UC Biologia Molecular Past Paper 2024-2025 PDF

UC Biologia Molecular| Ano lectivo 24_25 Parte II| Aula: 3 25 e 26 novembro Sumário: Splicing alternativo regulado. Diferentes tipos de splicing alternativo (SA/AS). O código de splicing. Enhancers e silenciadores de splicing- RNA binding proteins. De nição exão/ intrão. Factores que afectam o SA....

UC Biologia Molecular| Ano lectivo 24_25 Parte II| Aula: 3 25 e 26 novembro Sumário: Splicing alternativo regulado. Diferentes tipos de splicing alternativo (SA/AS). O código de splicing. Enhancers e silenciadores de splicing- RNA binding proteins. De nição exão/ intrão. Factores que afectam o SA. SA controla padrões complexos de desenvolvimento. Defeitos no splicing do pre mRNA e a sua associação com doenças. Edição do RNA e a sua associação com doenças genéticas. Regulated alternative splicing. Different types of alternative splicing (SA/AS). The splicing code. Splicing enhancers and silencers - RNA binding proteins. Exon/intron de nition. Factors affecting AS. AS controls complex patterns of development. Defects in pre mRNA splicing and their association with diseases. RNA editing and its association with genetic disorders. 1 S. Mendo| BM24_25  Splicing ‣ In humans, during transcription elongation at least 65% of introns are removed prom pre- mRNA within 5 minutes; ‣ Introns, must be ef ciently and precisely removed in order to ensure delity in gene expression ‣ splicing may not be ef cient ( 5-15% of expressed intron are retained with mRNA) ‣ some will be spliced post-transcriptionally in the nucleus ‣ others will be directed to nonsense-mediated decay (NMD) Prp8 (SF component of U5snRNP): is part of the fully assembled complex B, playing two crucial roles in splicing. maintaining conformation of active site, involved in substrate positioning ‣ Motifs of Prp8 facilitate interactions between pre-mRNA, snRNPs and splicing factors (SFs) 2 S. Mendo| BM24_25 fi fi Retinitis pigments: a disease caused (in part) by Prp8 gene mutations ‣ Causes blindness in humans (degeneration of photoreceptors in the retina), and affects 1/4000 individuals; ‣ X-linked, autosomal dominant or recessive; ‣ Prp8-RP mutants cause defects in spliceosome activation; ‣ In Prp8-linked RP, disease likely results not only from defects in spliceosome assembly; and activation, but also because of defects in splicing catalysis. 3 S. Mendo| BM24_25  Alternative splicing ‣ 95-100% of human genes pre- mRNA transcripts are alternatively spliced ‣ Each gene: 3 or more isoforms (~25K genes…90K proteins) https://bitesizebio.com/10148/what-is-alternative-splicing-and-why-is-it-important/ 4 S. Mendo| BM24_25  Di erent types of splicing events ✓Constitutive exons are always included in mRNA´s; ✓The splicing of some exons is regulated (may be included or not). ‣ Constitutive splicing: mRNA is spliced in exactly the same way, every time, by the spliceosome. Sen. Hepatoma Research 2018;4:37 DOI: 10.20517/2394-5079.2018.39 5 S. Mendo| BM24_25 ff  Alternative splicing resulting isoforms ✓Alternate promoters can change transcription initiation sites, resulting in different isoforms. Human Hepatocyte nuclear factor 4 alpha (HNF4A) isoforms http://atlasgeneticsoncology.org/Genes/HNF4AID44014ch20q13 ✓Microarray and RNA-seq showed that 95% human genes encode alternatively spliced mRNAs 6 S. Mendo| BM24_25  The power of (extreme) alternative splicing Dscam (down syndrome cell adhesion molecule- human homologue maps at cr XXI gene ( rst described in humans) contribute to the diversity of neurone connections important for normal brain development in fruit y Dscam gene (95 variable exons out of 115) can produce 38,016 different proteins! 