Coding Life: Reading the Instructions of the Cell

Gene Structure and Transcription

  • Covers building a eukaryotic gene, transcribing RNA from it, converting RNA to mRNA, and splicing mRNA molecules.

Building a Gene

  • Typical Eukaryotic Gene Structure:

    • 5' UTR (Untranslated Region)
    • EX1, IN1, EX2, IN2, EX3, IN3, EX4
    • 3' UTR
    • Enhancer (A), Promoter (B), Terminator (C)

  • Correct order of gene elements: Enhancer, Promoter, Terminator.

  • Proteins binding to the promoter region during transcription initiation:

    • General transcription factors
    • RNA polymerase

  • Regulatory transcription factors (in eukaryotic cells) bind to upstream sequences called enhancers, facilitating the recruitment of general transcription factors to the gene’s promoter, subsequently followed by the recruitment of the RNA polymerase complex.

Transcribing a Gene

  • Gene elements excluded from the pre-mRNA molecule:
    • Enhancer
    • Promoter
    • Terminator sequence
  • During pre-mRNA processing to mature mRNA:
    • Introns are removed.
    • A PolyA tail is added to the 3’ end.

Alternative Splicing

  • mRNA splice variants/isoforms from pre-mRNA:
    • Various combinations of exons (e.g., EX2-EX4-EX1, EX2-EX3-EX4-EX1, EX3-EX4-EX1, EX2-EX3-EX1, EX4-EX1) yield different mRNA molecules.

Wilson Disease Case Study

  • Symptoms include hepatitis-like liver damage, neuropsychological symptoms, and copper deposits (Kayser-Fleischer Rings) in the eyes.
  • The ATP7B gene:
    • Spans over 84,000 bp (84 Kb).
    • Coding sequence consists of 20 exons.
    • mRNA coding sequence of 4,398 bp, resulting in a protein of 1,465 amino acids.
  • ATP7B gene structure includes: 5’ UTR, EX1, IN1, EX2, IN2, EX3, IN3, EX4, 3’ UTR, Enhancer, Promoter, Terminator.

Gene Regulation in Wilson Disease

  • Role of ATP7B: Removes excess copper from the liver into the bile for excretion.
  • ATP7B is predominantly expressed in the liver, and its transcription is induced by high copper levels.
  • Combinatorial transcriptional regulation determines gene activity in specific cell types.
  • Metal Response Elements (MREs):
    • Located in the enhancer region of ATP7B, upstream (5’) of the transcription start site.
    • Short DNA sequences (7bp) recognized and bound by the metal-responsive transcription factor MTF-1 (an activator protein).
  • Transcription factor binding sequences (enhancer sequences like MREs) are described as ‘consensus sequences’, meaning they aren't always identical.
  • Multiple MREs typically cluster in an enhancer region to increase transcription.
  • High cellular copper levels cause MTF-1 to bind to MREs, inducing expression of ATP7B and other copper-responsive genes.
  • If MREs from the ATP7B enhancer region were deleted or MTF-1 was non-functional, ATP7B transcription levels would decrease.
  • MREs a and b are partial/perfect matches to the consensus MRE sequence.
  • A sequence that differs from the consensus sequence might be considered an MRE because it's the human reference sequence.

Mutations and Wilson Disease

  • A mutation in one of the MREs can cause Wilson disease, leading to a 15% decrease in ATP7B transcript levels.
  • Only one sequence is shown for the patient, whereas two sequences are shown for each parent because the patient is homozygous for the mutation, and the parents are heterozygous.
  • The patient likely inherited two copies of a rare mutation due to a founder effect.

Post-Workshop Activity: Splicing in Wilson Disease

  • Alternative splicing affects the inclusion of Exon(Ex) regions, such as Ex3 to Ex9, leading to variations in mRNA sequences.
  • Specific mutations, like those at gta/ggtagaacacaaaagat, can influence splicing outcomes.
  • Variations in Exon inclusion (e.g., Ex4-Ex5-Ex6-Ex7 vs. Ex4-Ex5-Ex9) lead to different transcript lengths and protein isoforms.