Molecular Biology Week 6 Lecture Notes on Messenger RNA Processing

Learning Objectives

• Understand how mRNA is spliced.
• Grasp the concept of alternative splicing.
• Learn about mRNAs protection via a 5’ methylated cap and a 3’ polyadenylated tail.

Eukaryotic vs. Bacterial Gene Expression

• Eukaryotic Gene Expression: More complex; occurs in different cellular compartments.

  • Eukaryotic genes contain non-coding DNA (introns) requiring splicing.

• Bacterial Gene Expression: Simpler management of genes.

  • RNA polymerase simultaneously transcribes genes and ribosomes translate mRNA into protein.

Introns and RNA Splicing

• Introns: Intragenic regions that aren’t translated (also known as intervening sequences, IVSs).
• Exons: Coding regions of genes that express functional sequences.
• Example: Human titin gene has 362 introns.
• RNA Splicing: The process that removes introns to produce a mature mRNA transcript.

Splicing Signals

• Nucleotide signals are consistent in nuclear mRNA precursors:
  • First two bases of introns: GU
  • Last two bases of introns: AG
• Consensus sequences at 5’- and 3’-splice sites extend beyond GU and AG motifs.
• Mutations can lead to abnormal splicing.

Mechanism of Splicing

• Step 1: The 2’-OH group of an adenosine nucleotide within the intron attacks the phosphodiester bond linking the first exon and the intron, forming a lasso.
• Step 2: The 3’-OH at the end of the first exon then attacks the bond between the intron and the second exon, creating an exon-exon bond and releasing the intron as a lasso.

Branchpoint Signal

• Specific consensus sequences determine the branchpoint in splicing.
  • Yeast: UACUAAC
  • Higher eukaryotes show variability.
• The branchpoint’s final adenine plays a crucial role in splicing.

Spliceosomes

• The spliceosome is the molecular machine that mediates splicing, consisting of:
  • Pre-mRNA
  • Five small nuclear ribonucleoproteins (snRNPs)
  • Additional proteins
• Major snRNPs: U1, U2, U4, U5, U6, each fulfilling specific roles in the splicing process.

Spliceosome Assembly and Function

• Spliceosomes assemble in a stepwise manner and undergo:
  • Assembly: Formation of the spliceosome.
  • Function: Execution of splicing.
  • Disassembly: Recycling of components post-splicing.
• Regulation of spliceosome assembly can influence gene expression.

Types of Splicing Factors

• U1 snRNP: Essential for recognition of the splice site.
• U6 snRNP: Binds to the intron’s 5’-end and plays a critical role in splicing.
• U2 snRNP: Binds to the conserved branchpoint sequence necessary for splicing and interacts with U6 to stabilize the splicing process.

Alternative Splicing

• Enables a single pre-mRNA to create different protein products from the same gene.
  • Example: Distinguishing between secreted and membrane-bound proteins.
• Alternative Splicing Patterns:
  • Cassette Exon
  • Mutually Exclusive Exons
  • Intron Retention
  • Alternative splice sites (5’ or 3’)
  • Utilization of different promoters.

Control of Splicing

• Splicing is influenced by exonic splicing enhancers (ESEs) and silencers (ESSs) that bind specific protein factors to control splicing outcomes.
• Transcripts of Drosophila's tra gene illustrate how splicing results in sex-specific protein products.

mRNA Processing Steps

• Eukaryotic mRNAs are capped at the 5’ end with a methylguanylate cap and polyadenylated at the 3’ end.
• Capping:
  • Occurs early during transcription.
  • Provides resistance to degradation and facilitates mRNA transport and translation.
• Polyadenylation:
  • Involves adding a long chain of adenine residues to stabilize mRNA and regulate export to the cytoplasm.

Summary of mRNA-Processing Events

• Splicing, capping, and polyadenylation occur concurrently during transcription.
• Splicing requires a spliceosome, consisting of snRNPs and other proteins.
• Alternative splicing expands the protein diversity produced from the eukaryotic genome, highlighting the complexity of gene expression regulation.