7 S. Mendo| BM24_25 fi  Size of exons vs introns? ✓In humans, Exon ~100-300nt; introns >thousands nt (50-110 kb) ✓Introns can contain pseudoexons which have a splice site How exons are recognised by the splicing machinery? 8 S. Mendo| BM24_25  How exons are recognised by the splicing machinery? ✓The recruitment of SFs is controlled by activating and inhibitory proteins; ✓The splicing sites possess binding sites for nRNABP embedded in the pre-mRNA, referred to as “splicing code”, which is required for gene expression ✓The splicing code is made up of silencers and enhancers which help the spliceosome distinguish between introns and exons and pseudoexons in pre-mRNA Activate splicing of associated exons: ‣ are present within exons: ESEs ‣ or introns: ISEs Repress exon splicing: ‣ are present within exons: ESSs ‣ or introns: ISSs SC35 and ASF/SF2: members of SR (ser/arg rich) family of SFs In interphase nucleus SR are found in subnuclear domains (speckles- storage depots for components of the splicing machinery); Exonic Splicing Enhancers (ESE) Exonic Splicing Silencers (ESS) has splicing suppression functions 9 S. Mendo| BM24_25  Schematic illustration of pre-mRNA splicing. ‣ Most RNA-binding protein in the nucleus exist as heterogeneous ribonucleoprotein (hnRNPs) particles ‣ After splicing has occurred, the proteins remain bound to spliced introns and target them for degradation The presence of the proteins (hnRNPs) bound to a pre-mRNA molecule serves as a signal that the pre-mRNA is not yet fully processed and therefore not ready for export to the cytoplasm. 10 S. Mendo| BM24_25  Splicing code is deciphered by RNA-BP ✓Strong exons have strong splicing codes (consensus sequences+ biding sites for splicing enhancers) ✓ Splicing activators such as SR proteins bind to exonic splicing enhancers (ESEs), and interact with and recruit U2AF and/or U1 snRNP to the 3′ and 5′ splice sites respectively. ✓ Splicing repressors such as hnRNP proteins inhibit splicing by binding to intronic splicing silencers (ISSs), interfering with the binding of U2AF to the 3′ splice site (Poli-Py tract). ✓ Alternatively, hnRNP proteins can bind to ISSs in the introns anking an exon (preventing other proteins from binding), where they engage in protein-protein interactions and loop out the intervening exon. Nature Structural & Molecular Biology (2009) 16(1):13- 11 DOI: 10.1038/nsmb0109-13 S. Mendo| BM24_25 fl  De ning the splicing that will operate: exon and intron de nition ✓Exon de nition marks which of the pre-mRNA sequences are exons; primarily in humans ✓Intron de nition: splicesosome assemble directly through cross-intron interactions 12 S. Mendo| BM24_25 fi fi fi  Factors contributing to Alternative Splicing (AS) regulation It seems logical that splice junctions ef ciently recognised by the spliceosome are always part of an mRNA, while those weakly recognised are not ef ciently handled. ✓Three factors contribute to AS regulation: 1.Changes in the expression of RNA binding proteins (that recognise de splicing code in pre-mRNA) 2. Changes in the upstream signalling pathway (may initiate outside the cell but control mRNA in the nucleus 3.Changes in the rate of transcription 13 S. Mendo| BM24_25 fi  Factors contributing to AS regulation 1. Changes in nuclear concentration of RNA binding proteins can activate splicing: e.g.: Meiosis-speci c splicing in yeast No splicing outside meiosis Outside meiosis Exon 1 1 Exon 2 Exon 1 Exon 2 No Mer2 protein Weak ISE Mer2 pre-mRNA Mer2 pre-mRNA 5´SS Meiosis Splicing in meiosis Activation complex U1 Mer1 protein Exon 1 1 Exon 2 Exon 1 Exon 2 Mer2 protein ISE Mer2 pre-mRNA Mer2 mRNA ‣ Mer2: encodes a protein important for meiotic recombination Mer1 stabilizes binding of ‣ 5´SS is inef ciently recognised by U1, except during meiosis U1snRNP with the 5´splice site ‣ Mer1: RNABP that