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mRNA Splicing

Disorders   Antibodies General Spliceosomes

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Pre-mRNA Splicing

• DNA gene structure
  • Exons
    • Internal exons average 145 nucleotides in length
    • Also contain regulatory elements
      • Required for efficient splicing of constitutive and alternative exons
      • Different sets of auxiliary & regulatory elements used by different genes
      • Types
        • ESEs: Exonic splicing enhancers
        • ESSs: Exonic splicing silencers
  • Introns
    • Average more than 10x longer than exons
    • Also contain regulatory elements
      • Modulate cell-specific use of alternative exons
      • Act by binding multicomponent regulatory complexes
      • Types
        • ISEs: Intronic splicing enhancers
        • ISSs: Intronic splicing silencers
  • Splice sites
    • Locations: At intron/exon borders
    • Types
      • 5' splice site
        • Sequence: AGguragu
        • U1 snRNP: Binds to 5' splice site
      • 3' splice site
        • Sequence: yyyyyyy nagG (y= pyrimidine)
      • Branch site
        • Sequence: ynyuray (r = purine)
        • U2 snRNP: Binds to branch site via RNA:RNA interactions between snRNA and pre-mRNA
    • Structure: Short; Contain typical "degenerate" sequences
• General functions of pre-mRNA splicing
  • Removal of introns
  • Alternative splicing
    • Definition: Joining of different 5' and 3' splice sites
    • General features
      • ~80% of alternative splicing results in changes in the encoded protein
      • Up to 59% of human genes express more than one mRNA by alternative splicing
    • Functional effects: Generates several forms of mRNA from single gene
    • Allows functionally diverse protein isoforms to be expressed according to different regulatory programs
    • Structural effects: Insert or remove amino acids; Shift reading frame;; Introduce termination codon
    • Gene expression effects: Removes or inserts regulatory elements controlling translation, mRNA stability, or localization
    • Regulation
      • Cell specific
      • Splicing pathways modulated according to: Cell type; Developmental stage; Gender; External stimuli

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Spliceosomes ¹

Spliceosomes: General Location: Nucleus Function Process pre-mRNA by splicing out intronic nucleic acids Producing mRNA which is then translated to protein in ribosomes Mechanisms of action Recognition of the intron/exon boundaries Via splice sequences & multiple other weak signals Catalysis of cut-and-paste reactions that remove introns and join exons Contents Five small nuclear RNAs (snRNAs or U(uridine-rich)-RNAs) Each snRNA is associated with specific factors Complex forms a small nuclear ribonucleoprotein particle (snRNP) Proteins: > 100 snRNP components Namesake snRNA (e.g. U1 snRNP has U1 snRNA) snRNP RNAs: U1 ; U2 ; U4; U5; U6 Differ from mRNAs in cap structure: 2,2,7-trimethyl guanosine "Smith" (SM) core proteins common to all snRNPs B & B' , D1 , D2 , D3 , E , F, G Bind AU4-6G sequence Bind snRNA first Targets of autoantibodies (anti-Sm) in systemic lupus Major targets: Sm-B/B' and D proteins Antibodies precipitate U1, U2, U4/U6 & U5 particles snRNP-specific proteins U1: 70k , A , C Target of selective autoantibodies (anti-nRNP) In mixed connective tissue disease Protein target usually U1-70k or U1A U2: A' , B'' Target of autoantibodies in scleroderma U5: 25K, 1BP Brain specific: SNRNP-N Other SMN: Deficient in spinal muscular atrophy GEMIN SIP1 (Gemin 2): Interacts with SMN GEMIN5 Functional roles for each snRNP U1 Initially bound to 5'-splice site Released on recruitment of U4/U5/U6 ? Determines which 5'-splice site is used U2 Initially binds branchpoint recognition sequence Forms two duplexes with U6 Brings intron 5'-splice site close to the branchpoint U4 Initially complexed with U5 and U6 Keeps U6 in an unfolded conformation Released after delivering U6 to the 5'-splice site ? U6 chaperone U5 Initially complexed with U4 and U6 Binds exon sequences Location Upstream of 5'-splice site Downstream of 3'-splice site ? Guide sequence for holding exons together U6 Initially complexed with U4 and U5 Displaces U1 from 5'-splice site Forms duplex with intron sequences Complexes with U2 Brings intron 5'-splice site close to branchpoint ? Catalytic activity Spliceosomes: Illustrations Synthesis Mechanism

