RNA Splicing

Splicing

• RNA molecules, in complex with proteins, perform key steps in splicing, rather than proteins alone.
• Cis elements involved in splicing include:
• 5' splice site (donor site)
• 3' splice site (acceptor site)
• Branch point
• Polypyrimidine tract
• Trans-acting elements can act on any different splice site in the genome.

Alternative Splicing

• Increases the complexity of the protein that can be generated from available genomic coding sequences.
• Regulated by cis-acting elements, trans-acting factors, transcription, and chromatin structure.
• Alternative splicing is a key regulatory mechanism.

Splicing Activators and Repressors

• SR Proteins:
• Have extensive repeats of serine and arginine.
• Are RNA binding proteins.
• Bind to sequences in exons, recruiting components of the core splicing machinery and activating splicing.
• hnRNPs (heterogeneous nuclear ribonucleoproteins):
• Are RNA binding proteins.
• Bind to sequences in exons or introns.
• Interfere with the binding of core splicing machinery, repressing splicing.

Trans Splicing

• Involves splicing between two pre-mRNA molecules.
• Involves the same signals and components as cis splicing.
• Occurs in a variety of organisms, including Drosophila and C. elegans.
• In mammalian cells, can be associated with cancer.

Nonsense-Mediated mRNA Decay (NMD)

• Is a RNA quality control mechanism.
• Eliminates transcripts with premature termination codons (PTCs).
• Premature stop codons can arise from mutations, defects in splicing or transcription.
• Is a gene regulatory mechanism that also targets non-mutant transcripts.

Exon Junction Complex (EJC)

• Is deposited 24 nucleotides upstream of spliced junctions during splicing.
• Is removed by the first round of translation and recycled back into the nucleus.
• The core consists of 4 proteins: eIF4A3, MAGOH, RBM8A, and BTZ.

Post-transcriptional Control by Non-coding RNAs

• Non-coding RNAs include:
• "House-keeping" RNAs (e.g., rRNAs and tRNAs)
• Regulatory RNAs, including small ncRNAs (200 nucleotides)
• Main mode of action: scaffold to recruit proteins, DNA, and/or RNA.

Post-translational Control

• Protein folding involves:
• Polypeptide chain folding into a unique 3D conformation.
• Final secondary structures, including α-helices and β-sheets.
• Most hydrophobic residues are hidden in the interior core.
• Co-translational folding occurs within the polypeptide exit tunnel and at the surface of the ribosome.

Molecular Chaperones

• Molecular chaperones recognize incorrect configurations due to the exposure of hydrophobic surfaces.
• Heat shock proteins (HSP60 and HSP70) are molecular chaperones.
• HSP70:
• Functions early, sufficient for many proteins.
• ATP-bound HSP70 binds to exposed, short stretches of hydrophobic amino acids and hydrolyses ATP to ADP.
• Provides opportunity to fold correctly, prevents mis-folding, and formation of aggregates.
• Releases the protein when ATP has been exchanged for ADP.
• HSP60 (chaperonin):
• Acts later, provides a barrel-shaped isolation chamber.
• Substrate captured via hydrophobic entrance (helps with unfolding).
• Binding of ATP and sealing with cap releases protein into chamber.
• Protein re-folding in isolation.
• ATP hydrolysis weakens binding of cap.
• Binding of new ATP ejects cap and releases protein.

Marking Proteins for Degradation - Ubiquitination

• Ubiquitin is a small, 76 amino acid protein.
• Ubiquitin is heat-stable and highly evolutionarily conserved.
• Ubiquitin is expressed in all eukaryotic cells.
• Glycine 76 binds to a lysine residue on the substrate.