6# mRNA Degradation

Importance of mRNA Degradation

• A critical aspect of post-transcriptional gene regulation
• Steady-state mRNA levels result from a balance between synthesis and degradation
• Plays a role as significant as mRNA synthesis in gene expression control

mRNA vs. Protein Stability

• Studies show wide variation in both mRNA and protein abundance and half-lives
• mRNA and protein abundance are somewhat correlated
• Stabilities (half-lives) of mRNAs and corresponding proteins are not well correlated

Diversity of mRNA Half-Lives

• Range from minutes to tens of hours
• In mouse fibroblasts, median mRNA half-life is ~9 hours
• For some mRNAs, synthesis takes longer than their functional lifetime

Measuring mRNA Half-Life

• Method involves using a transcription inhibitor (e.g., α-amanitin) to stop RNA Pol II activity
• mRNA decay is monitored over time
• RNA extraction uses reagents like TRIzol to isolate RNA
• Quantification methods include Northern blot, RT-qPCR, or RNA-seq
• Data analysis involves plotting mRNA levels over time, fitting data to an exponential decay curve to determine half-life

Key Takeaway on mRNA Degradation

• Essential and highly variable
• Fundamental aspect of post-transcriptional gene regulation

Role in Gene Expression

• A key regulatory step in controlling mRNA levels and protein production
• Allows cells to adjust gene expression post-transcriptionally under various conditions

Global (Constitutive) mRNA Turnover

• All mRNAs undergo continuous synthesis and degradation
• Global turnover rate is influenced by mRNA decay factors, including nucleases and associated proteins
• Maintains a dynamic balance of the cellular mRNA pool

Regulated Turnover of Specific mRNAs

• Targeted degradation of individual mRNAs occurs in response to signals
• Enables fine-tuning of specific mRNA levels
• Regulated by RNA-binding proteins and microRNAs
• Often coupled with translational repression, where degradation and inhibition of translation co-occur

mRNA Quality Control (Surveillance)

• Mechanisms detect and degrade defective mRNAs, such as those lacking stop codons, with premature stop codons, or other translation-impairing defects
• Closely linked to translation, translation errors often trigger degradation

Key Takeaway on mRNA Degradation Process

• Not just passive decay
• An active, regulated process integrated with translation
• Essential for fine-tuning gene expression and maintaining mRNA integrity

Overview of Constitutive mRNA Degradation Pathways

• Constitutive mRNA turnover ensures continuous removal and replacement
• Initiated by deadenylation, which is the 3′ poly-A tail removal

Step 1: Deadenylation

• Catalyzed by deadenylase enzymes
• Produces an intermediate with a short poly-A tail, but an intact 5′ cap
• Rate-limiting and entry point for further degradation

5′ to 3′ Decay Pathway

• Decapping by DCP1–DCP2 complex removes the 5′ cap
• Leaves a 5′ monophosphate, a substrate for XRN1, a 5′→3′ exoribonuclease
• XRN1 degrades mRNA from the 5′ end

3′ to 5′ Decay Pathway

• The exosome complex (multi-protein) degrades the deadenylated 3′ end
• It proceeds in the 3′→5′ direction

Endonucleolytic Cleavage Pathway

• Endonucleases cleave the mRNA internally
• Produces a 3′ fragment lacking a poly-A tail, which is degraded by the exosome (3′→5′) and a 5′ fragment with a 5′ monophosphate, degraded by XRN1 (5′→3′)
• Entry point for specialized degradation pathways, including Nonsense-mediated decay (NMD), No-go decay (NGD), and siRNA-mediated RNA interference (RNAi), where initial cleavage is called slicing

Key Concepts of mRNA Degradation

• Deadenylation is the first and crucial step in canonical decay
• Degradation is directional and enzyme-specific, XRN1 for 5′→3′ and Exosome for 3′→5′
• Endonucleolytic cleavage creates direct entry points for exonucleases and is used in surveillance and silencing mechanisms

Discovery in Budding Yeast

• Early understanding of mRNA degradation comes from yeast studies
• They helped define the canonical degradation pathways

Main Enzymes Identified in Yeast

• Deadenylation: Main enzyme is CCR4
• Decapping: Heterodimer of DCP1 and DCP2, with DCP2 as the catalytic subunit
• 5′→3′ Exonuclease: XRN1, acts on RNAs with a 5′ monophosphate

Conservation Across Eukaryotes

• Homologs of DCP1, DCP2, and XRN1 are found in human cells and higher eukaryotes
• Enzyme names and functions are often retained in higher organisms

Pathway Dominance Varies by Organism

• In yeast, the 5′→3′ pathway (XRN1-dependent) is dominant
• In higher eukaryotes, dominance between 5′→3′ (XRN1 pathway) and 3′→5′ (Exosome pathway) varies by cell type and conditions
• No universal rule exists, both pathways may be active in different biological contexts

Summary of mRNA Degradation

• The core components of mRNA degradation were discovered in yeast and evolutionarily conserved
• The dominant pathway (5′→3′ vs 3′→5′) varies across organisms and conditions

Deadenylation: The First Step

• It refers to the removal of the poly-A tail, initiating degradation
• Three main deadenylase complexes are CCR4-NOT complex (major deadenylase), PAN2–PAN3 complex (catalyzes initial shortening), and PARN (specialized deadenylase)

3′ → 5′ Degradation: The Exosome

• A 10-subunit multi-protein complex
• Functions in cytoplasmic mRNA degradation and nuclear RNA processing
• The structure forms a central channel leading RNA to the exonuclease active site
• Regulated by RNA helicases and cofactors that help recruit and insert mRNAs into the exosome
• Ensures regulated access and degradation of deadenylated mRNAs

5′ → 3′ Degradation: XRN1

• Key 5′→3′ exonuclease
• Requires a 5′ monophosphate to function
• Blocked by 5′ caps or triphosphates
• Substrate specificity is ensured by a positively charged pocket that recognizes the 5′ phosphate
• Its mechanism is processive, each nucleotide cleavage leaves a 5′ monophosphate, allowing continuous degradation, and XRN1 stays bound and processes the mRNA without dissociating

Fidelity and Irreversibility

• Only decapped mRNAs can be degraded by XRN1
• Decapping commits the mRNA to degradation, and this process is irreversible
• This ensures precision and control in mRNA turnover

Key takeaways from mRNA Degradation

• Precisely regulated through structural and enzymatic specificity
• Begins with deadenylation and proceeds via the exosome or XRN1, depending on terminal modifications
• Enzyme design ensures fidelity, directionality, and irreversibility

Decapping Enzyme Complex

• Catalyzed by DCP1–DCP2 heterodimer, where DCP2 is the catalytic subunit and DCP1 is a co-activator required for full activity
• Activated and recruited by decapping activators (e.g., EDC4, EDC3, DHH1) and RNA-binding proteins that recognize deadenylated mRNAs

Link Between Decapping and Translation

• Actively translated mRNAs are protected from decapping and deadenylation by the 5′ cap, poly-A tail, and poly-A binding protein (PABP)
• PABP stabilizes the poly-A tail, enhances translation initiation, and prevents early degradation
• Translation competes with degradation, ongoing translation stabilizes mRNA, and reduced translation makes mRNA degradation easier

Mechanism of mRNA Destabilization

• PABP dissociates, reduces translation efficiency
• This promotes recruitment of decapping co-activators (e.g., DHH1, LSM1–7 complex) and assembly of decapping machinery at the 5' end
• Leads to decapping, then degradation by XRN1 (5'→3') or exosome (3'→5')