1# Eukaryotic mRNA: Processing and Translation

• Focus includes generation of eukaryotic mRNAs in cell nuclei, mRNA processing, export to cytoplasm, and use for translation.
• Module focuses on processes after mRNA transcription, including mRNA processing, translation into proteins, and protein functions in cells.

Central Dogma of Molecular Biology Summary

• Genetic information is stored in double-stranded DNA, then transcribed into single-stranded mRNA.
• mRNA sequence is read in triplets (codons) that code for amino acids, resulting in protein synthesis.
• Proteins are functional molecules resulting from this flow of genetic information.
• Process describes transfer of genetic information from DNA to mRNA to protein.
• This mechanism operates in both prokaryotic and eukaryotic cells.

Prokaryotic Cells vs Eukaryotic Cells

• Course focuses on eukaryotic cells, so it is essential to understand the differences between prokaryotic and eukaryotic cells because these differences impact how gene expression is organized and regulated.

Prokaryotic Cells (Bacteria)

• No nucleus: DNA and ribosomes share same cellular compartment (cytoplasm).
• Transcription and translation occur simultaneously.
• As soon as mRNA is transcribed by RNA polymerase, ribosomes can immediately start translating it in a process called co-transcriptional translation.

Eukaryotic Cells

• Nucleus separates DNA from cytoplasm where ribosomes are located.
• Transcription and translation are separated in space and time.
• Transcription occurs in the nucleus.
• mRNA must be processed and exported to the cytoplasm before translation.
• There is more regulatory complexity due to physical and temporal separation allows for many regulatory steps in gene expression before mRNA translation.
• Eukaryotic cells' increased complexity contributes to the sophisticated regulation of gene expression.

Non-Coding RNAs (ncRNAs) and Gene Regulation Roles

• Genome contains regions producing non-coding RNAs (ncRNAs) that regulate genes.
• Only ~3% of the human genome encodes proteins, while a large portion of non-coding DNA is transcribed into RNA.

Non-Coding RNAs Functions

• Act as molecular guides, binding to other nucleic acids (mainly mRNA) to influence their function.
• ncRNAs regulate metabolism, cell differentiation, development, immunity, and cancer.
• RNA interference & microRNAs help maintain genome stability, control gene expression, and act as antiviral defense.
• These regulatory roles of ncRNAs and their impact on biological functions will be explored.

Key Questions on Eukaryotic Gene Expression Regulation

• This module explores how eukaryotic gene expression is regulated beyond transcription, identifying the key control points and their interactions.

Regulation Beyond Transcription

• Translation regulation determines how much protein is synthesized from an mRNA.
• mRNA stability controls amount of mRNA available for translation.
• Protein stability regulates functional lifespan of proteins in cell.

Balancing mRNA Production and Degradation

• Gene expression is influenced by a dynamic balance between mRNA synthesis and degradation.
• Increasing mRNA levels is accomplished through higher transcriptional output and enhanced processing or stabilization of mRNA.
• Decreasing mRNA levels is accomplished through faster mRNA degradation and reduced transcription or processing efficiency.

mRNA and Protein Levels Regulation

• Every step in the mRNA lifecycle, from transcription to degradation, serves as a control point in gene expression because these processes are interconnected and mRNA levels and protein output are finely tuned through multiple regulatory mechanisms.

Regulation of mRNA and Protein Levels in Eukaryotic Cells

• Gene expression in eukaryotic cells is regulated beyond transcription, including mRNA processing, stability, localization, and translation, as well as protein folding, modification, and degradation.
• These steps ensure precise control over protein production and function.

Key Regulatory Steps

• mRNA Processing & Transport: Splicing, capping, and polyadenylation influence mRNA stability and translation efficiency.
• Nuclear export ensures processed mRNA reaches the cytoplasm.
• mRNA localization within the cytoplasm affects translation and cellular function.
• Translation & Quality Control is regulated to determine how much protein is synthesized. Quality control mechanisms prevent errors in protein production.
• mRNA and Protein Stability: mRNA degradation rates influence steady-state mRNA levels and protein stability and degradation regulate functional protein availability.

Diversity in mRNA and Protein Lifetimes

• Studies on cellular mRNA and protein abundances reveal significant variability.
• In mouse fibroblasts, median values show mRNA levels are ~17 copies per cell with a half-life of ~9 hours, and protein levels are ~50,000 copies per cell with a half-life of ~46 hours.
• Some mRNAs and proteins are highly abundant, while others exist in very low quantities or are not expressed at all.

mRNA vs. Protein Abundance

• While mRNA abundance generally correlates with protein abundance, the correlation is weak (R² ~ 0.4).
• Outliers exist because highly abundant mRNAs produce little protein and low-abundance mRNAs produce significant protein levels.
• mRNA and protein stabilities are not correlated, as their degradation pathways are distinct.

