Learning Objective: By the end of this session, the learner will be able to explain the steps involved in eukaryotic mRNA transcription and processing—including promoter recognition, transcription by RNA polymerase II, RNA modifications, intron removal, and alternative splicing—and relate these processes to clinical correlations.

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Eukaryotic genes contain exons (coding regions) interrupted by introns (non-coding regions). Both are transcribed into a primary RNA transcript (hnRNA), which undergoes processing to form mature monocistronic mRNA that is exported to the cytoplasm for translation.

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Transcription of Eukaryotic Genes

Promoter Recognition

• Transcription begins when RNA polymerase II binds the promoter with the aid of transcription factors (e.g., TFIID).
• Key promoter consensus sequences:
  • TATA (Hogness) box at −25
  • CAAT box at −70

Initiation & Elongation

• RNA polymerase II unwinds DNA.
• Template strand is read 3′ → 5′.
• RNA is synthesized 5′ → 3′.
• The primary transcript (hnRNA) includes both exons and introns.

Termination

• Eukaryotic transcription termination signals are not well understood for RNA polymerase II.

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Activity

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Processing of Eukaryotic Pre-mRNA

5′ Capping

• Addition of 7-methylguanosine (m⁷G) to the 5′ end.
• Functions:
  • Protects mRNA from degradation
  • Required for ribosome binding

Polyadenylation (Poly-A Tail)

• Occurs at the 3′ end.
• Involves poly-A polymerase.
• Stabilizes mRNA & facilitates export.

RNA Splicing

• Introns are removed by spliceosomes (snRNPs / snurps).
• Splicing occurs at:
  • 5′ donor site
  • 3′ acceptor site
• Intron removed as a lariat.
• Neighboring exons joined to form the coding sequence.

Clinical Note

Mutations in splice sites → abnormal mRNA → diseases (e.g., β-thalassemia).

hnRNA and Final Export

• All intermediates (uncapped, partially spliced, etc.) = hnRNA.
• Fully processed mRNA is exported to the cytoplasm for translation.

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Activity

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Alternative Splicing

Alternative splicing allows a single pre-mRNA transcript to be spliced in multiple ways, producing different protein isoforms from the same gene.

Examples

• Tropomyosin and Troponin T isoforms in muscle.
• Immunoglobulin isoforms:
  • Membrane-bound Ig in resting B cells
  • Secreted Ig in activated B cells

Biological Importance

• Greatly increases protein diversity.
• Human genome: ~20,000–25,000 genes
• Human proteome: >100,000 protein variants

Tissue-Specific Splicing

• Different tissues may splice the same gene differently → tissue-specific protein expression.

Detection

• Alternative splicing can be identified using Northern blotting.

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Summary Table – RNA Polymerases

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Activity