binds to the intronic splicing enhancer (ISE) 14 S. Mendo| BM24_25 fi fi  Factors contributing to AS regulation 2. Changes in the cellular environment can trigger changes in the activity of nRNABP to which transcripts with appropriate cis-sequences respond. Cells respond to stress (UV irradiation, osmotic shock) by activating cascades of proteins phosphorylation ‣ hnRNP A1 (splicing repressor protein) concentrates in the nucleus, where is regulates splicing events; ‣ Yet, it shuttles between the nucleus and the cytoplasm; P blocks its entrance back into the nucleus; ‣ As a result A1 continues to be exported from the nucleus, accumulating in the cytoplasm, leading to changes in splicing patterns 15 S. Mendo| BM24_25  Factors contributing to AS regulation 3. How can transcription affect alternative pre-mRNA splicing? ‣ Elongation time of polymerase ‣ Long introns lead to a time lag in exon synthesis, thus spliceosome will have no choice for other competing exons further downstream Alberto R Kornblihtt 2006 STRUCTURAL & MOLECULAR BIOLOGY| VOLUME 13 NUMBER 1 16 S. Mendo| BM24_25  Biological importance of alternative splicing (AS) AS can affect ‣ Protein domains: inserting new peptide information ‣ Protein modi cations: including or excluding peptide sequences that undergo phosphorylation ‣ Protein-protein interactions: inserting information for interaction domains ‣ Produce dramatically di erent proteins. E.g.: male and female isoforms of Doublesex TFs that control sex differentiation in Drososphila ‣ Female sex determination starts with production of Sex-lethal RNA BP (SxL); males don’t do this protein. https://doi.org/10.1016/j.cub.2007.03.012 Male TRA isoform has STOP codons, thus not functional 17 S. Mendo| BM24_25 fi ff Biological importance of alternative splicing (AS) Exon 3 of sxl gene contains stop codon; this exon is included in the mRNA produced by males, but is skipped in females 18 S. Mendo| BM24_25  AS is crucial and shapes many cellular processes Alternative splicing has a key role in many other cellular processes ‣ E.g.: AS in nervous system In the absence of Alternative NOVA-1 the glycine splicing between receptor and other two mutually exons are misregullated exclusive exons represses 3A, so, 3B is included leads to apoptotic cell 3A and 3B death of motor neurons. ✓ NOVA is an RNABP; in humans NOVA 1 and 2 (expressed in different parts of the brain, thus their products can not complement each other) ✓ NOVA proteins are involved in shaping the synapse and thus are important for neuronal splicing. ✓ Knock out of encoding genes leads to a dramatic phenotype in mice (misregulation, apoptotic cell death of neurons, thus mice die soon after birth) ✓ NOVA regulates splicing of introns encoding proteins involved in synapse function ✓GlyR⍶2 (encode a subunit of the Gly receptor in the brain pre-mRNA and is regulated by NOVA ✓NOVA (neuro-oncological ventral antigen 1) proteins have a connection with cancer: people suffering from this types of cancer develop antibodies against NOVA proteins, initially leading the tumour to shrink; later this autoantibodies attack neuronal cells, leading to neurological deffects, trembling, and causing paraneoplastic disorders. 19 S. Mendo| BM24_25  Mutations a ecting the splicing code ✓ Point mutation which affect RNA splicing signal ✓ creation of new 5´ splice site: mutation causing premature ageing syndrome (HGPS) 20 S. Mendo| BM24_25 ff  RNA splicing defects and disease A high number of point mutations leading to disease caused defects in mRNA splicing ‣ Mutations in splice site sequences ‣ Mutations in splicing control sequences (splicing enhancers: ESE/ ESS, and silencers: ISE and ISS)-alter the splicing code J Med Genet: rst published as 10.1136/jmg.2004. 