Key Takeaways on mRNA and Protein Levels

• Gene expression regulation is multi-layered, involving transcription, mRNA processing, translation, and protein turnover.
• mRNA and protein levels vary greatly, with diverse lifetimes and abundances.
• Post-transcriptional regulation significantly influences protein output, beyond just mRNA levels.
• mRNA stability does not predict protein stability, highlighting independent regulatory mechanisms.
• This complexity allows cells to fine-tune gene expression based on their needs, adapting to different physiological conditions.

Eukaryotic Cells vs. Prokaryotic Cells mRNA Structure and Processing

• mRNA molecules in both prokaryotic and eukaryotic cells share a general structure but differ in how translation is initiated.

General mRNA Structure

• Consists of three key regions: Open Reading Frame (ORF), 5′ Untranslated Region (5′ UTR), and 3′ Untranslated Region (3′ UTR)

Open Reading Frame (ORF)

• Protein-coding region, spanning from the start codon (AUG) to the stop codon.

5′ Untranslated Region (5′ UTR)

• Region between the 5′ end of the mRNA and the start codon that plays a role in translation regulation.

3′ Untranslated Region (3′ UTR)

• Region between the stop codon and the 3′ end of the mRNA that is important for mRNA stability and translation efficiency.

Translation Initiation Differences

• Prokaryotic mRNAs use the Shine-Dalgarno sequence (ribosome binding site) which precedes the start codon and base-pairs with the 16S rRNA of the small ribosomal subunit to help define the start site for translation.
• Eukaryotic mRNAs translation initiation relies on scanning, where the ribosome binds the 5′ cap and moves downstream to find the first AUG, and the Kozak sequence is a consensus motif that surrounds the AUG start codon, enhancing translation efficiency.

Key Takeaways on Eukaryotic Cells vs. Prokaryotic Cells mRNA Structure and Processing

• mRNAs in both prokaryotes and eukaryotes have UTRs and an ORF.
• Prokaryotic translation is guided by the Shine-Dalgarno sequence, while eukaryotic translation relies on ribosome scanning and the Kozak sequence.
• These differences influence gene expression regulation in both cell types.

Eukaryotic mRNA Structure and Function

• Eukaryotic mRNAs have distinct features that regulate their stability, translation, and function. including the 5′ cap, 3′ poly(A) tail, and untranslated regions (UTRs).

Key Features of Eukaryotic mRNA

5′ Cap (7-methylguanosine, m⁷G)

• Added co-transcriptionally during mRNA synthesis.
• Protects mRNA from 5′ to 3′ exonucleases.
• Recognized by eIF4E, a translation initiation factor.

3′ Poly(A) Tail

• A non-templated stretch of adenosine nucleotides (A's) added post-transcriptionally by poly(A) polymerase (PAP).
• Enhances mRNA stability and translation efficiency by interacting with poly(A)-binding protein (PABP).
• Tail length varies (e.g., ~200 nucleotides in mammals, ~50 in yeast).

Untranslated Regions (UTRs)

• 5′ UTR influences translation efficiency based on length and secondary structure.
• 3′ UTR contains regulatory elements affecting translation, stability, and localization.
• UTRs can be long in eukaryotes (sometimes longer than the coding region), allowing for complex regulation.

Key Takeaways on Eukaryotic mRNA Structure and Function

• 5′ Cap and 3′ Poly(A) Tail protect mRNA from degradation and are essential for efficient translation.
• UTRs play regulatory roles, impacting translation, stability, and localization.
• Eukaryotic mRNAs undergo complex post-transcriptional modifications, ensuring precise gene expression control.

Eukaryotic mRNA 5′ Cap Structure and Function

• The 5′ cap is a 7-methylguanosine (m⁷G) structure attached to the first nucleotide of mRNA via a 5′-5′ triphosphate linkage.
• It plays a crucial role in mRNA stability and translation initiation.

Key 5′ Cap Features

• Chemical Structure of guanosine is methylated at the N7 position, giving it a positive charge, which influences protein recognition, and the first one or two nucleotides may also be 2′-O-methylated, enhancing translation efficiency.
• Functions include protecting mRNA from 5′-3′ exonucleases (preventing degradation), and being essential for translation initiation, enabling ribosome recruitment.
• Recognition by EIF4E, a translation initiation factor that binds the m⁷G cap involving aromatic stacking with tryptophan residues in EIF4E and charge interactions with a glutamate residue ensuring cap specificity.

Key Takeaways on Eukaryotic mRNA 5′ Cap Structure and Function

• The 5′ cap protects mRNA and is required for translation initiation.
• EIF4E recognizes the cap through specific molecular interactions.
• Methylation of the cap and nearby nucleotides enhances translation efficiency.