Pre-mRNA splicing U1 binds to 5' splice site U2 binds to branchpoint recognition sequence U4-U5-U6 complex binds to 5' splice site   U1 is displaced U6 binds to U2 5'-splice site near branchpoint   U5 binds exon sequences Intron is removed

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Pre-mRNA splicing: Human disorders ²

Mutation effects: General principles

• Splicing element types disrupted
  • cis-element
    • Affects expression of a single gene
    • Can affect use of constitutive or alternative splice sites
    • Disrupted constitutive splicing: Most often results in loss of gene expression due to aberrant splicing
    • Inactivation or activation of alternatively used splice sites
      • May force expression an alternative splicing pattern: Natural mRNA is expressed
      • But expression in inappropriate tissue or developmental stage might result in disease
  • trans effect
    • Changes multiple genes
    • Disruption types
      • Disorders of basal splicing machinery
      • Disorders of splicing regulation
• mRNA types produced
  • Expression of unnatural mRNAs: Aberant splicing creates unnatural splicing patterns
  • Inappropriate expression of natural mRNAs: Disrupts use of alternatively used splice sites

Mutation effects: Disorders

• Cis effects: Mutations that disrupt use of alternative splice sites
  • Familial isolated growth hormone deficiency type II: Short stature
    • Mutations cause increased alternative splicing of exon 3
    • Mechanism: Disruption one of three splicing elements, ISE, ESE, or 5' splice site
  • Frasier syndrome: Mutations inactivate 5' splice site, resulting in shift of isoforms
  • Frontotemporal dementia & Parkinsonism linked to Chromosome 17 (FTDP-17)
    • Mechanism: Most mutations alter regulatory elemants; Cause disease by increasing inclusion of exon 10 of MAPT gene
  • Atypical cystic fibrosis
    • Polymorphisms in intron 8 affect splicing of exon 9: Variable disease manifestations
• Trans effects: Mutations that affect basal splicing machinery
  • Retinitis pigmentosa
    • Dominant RP syndromes: PRPF31; HPRP3; PRPC8
    • Genes involved in function of U4/U6 · U5 tri-snRNP
  • Spinal muscular atrophy
    • SMN2 gene does not completely compensate for loss of SMN1 function
      • One nucleotide substitution disrupts an ESE in exon 7: Causes exon 7 to be skipped in most SMN2 mRNAs
    • SMN1 is an assembler of SnRNP complexes
      • U1, U2, U4, & U5 snRNPs
      • SMN is required for the cytoplasmic assembly of the snRNPs
• Trans effects: Mutations that affect regulators of alternative splicing
  • Myotonic dystrophy: DM1; DM2
    • RNAs with long tracks of CUG or CCUG repeats are transcribed from expanded DMPK and ZNF9 alleles
    • Repeat-containing RNAs accumulate in discrete nuclear foci
    • Expanded RNA repeats: Disrupt function of RNA-binding proteins
      • CUG-BP: May be associated with disrupted RNA processing of cTNT, IR & ClC-1
      • Muscleblind: Associated with nuclear foci
    • 5 pre-mRNAs undergo aberrantly regulated splicing in DM1: Switch to embryonic pattern
      • Cardiac troponin T (cTNT): ? Related to cardiac disease
      • Insulin receptor (IR): Lower signalling isoform; Correlates with insulin resistance in DM
      • Muscle-specific chloride channel (ClC-1): Loss of function; Probably related to myotonia in DM
      • Tau
      • Myotubularin-related 1
  • Neoplasms
• RNP protein disorders
  • HNRNPA2B1: Myopathy + Pagets ± Motor neuron disease or Cognitive disorders
  • HNRNPA1: IBMPFD3
  • HNRNPDL: LGMD 1G
• RNA processing disorders ³
  • Primary
    • TDP-43: ALS
    • FUS: ALS
    • RNU12: SCAR33
    • SMN: Spinal muscular atrophy
    • SNUPN: Myopathy + Contractures
    • TOE1: PCH7
  • Secondary
    • MBNL, Especially 1 & 2: Myotonic dystrophy 1
    • CUGBP1: Myotonic dystrophy 1

Auto-Antibodies

• U1snRNP
• U2snRNP
• U3snRNP
• U5snRNP

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References
1. Clin Exp Rheum 1997;15:549-560
2. Genes & Devel 2003;17:419–437
3. Chembiochem 2024 Mar 9:e202300864

3/11/2024