mRNA Processing and Splicing Mechanisms

Clinical Case: $\beta$-Thalassemia Intermedia

• Patient Profile: Anne Niemick, a 4-year-old girl of Mediterranean ancestry, diagnosed with $\beta^+$-Thalassemia intermedia, an autosomal recessive disorder.

• Clinical Presentation: Severe anemia (Hemoglobin level of $6.2\,g/dL$), slate-gray complexion, shortness of breath, and growth below the 20th percentile.

• Biochemical Basis: The condition is commonly caused by mutations affecting mRNA splicing in the $\beta$-globin gene, preventing the production of functional $\beta$-globin protein.

Overview of mRNA Processing

• Synthesis: RNA Pol II synthesizes a primary mRNA transcript (pre-mRNA) in the nucleus.

• Modifications: The transcript undergoes three essential modifications to become functional:

  • 5' Capping.

  • 3' Polyadenylation (addition of a poly-A tail).

  • RNA Splicing (removal of introns).

5' Cap Addition

• Timing: Occurs after the synthesis of $20-30$ nucleotides.

• Enzymes: The Cap-synthesizing complex (CSC) includes Phosphohydrolase, guanylyltransferase, guanine-7 methyltransferase, and $2'\text{-O-methyltransferase}$.

• Mechanism:

  • GTP binds to the $+1$ nucleotide via an unusual $5'-5'$ linkage.

  • Methyl groups from S-adenosylmethionine (AdoMet) are added to guanine at N-7 and the $2'$ hydroxyls of the first two nucleotides.

• Transition: CSC is eventually replaced by the cap-binding complex (CBC).

3' Poly(A) Tail Addition

• Signal: Transcription continues past the cleavage signal sequence $5'\text{ AAUAAA }3'$.

• Process:

  • Polyadenylation factors and endonucleases catalyze the cleavage of the mRNA.

  • Poly (A) polymerase (PAP) synthesizes a tail of $80$ to $250$ nucleotides.

  • Substrate: PAP uses ATP for tail synthesis.

RNA Splicing and the Spliceosome

• Components:

  • Exons: Coding sequences expressed as protein.

  • Introns: Non-coding sequences characterized by a $5'$ splice site ($5'\text{ GU }3'$), a $3'$ splice site ($5'\text{ AG }3'$), and a branch point A.

• Splicing Factors: Small nuclear RiboNucleoProteins (snRNPs) composed of snRNAs ($U1$, $U2$, $U4$, $U5$, $U6$) and nuclear proteins.

• Spliceosome Assembly:

  • $U1$ binds the $5'$ GU site; $U2$ binds branch point A.

  • $U4/U6$ complex and $U5$ join to form the inactive spliceosome.

  • Activation: $U4$ and $U1$ are released; $U6$ pairs with the $5'$ splice site and $U2$; binding of the nineteen complex (NTC) occurs.

• Catalysis: Two transesterification reactions occur, where snRNAs act as ribozymes, releasing the intron as a "lariat" structure.

Clinical Relevance of Splicing

• Genetic Diseases: Approximately $15\%$ of all genetic diseases result from mRNA splicing mutations.

• Lupus: An autoimmune disease involving the generation of antibodies against the body's own spliceosome protein components.