene ♂ ♀ alternative 3’ splice site Tra protein produced in females but not males Tra is a splicing enhancer at the dsx gene stop codon Promotes inclusion of a different exon in females vs. males Dsx is a transcription factor that controls sexspecific genes Male and female proteins have 400 aa in common, followed by regions that are male- or femalespecific male-specific female-specific Alternative splicing & pluripotency Pluripotent cells can give rise to all tissues and cell types FOXP1 transcription factor exhibits alternative splicing in stem cells vs. differentiated cells Changes composition of DNA-binding domain Changes target site specificity Splicing & Human disease ~ 15% of point mutations that cause disease do so by altering splicing recognition sequences Disrupt normal splice sites or create new ones Alter binding sites for splicing enhancers or silencers Mutations may also affect splicing machinery, repressor, or activator proteins Mutations are often in introns and not detected by sequencing exons only E.g. Mutation in first intron of β-globin gene creates a 3’ splice site that is used instead of the normal splice site, leading to β-thalassemia correcting splicing defects Antisense oligonucleotides may be used therapeutically to alter splicing pattern e.g. SMN1 is required for maturation of snRNPs Mutations cause Spinal muscular atrophy (SMA) SMN1 differs from SMN2 by inclusion of exon 7 Splicing repressor hnRNP blocks inclusion Targeting antisense oligonucleotides to hnRNP binding site may allow inclusion of exon 7 into SMN2 Evolution of introns Introns are observed in eukaryotes but not prokaryotes Introns early model: Introns existed in all organisms, but were lost from bacteria Stream-lined genome allows increased rate of chromosome replication and cell division Rapidly growing unicellular eukaryotes (e.g. yeast) have far fewer introns than complex eukaryotes Introns late model: Introns never existed in bacteria and arose later in evolution Inserted into previously-existing genes, perhaps by a transposon-like mechanism Exon shuffling Creation of new genes by reshuffling exons Exons are short and introns are long Recombination likely to be in introns, shuffling exons rather than disrupting them Splicing machinery ensures exons are largely transferrable Evidence for this process: Borders between exons and introns often coincide with boundaries between domains Many genes contain exons that have duplicated & diverged, or duplicated & form repeating units within the protein Exon shuffling Evidence (cont.) Related exons are sometimes found in otherwise unrelated genes Numerous examples of proteins made up of highly related domains used in various combinations e.g. LDL receptor gene contains exons closely related to exons of C9 complement gene, and other exons closely related to EGF-precursor gene RNA Editing Targeted change of mRNA sequence Mechanisms: Site-specific deamination Guide RNA directed insertion or deletion of uridines Alters coding information in the mRNA Deamination Targeted deamination of cytosine or adenine e.g. Cytidine deaminase converts a C in apolipoprotein B mRNA to a U in intestinal cells only Converts CAA to UAA (stop codon) Produces full-length protein in liver, but truncated protein in intestine end Deamination APOBEC3G (A3G) is a related cytidine deaminase with antiviral activity Targets HIV cDNA and changes C to U Damages viral genome HIV produces Vif, which directs degradation of A3G and protects virus Deamination also occurs by converting adenine to inosine Enzyme = Adenosine deaminase acting on RNA (ADAR) A to I editing is required for mRNA encoding ion channel in mammalian brains Failure to edit impairs brain development Guide RNAs Observed in mitochondria of trypanosomes Addition or deletion of Us alters reading frame Modification is required to generate the correct reading frame Directed by a guide RNA that interacts with target RNA through an anchor sequence Sequence of guide RNA determines U insertion pattern U insertion RNAs interact and region where U will be inserted loops out Endonuclease cuts mRNA opposite loops Us are transferred into the gap Gap sealed by ligase RNA export Fully processed mRNA (capped, spliced, and polyadenylated) is transported to the cytoplasm Carefully regulated active process Distinguishes between mRNA, damaged & misprocessed RNAs, and liberated introns mRNA destined for cytoplasm is identified by the collection of proteins bound to it Poly-A binding protein, SR proteins, exon-exon junction proteins, etc. Export through nuclear pore by active transport Discarded proteins are imported back to nucleus Lecuyer et al, Cell, 2007 end