Lecture Notes: RNA Degradation, Silencing, and Splicing

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Your Name

Published

February 5, 2025

Introduction

In this lecture, we will explore RNA metabolism, focusing on RNA degradation, translational control, and splicing. We will examine:

Mechanisms of RNA degradation in prokaryotes and eukaryotes, including key enzymes and regulatory sequences.
RNA silencing and translational control, and their impact on gene expression and cellular functions.
The process of RNA splicing, including constitutive and alternative splicing, the spliceosome machinery, and associated human pathologies.

Understanding these processes is crucial for comprehending gene regulation and cellular biology.

RNA Degradation Mechanisms

RNA degradation is a crucial regulatory process that controls RNAlifespan and protein expression, essential for cellular homeostasis and responses to environmental changes.

Prokaryotic RNA Degradation: The Degradosome

In prokaryotes, RNAdegradation is rapid and often co-transcriptional, primarily mediated by the degradosome, a multi-enzyme complex.

Degradosome Components:
- Ribonuclease E (RNase E): An endoribonuclease initiating degradation with non-specific internal cleavages.
- Polynucleotide Phosphorylase (PNPase): A 3’ to 5’ exoribonuclease degrading RNA fragments.
- RNA Helicases: Unwind RNA secondary structures to facilitate ribonuclease access.
Mechanism: Degradation is initiated when ribosome-free RNAregions become accessible. RNase E introduces cuts, followed by 3’ to 5’ degradation by PNPase, resulting in short mRNAhalf-lives in prokaryotes.
Regulation: Prokaryotic RNAdegradation is generally non-specific and not tightly regulated.

Eukaryotic RNA Degradation: Regulated Turnover

Eukaryotic RNAdegradation is a regulated process critical for controlling protein expression. mRNAhalf-life is a key regulatory parameter, influenced by specific RNAsequences.

Destabilizing Sequences: AU-Rich Elements (AREs)

AU-Rich Elements (AREs) are -rich sequences in the 3’ UTR of mRNAthat signal for rapid degradation by recruiting destabilizing enzymes.

Deadenylation-Dependent Decay

The primary eukaryotic mRNAdegradation pathway is deadenylation-dependent decay, initiated by shortening of the 3’ poly(A) tail by deadenylases like the CCR4-NOT complex (including CCR4 and PARN). Deadenylation destabilizes mRNAby disrupting the protective loop formed by the poly(A) tail and 5’ cap.

Decapping and 5’-3’ Degradation

Following deadenylation, the 5’ cap is removed by the decapping enzyme DCP1. The uncapped mRNAis then degraded 5’ to 3’ by the exoribonuclease XRN1, the primary enzyme in this pathway.

Nonsense-Mediated Decay (NMD)

Nonsense-Mediated Decay (NMD) is a surveillance pathway in eukaryotes that specifically degrades mRNAcontaining premature termination codons (PTCs) or nonsense codons (UAA, UGA, UAG). NMDprevents the production of truncated proteins from aberrant mRNA.

Mechanism of NMD

NMDis triggered during translation when a ribosome encounters a PTC.

Role of Exon Junction Complexes (EJCs) in NMD

Exon Junction Complexes (EJCs), deposited at exon-exon junctions during splicing, are crucial for NMD.

During the first translation round, ribosomes typically displace EJCs.
If a PTC is upstream of the last EJC, translation terminates prematurely, leaving EJCs on the mRNA.
These post-termination EJCs are recognized by UPF proteins (surveillance factors).
The mRNAis then targeted for degradation in P-bodys via the 5’ to 3’ pathway.

This mechanism ensures degradation of mRNAwith PTCs, preventing synthesis of truncated proteins and conserving cellular energy.

Major Eukaryotic mRNA Degradation Pathways

Eukaryotes utilize two main mRNAdegradation pathways, distinguished by directionality and subcellular location: 5’-3’ and 3’-5’ degradation.

5’-3’ Degradation Pathway

The 5’-3’ degradation pathway is the primary route for normal mRNAturnover in eukaryotes, occurring mainly in cytoplasmic P-bodies.

Cytoplasmic P-bodies

P-bodies are cytoplasmic granules where mRNAdegradation and translational repression are concentrated.

Pathway Steps

Deadenylation: Initial shortening of the poly(A) tail.
Decapping: Removal of the 5’ cap by DCP1.
5’-3’ Exonucleolytic Degradation: Degradation of the mRNAbody by XRN1.

