biol3010 2/24/25

Exam Information

• Exam 2 is scheduled for one week from today.

• Opportunity for discussion tonight with Daniel about recent topics.

Overview of Gene Splicing

• Eukaryotic genes contain introns that must be removed through splicing to create mature RNA.

• The spliceosome is the molecular machinery responsible for splicing.

• The spliceosome recognizes specific sequences at intron-exon boundaries during transcription by RNA Polymerase II.

Intron-Exon Boundaries

• Key sequences include:

  • 5' end of intron: Typically has guanine followed by any purine.

  • Splice acceptor site: Always includes an A, with most often an AG following.

  • Branch site: Commonly has specific nucleotide sequences.

Importance of Alternative Splicing

• Complexity in organisms arises not solely from gene number but also from the processing of those genes.

• Humans have around 20,000 protein-coding genes that can produce over 100,000 proteins through alternative splicing.

• Example: Different combinations of retained or excluded exons result in unique proteins.

Diseases Linked to Splicing Defects

• Several diseases arise from mutations affecting the splicing of the laminin A gene.

• Types of diseases associated with laminin A mutations:

  • Limb-girdle muscular dystrophy due to retention of intron 9 leading to a premature termination codon (PTC).

  • Familial partial lipid dystrophy type 2 caused by intron 8 retention due to a similar mutation.

  • Hutchinson-Gilford Progeria syndrome caused by mutations in exon leading to faulty splicing between exons 11 and 12.

  • Dilated cardiomyopathy caused by different mutations in laminin A.

Alternative Transcription and Splicing Control

• Genes can produce many different mRNAs.

  • Example: The Kcnma1 gene can lead to over 500 different mRNAs.

• Alternative splicing mechanisms include:

  • Exon skipping.

  • Alternative splice site selection (5' and 3' splice sites).

Factors Influencing Splicing Outcomes

• Splice site strength and conservation affect splicing accuracy.

• Cis-regulatory sequences in pre-mRNA aid or hinder splicing.

• Proteins that bind to pre-mRNA can promote or inhibit the splicing process:

  • Serine-rich proteins enhance splicing.

  • HNRNPs can suppress splicing outcomes.

Definitions of Importance

• ISS: Intronic splicing suppressor.

• ISE: Intronic splicing enhancer.

• ESE: Exonic splicing enhancer.

• ESS: Exonic splicing suppressor.

Polyadenylation and mRNA Stability

• Addition of the poly-A tail is critical for mRNA stability and processing.

• The poly-A tail is added post-transcriptionally and helps protect mRNA from degradation.

  • Regulated by the polyadenylation signal (AAUAA) and endonucleases.

  • Poly-A polymerase adds adenine nucleotides to form the tail.

Functions of the Poly-A Tail

• Enhances transcript stability.

• Facilitates export from the nucleus to the cytoplasm.

• Promotes translation by binding to ribosomes and stabilizing mRNA.

Binding Proteins

• Different proteins interact with the poly-A tail in the nucleus and cytoplasm.

  • Nuclear: Important for tail extension and processing.

  • Cytoplasmic: Stabilize the mRNA and assist in translation.

Early Experiments on the Genetic Code

• Characteristics of the genetic code:

  • Composed of triplet codons, with 61 coding for amino acids and 3 for stop signals.

  • Non-overlapping and degenerate nature.

Charles Yanofsky's Experiment

• Investigated the trpA gene in E. coli by creating mutations and studying the resultant proteins.

• Found that gene sequences corresponded collinearly to the protein sequences.

• Defined missense and nonsense mutations and their implications for protein synthesis.

• Proposed that adjacent nucleotide mutations affect the same amino acid, demonstrating the collinearity of codons and amino acids.