029538 on 30 September 2005 21 S. Mendo| BM24_25 fi RNA splicing defects and disease 22 S. Mendo| BM24_25 Hutchinson-Gilford progeria syndrome (HGPS) 23 S. Mendo| BM24_25  Genetic mutations create a new (5´) splice site ‣ Lifespan ~13 years ‣ ageing phenotype (wizened skin, osteoporosis, baldness, cogged arteries ‣ Due to (dominant) mutation within the LMNA gene (prelaminA) ‣ Complete deletion of LMNA causes a form of muscle dystrophy ‣ Subtle point mutations in LMNA cause HGPS, creating a new 5´SS within LMNA exon 11. Part of exon 11 will be missed. ‣ Lower levels of LMNA splicing defects contribute to normal ageing 24 S. Mendo| BM24_25  Abnormal processing of lamin A in HGPS The mutation in exon 11: ‣ activates a cryptic splice site leading to deletion of 50 aa residues from the precursor protein, including the nal Zmpste24 cleavage site (Protease) Premature ageing e ects ✓ addition of a 15-carbon isoprenoi ✓ facilitates their membrane association and also promotes protein-protein interaction. 25 S. Mendo| BM24_25 ff d fi  Mutation of an exonic splicing enhancer (ESE) leads to breast cancer ✓BRCA1 are normally expressed in the cells of breast ✓Involved in DNA damage repair (error-free repair of DNA double-strand breaks) or in destroying cells if DNA cannot be repaired. ✓Non repaired damages increases the risk for breast cancer ✓The BRCA1 protein associates with RNA polymerase II, and through the C-terminal domain, also interacts with histone deacetylase (transcription repression) complexes. ✓Point mutations prevent exon recognition by the spliceosome and thus exon skipping ‣ Exon 18 ESE (exon splicing enhancer ) is a critical site to which SR binds to correctly recognise the exon (this site is altered) ‣ Exon 18 is not spliced into the mRNA ‣ BRCA1 is not functional 26 S. Mendo| BM24_25  Mutations a ecting components of the spliceosome ‣ Removal of introns from pre-mRNA precursors (pre-mRNA splicing) is a necessary step for the expression of most genes in multicellular organisms, and alternative patterns of intron removal diversify and regulate the output of genomic information. ‣ Mutations in genes encoding spliceosome components (snRNAs and splicing proteins) have a dramatic e ect to the cell ✓ Disease-associated mutations in the speci c protein and snRNA components of the minor spliceosome. E.g.: Microcephalic osteodysplastic primordial dwar sm 27 S. Mendo| BM24_25 fi fi ff ff  Splicing changes can alter the properties of cancer cells ✓Changes in splicing cause different mRNA´s to be made. ✓Tumors undergo metabolic reprogramming to support the synthesis of new macromolecules required for rapid cell division. ✓Metabolic transformation of cancer cells encompasses multiple interconnecting metabolic networks with feedback loops and crosstalk acting to provide plasticity and help the cells survive the steep and localized nutrient and oxygen gradients present in the harsh tumor microenvironment ✓The glycolytic pathway sustains tumors through the generation of metabolites that bene t cell proliferation. ‣ Cancer cells switch from oxidative metabolism in mitochondrial respiration to break down sugars in the cytoplasm by glycolysis- Warburg e ect (“Anaerobic glycolysis”) ‣ Favor a specialised fermentation over the aerobic respiration pathway Mutually exclusive alternative splicing of exon 9 and exon 10 PKM works slowly; accumulation of upstream components in glycolysis PKM: pyruvate kinase M which are used by cancer cells 28 Constitutively exists S. Mendo| BM24_25 ff  Splicing changes can alter the properties of cancer cells Cancers 2018, 10, 458; doi:10.3390/cancers10110458 29 S. Mendo| BM24_25  RNA editing in mammals ‣ Post transcriptional modi cation occurring in ds RNA, catalyzed by adenosine deaminases acting on the