Eukaryotic mRNA Poly(A) Tail Structure and Function

• The poly(A) tail, a stretch of adenosine nucleotides at the 3′ end of eukaryotic mRNAs, plays a crucial role in mRNA stability and translation efficiency.

Key Poly(A) Tail Functions

• mRNA Protection by shielding mRNA from 3′-5′ exonucleases, preventing degradation, and mRNA degradation begins with poly(A) tail removal, marking it for decay.
• Translation Regulation: Essential for efficient mRNA translation.
• Poly(A)-binding protein (PABP) binds the poly(A) tail and interacts with EIF4G, a translation initiation factor.
• This circularizes the mRNA, linking the 5′ cap and 3′ poly(A) tail, promoting ribosome recycling and efficient translation.
• Recognition by PABP involves PABP recognizing adenine bases using RNA recognition motifs (RRMs) while base-stacking and hydrogen bonding provide both affinity and specificity for the poly(A) tail.

Key Takeaways on Eukaryotic mRNA Poly(A) Tail Structure and Function

• The poly(A) tail protects mRNA and enhances translation efficiency.
• Poly(A)-binding protein (PABP) interacts with EIF4G, enabling mRNA circularization for efficient translation.
• Poly(A) tail removal is the first step in mRNA degradation, controlling mRNA stability.

Eukaryotic mRNA Processing and Regulation Key Aspects

• This lecture covers how eukaryotic gene expression is regulated beyond transcription, focusing on mRNA processing, stability, and translation control.

3′ UTR: A Regulatory Hub

• Plays a crucial role in mRNA stability, translation efficiency, and localization.
• Contains cis-acting elements that bind trans-acting factors such as RNA-binding proteins (RBPs) and Non-coding RNAs (e.g., microRNAs).
• These interactions regulate translation initiation, mRNA degradation, and poly(A) tail length.
• Serves as a control hub, influencing gene expression at multiple levels.

RNA-Binding Proteins (RBPs) and Gene Regulation

• RNA-binding proteins (RBPs) recognize specific RNA sequences or structures to regulate gene expression and contain specialized domains, such as RNA Recognition Motif (RRM), KH Domains, and Zinc Finger Domains.
• These proteins influence mRNA stability, transport, and translation.

mRNA Processing: Splicing and Maturation

• Eukaryotic mRNAs are first transcribed as precursor mRNAs (pre-mRNAs) containing introns.
• Introns must be removed through pre-mRNA splicing, which joins exons to form the mature mRNA, ensuring that only protein-coding sequences remain for translation.

Eukaryotic Transcription Process

• Transcription of mRNA is carried out by RNA polymerase II, which undergoes Initiation where transcription factors recruit RNA polymerase to the promoter, Elongation where mRNA is synthesized using the DNA template, and Termination where transcription stops, and RNA polymerase dissociates.
• Regulation via Phosphorylation is accomplished through The C-terminal domain (CTD) of RNA polymerase II undergoing differential phosphorylation, controlling mRNA processing.

Co-Transcriptional mRNA Modifications

• 5′ Capping: The 5′ cap (7-methylguanosine) protects mRNA from degradation and facilitates ribosome binding via EIF4E, ensuring efficient translation.
• 3′ Polyadenylation: The poly(A) tail stabilizes mRNA and enhances translation.
• Cleavage and polyadenylation factors (CPSF, CSTF) recognize polyadenylation signals to process the 3′ end.

Alternative Polyadenylation and Its Impact

• Most human genes have multiple polyadenylation sites, producing mRNA isoforms with different 3′ UTR lengths.
• Short 3′ UTRs (common in cancer cells and proliferating cells) lead to less regulation, increased stability.
• Long 3′ UTRs (in differentiated cells) lead to more regulatory elements, leading to controlled gene expression.

Transcription Termination Mechanism

• RNA polymerase continues transcribing past the polyadenylation site, generating an uncapped RNA fragment.
• Exonuclease XRN2 degrades the downstream RNA, aiding in polymerase release (Torpedo Model).

Histone mRNAs Exception

• Unlike most polyadenylated mRNAs, histone mRNAs are processed differently using a stem-loop structure at the 3′ UTR instead of a poly(A) tail.

Key Takeaways on Eukaryotic mRNA

• Gene expression is regulated beyond transcription, involving multiple post-transcriptional processes.
• mRNA processing steps (capping, splicing, and polyadenylation) are crucial for stability and translation efficiency.
• Alternative polyadenylation generates mRNA isoforms with different regulatory properties.
• RNA-binding proteins and cis-regulatory elements in the 3′ UTR fine-tune mRNA fate.
• Histone mRNAs are an exception to the polyadenylation rule.
• This knowledge is fundamental for understanding how gene expression is tightly controlled in eukaryotic cells.