2# Pre-mRNA Splicing and Processing

Introduction

• Pre-mRNA splicing is a key process in eukaryotic cells enabling gene expression regulation and transcriptome diversification.
• The lecture provides a foundation for understanding alternative splicing regulatory mechanisms.

The Central Dogma and mRNA Processing

• Genetic information flows from DNA to mRNA (transcription in the nucleus) to protein (translation in the cytoplasm).
• Eukaryotic cells separate transcription and translation, allowing for intermediate processing steps like pre-mRNA splicing.
• Eukaryotic mRNAs feature a protein-coding open reading frame (ORF) flanked by untranslated regions (UTRs).
• UTRs contain regulatory elements affecting translation efficiency, stability, and localization.
• mRNAs have terminal modifications including a 5' cap (7-methylguanosine) and a 3' poly(A) tail.
• The 5' cap protects mRNA from degradation and helps initiate translation.
• The 3' poly(A) tail, added by poly(A) polymerase, enhances mRNA stability and translation efficiency.
• The length of the poly(A) tail correlates with mRNA stability and translation.

Pre-mRNA Splicing: Concept and Discovery

• Pre-mRNA splicing removes non-coding introns and joins exons to form mature mRNA.
• This was first observed in the late 1970s when viral mRNA sequences differed from genomic DNA.
• Electron microscopy showed looped-out structures in hybridized mRNA-DNA complexes, confirming introns' presence.
• The 1993 Nobel Prize was awarded for the discovery of introns.

Mechanism of Splicing

• Splicing involves two transesterification reactions facilitated by the spliceosome.
• A transesterification reaction involves a phosphoester reacting with a hydroxyl group, resulting in another phosphodiester and another alcohol.
• Branching occurs in the first step via a 2' hydroxyl of an adenosine residue (at the branch point) attacking the 5' splice site.
• This cleaves the 5' exon-intron junction and creates a lariat intermediate.
• In the second step, the 5' exon's free 3' hydroxyl attacks the 3' intron-exon junction which joins exons and removes the intron.
• These reactions are catalyzed by magnesium ions (Mg2+), which stabilize the transition state.

Splice Site Recognition and Intron Structure

• Splice sites are marked by conserved sequences in the pre-mRNA.
• The 5' splice site typically contains a GU dinucleotide.
• The 3' splice site is characterized by an AG dinucleotide.
• The branch point has a conserved adenosine residue, which is key for the first catalytic step.
• A polypyrimidine tract is found between the branch point and the 3' splice site.
• In humans, around 97% of protein-coding genes have introns, averaging 8-9 introns per gene.
• Introns make up about 25% of the human genome.
• Intron sizes range from a few nucleotides to over one megabase.
• Simpler eukaryotes like yeast have introns in only about 5% of genes, typically as single, short sequences.

The Spliceosome: A Dynamic Molecular Machine

• The spliceosome is a ribonucleoprotein complex that catalyzes pre-mRNA splicing.
• It consists of five small nuclear ribonucleoprotein particles (snRNPs): U1, U2, U4, U5, and U6.
• Each snRNP includes a specific small nuclear RNA (snRNA) and associated proteins.
• U1 recognizes the 5' splice site.
• U2 binds to the branch point.
• U4-U5-U6 assemble to form the active spliceosome.
• The spliceosome undergoes conformational rearrangements, driven by ATP-dependent RNA helicases.
• U1 is displaced by U6 during spliceosome activation, and U6 base-pairs with the 5' splice site.
• Interactions between U2 and U6 establish the catalytic core, positioning the branch point.
• Remodeling follows branching to accommodate the second transesterification step, leading to exon ligation and intron release as a lariat.

ATP-Dependent Helicases and Spliceosome Remodeling

• Spliceosome assembly and catalysis require energy from ATP-dependent RNA helicases.
• RNA helicases unwind RNA structures, displace proteins, and drive conformational changes.
• Helicases like Brr2, Prp2, and Prp16 coordinate splicing cycle steps.

The Exon Junction Complex and mRNA Surveillance

• The exon junction complex (EJC) is deposited at splice junctions after exon ligation.
• The EJC marks successful splicing and functions in nuclear export, translation regulation, and nonsense-mediated mRNA decay (NMD).
• The EJC distinguishes mature mRNAs from incompletely processed transcripts.

Alternative Splicing and Transcriptome Diversity

• Alternative splicing generates multiple mRNA isoforms from a single gene, increasing proteomic diversity.
• Variations in splice site selection leads to exon skipping, alternative 5' or 3' splice site usage, mutually exclusive exon inclusion, or intron retention.
• Alternative splicing allows for gene expression fine-tuning in different tissues and developmental stages.
• Over 90% of human genes undergo alternative splicing.

Summary

• Pre-mRNA splicing removes introns and joins exons to create mature mRNA.
• The spliceosome orchestrates this process through intricate molecular interactions and ATP-dependent remodeling.
• Splicing regulation is key for gene expression control and alternative splicing.
• Alternative splicing significantly contributes to transcriptome diversity.