RNA (ADAR) protein family ‣ A wide spread mechanism for changing of gene-speci c codons and thus protein (recoding) structure and function. ‣ Plays important roles in regulating diverse processes, including neurotransmission and lipid metabolism. ‣ Approximately 85% of pre-mRNAs are estimated to be edited in humans. This preferentially occurs in the non-coding region of mRNA, especially in the 3′untranslated region (UTR)- Alu elements- and introns. Two classes of editing enzymes ‣ Generate inosine (I) from adenosine (A); thus A will pair with G ‣ Generates uridine (U) from cytidine C 30 S. Mendo| BM24_25 fi fi RNA editing defects and neuro bromatosis type 1 (a form of cancer) ‣ C to U editing creates a premature STOP codon, which results in a truncated form of the protein NF1(neuro bromin) that lacks tumor supressor functions Neuro bromatoses (not a single medical disorder) are a group of genetic disorders that cause tumors to form on nerve tissue (including the brain, spinal cord and nerves) 31 S. Mendo| BM24_25 fi fi fi  RNA editing in mammals and neuronal disorders ‣ Could a ect: splicing, RNA localization, RNA stability and, ultimately, translation ‣ Particularly abundant in brain tissues (receptors and ion channels) ‣ Editing within coding regions occur in a number os neurological disorders (in ammation, epilepsy, depression, cancer, amyotrophic lateral sclerosis - ALS). ✓ALS or Lou Gehrig's disease, is a neurological disorder that a ects motor neurons, the nerve cells in the brain and spinal cord that control voluntary muscle movement and breathing. ‣ A ects 0.8-7.3 /100000 individuals ‣ 5-10% cases are familial ✴ defects in >30 genes (SOD1, ALS2; senataxin, involved in key cellular processes: oxidative stress, protein misfolding, axonal transport) ‣ Sporadic ALS (non hereditary) accounts for >90% of cases ✴ ine cient RNA editing of ⍺-AMPA receptors (subtype of glulatamate receptors) ✴ high concentrations of glutamate can cause cell death due to excessive activation of ⍺-AMPA receptors ✴Ca2+ in ux-triggered excitotoxicity (excessive release of glutamate from axon terminals) through Ca2+-permeable (CP)-AMPA receptors (AMPARs) 32 S. Mendo| BM24_25 ff ffi fl ff ff  RNA editing in mammals and neuronal disorders ‣ Almost all GluR2 mRNA undergo RNA editing catalysed by ADAR2: Glutamine (Q) to Histidine (R) in transmembrane domain 2 (M2) DOI:10.3389/fnmol.2012.00034 Ca 2+ permeability of AMPA receptors and GluR2 subunit. AMPA comprise GluR1, GluR2, GluR3, GluR4. Ca 2+ permeability of AMPA differs depending on the presence of GluR2. Receptors lacking GluR2 or containing unedited GluR2 (Q at Q/R site) are Ca 2+ DOI 10.1007/s00109-004-0599-z permeable Structure of GluR2: The Q/R site is located in the putative second membrane domais (M2) In ALS patients the de ciency in RNA editing is due to an age -dependent reduction of ADAR2 deaminase activity 33 S. Mendo| BM24_25 fi SUMMARY ‣ AS produces many different mRNA isoforms, generating proteomic diversity ‣ Different kinds of splicing can occur ‣ AS can be positively regulated or repressed ‣ Different species have different kinds of AS ‣ AS plays a role in all eukaryotes in cellular differentiation ‣ Major factors that affect AS are the concentration of nuclear RNA-BP, the speed of transcription and the activity of cellular signalling pathways. ‣ Alternative splicing can control complex patterns of development. ‣ Diseases can be caused by mutations in the splicing code, in the spliceosome machinery. ‣ RNA editing plays important roles in regulating a diversity of processes, including aspects of neurotransmission. ‣ Excitotoxicity is one of the primary mechanisms of cell loss in a variety of diseases of the central and peripheral nervous systems. 34 S. Mendo| BM24_25