explain that, in eukaryotes, the RNA molecule formed following transcription (primary transcript) is modified by the removal of non-coding sequences (introns) and the joining together of coding sequences (exons) to form mRNA

Protein Synthesis – RNA Processing in Eukaryotes (Cambridge 9700 – Topic 6.2)

Learning Objective

Explain that, in eukaryotes, the RNA molecule produced by transcription (the primary transcript or pre‑mRNA) is modified in the nucleus to become a mature messenger RNA (mRNA) that can be exported to the cytoplasm for translation.

Where does RNA processing occur?

All three modifications – 5′ capping, splicing, and 3′ poly‑A tail addition – take place in the nucleus** while the pre‑mRNA is still being synthesised (co‑transcriptionally). The mature mRNA is then transported through nuclear pores to the cytoplasm.

Key Terms

• Primary transcript (pre‑mRNA) – the initial RNA copy of a gene made by RNA polymerase II.
• Introns – non‑coding sequences removed during processing.
• Exons – coding sequences that remain in the final mRNA.
• Spliceosome – a large ribonucleoprotein complex composed of five small nuclear ribonucleoproteins (snRNPs U1, U2, U4, U5, U6) and associated proteins that carries out splicing.
• 5′ Cap – a 7‑methylguanosine added to the 5′ end by a capping enzyme complex (RNA 5′‑phosphatase + guanylyltransferase).
• Poly‑A tail – a stretch of ~200 adenine residues added by poly‑A polymerase.
• Alternative splicing – the regulated inclusion or exclusion of exons, producing different mRNA isoforms from one gene.

Chronological Steps of RNA Processing (cap → splice → poly‑A)

1. 5′ Capping (co‑transcriptional)

   • Enzyme complex: RNA 5′‑phosphatase removes the γ‑phosphate of the first nucleotide; guanylyltransferase adds a GMP via a 5′‑5′ triphosphate bond; a methyltransferase then methylates the guanine to give a 7‑methylguanosine cap.
   • Functions:

     • Protects the transcript from 5′‑exonucleases.
     • Required for ribosome recruitment – recognised by the cap‑binding protein eIF‑4E.

2. Splicing (co‑transcriptional)

   • Recognition of conserved splice‑site sequences:

     • 5′ splice site: GU at the start of the intron.
     • Branch‑point: an adenine (A) ~20–50 nt upstream of the 3′ splice site.
     • 3′ splice site: AG at the end of the intron.

   • Two trans‑esterification reactions catalysed by the spliceosome:

     1. The 5′ splice site attacks the branch‑point A, forming a lariat‑shaped intron.
     2. The free 3′‑hydroxyl of the upstream exon attacks the 3′ splice site, joining the two exons and releasing the intron lariat.

   • The intron lariat is rapidly degraded; the exons are ligated to give a continuous coding sequence.
   • Splicing is tightly coupled to transcription – the spliceosome assembles on the nascent transcript as it emerges from RNA polymerase II.

3. 3′ Poly‑A Tail Addition (post‑transcriptional)

   • After transcription of a short downstream “poly‑U” stretch, poly‑A polymerase adds ~200 adenine residues to the 3′ end.
   • Functions:

     • Protects the mRNA from 3′‑exonucleases.
     • Facilitates nuclear export (interaction with export factors).
     • Enhances translation initiation – poly‑A‑binding proteins (PABPs) bind the tail and interact with the cap‑binding complex, circularising the mRNA and promoting ribosome recruitment.

Comparison of Primary Transcript and Mature mRNA

Spliceosome Composition

The spliceosome is built from five snRNPs (U1, U2, U4, U5, U6) together with numerous non‑snRNP proteins. The snRNPs recognise the GU‑AG splice sites and the branch‑point A, positioning the catalytic core for the two trans‑esterification steps.

Alternative Splicing – Generating Protein Diversity

• Regulated inclusion or exclusion of exons, or retention of introns, creates multiple mRNA isoforms from a single gene.
• Key regulatory elements:

  • Exonic/intronic splicing enhancers (ESE/ISE) and silencers (ESS/ISS).
  • RNA‑binding proteins: SR proteins (enhancers) and hnRNPs (silencers).
  • Cell‑type‑specific expression of these regulatory proteins.

• Example: The human fibronectin gene produces a plasma‑type isoform (exon 6 skipped) and a cellular‑type isoform (exon 6 included) depending on tissue‑specific splicing factors.

Biological Significance of RNA Processing

• Ensures that only coding information is retained in the mRNA.
• Protects the transcript from nuclease degradation (5′ cap and poly‑A tail).
• Provides a major point of gene regulation – especially through alternative splicing.
• Prepares the mRNA for efficient nuclear export and for recognition by the translation machinery.

Suggested Diagram (for classroom use)

A schematic showing a gene with exons (boxes) and introns (lines), the primary transcript, and the mature mRNA after 5′ capping, splicing (exons joined), and poly‑A tail addition. Include labels for the capping enzyme complex, spliceosome, and poly‑A polymerase.

Quick Revision Checklist

• List the three main nuclear modifications that convert a primary transcript into mature mRNA.
• State the conserved splice‑site sequences (5′ GU … branch‑point A … 3′ AG) and describe the two trans‑esterification steps.
• Identify the enzymes/complexes responsible for each modification:

  • 5′ capping – RNA 5′‑phosphatase + guanylyltransferase (plus methyltransferase).
  • Splicing – spliceosome (snRNPs U1‑U6).
  • Poly‑A tail – poly‑A polymerase.

• Explain two functions of the 5′ cap and two functions of the poly‑A tail.
• Describe how alternative splicing increases protein diversity.
• Recall where (nucleus) and when (co‑transcriptionally) each modification occurs.