3’-5’ Degradation Pathway

The 3’-5’ degradation pathway is active in the nucleus and cytoplasm, utilizing the exosome complex.

Nuclear Exosome Complex

The exosome is a multi-subunit complex with 3’-5’ exoribonuclease activity, present in both the nucleus and cytoplasm. In the nucleus, it is crucial for degrading introns, rRNAs, and improperly processed mRNA. Decapping is not required for 3’-5’ degradation.

Roles of 3’-5’ Degradation

Intron Turnover: Degradation of excised introns in the nucleus.
rRNA Turnover: Quality control and turnover of ribosomal RNA.
Aberrant mRNA Degradation: Nuclear degradation of improperly processed mRNA(e.g., unspliced mRNA) to prevent cytoplasmic translation of non-functional mRNA.

RNA Silencing and Translational Control

Beyond degradation, cytoplasmic mRNAcan be translationally silenced and stored, providing sophisticated control over gene expression for temporal and spatial regulation of protein synthesis.

Cytoplasmic Fate of mRNA: Translational Silencing and Storage

Cytoplasmic mRNAis not always immediately translated or degraded. It can be stored in a translationally inactive form, a process known as translational silencing. This allows for:

Temporally Controlled Translation: mRNAcan be stockpiled and translated only when needed, crucial during development and in response to cellular signals. Storage in ribonucleoprotein complexes prevents ribosome binding.
Spatial Control and Inheritance: mRNAcan be localized to specific subcellular locations and even transmitted to daughter cells, influencing cell function and potentially heritable traits.

mRNA Transport and Subcellular Localization

mRNAlocalization ensures protein synthesis occurs at the correct place and time, often involving ribonucleoprotein granules (RNPs) and cytoskeletal motor proteins.

Ribonucleoprotein Granules (RNPs)

RNPs are mRNA-protein complexes that protect mRNAfrom degradation and facilitate transport and translational control. P-bodys are a type of RNP involved in degradation and silencing.

Cytoskeletal Transport Mechanisms

mRNAtransport utilizes the cytoskeleton and motor proteins:

Actin-based transport: Myosin motors on actin filaments mediate short-range transport, e.g., maternal RNAin yeast budding.
Microtubule-based transport: Dynein and kinesin motors on microtubules facilitate long-range transport, e.g., dynein for maternal RNAin Drosophila oocytes.

Localized Protein Synthesis and Cellular Functions

Localized mRNAtranslation enables precise protein synthesis where needed, impacting:

Cell Migration: Localized beta-actin mRNAtranslation at pseudopods in fibroblasts.
Cell Polarity and Asymmetric Division: Establishment of cell polarity and asymmetric divisions.

Maternal mRNA and Embryonic Asymmetry

Maternal mRNA deposited in oocytes are critical for early embryonic development, establishing initial asymmetries in the zygote.

Maternal mRNA Deposition

Oocytes accumulate maternal mRNAduring oogenesis, often translationally silenced until fertilization.

Drosophila Model: Bicoid and Oscar

In Drosophila, maternal mRNAlike Bicoid and Oscar establish the anterior-posterior axis:

Bicoid mRNA: Localized anteriorly, encodes a transcription factor for anterior development.
Oscar mRNA: Localized posteriorly, essential for germ cell and posterior axis formation.

Nurse cells synthesize these mRNAand transport them to the oocyte. Dynein-mediated transport along microtubules positions Bicoid and Oscar mRNAat opposite poles, initiating embryonic asymmetry.

3’ UTRs as Localization Signals

3’ in mRNAoften contain cis-regulatory elements that act as localization signals, recognized by RNA-binding proteins (RBPs). These RBPs mediate transport via interactions with cytoskeletal motors, determining mRNAdestination within the cell.

RNA Splicing: Intron Removal and Exon Ligation

RNA splicing is a fundamental eukaryotic process for gene expression, removing non-coding introns from precursor mRNA(hnRNA) and ligating coding exons to form mature mRNA.

Introduction to Splicing

hnRNA Maturation

Splicing is essential for the maturation of heterogeneous nuclear RNA (hnRNA), the primary transcript, into functional mRNA. hnRNAcontains both introns and exons; splicing excises introns, yielding translatable mRNA.

Constitutive and Alternative Splicing

Splicing occurs in two main forms:

Constitutive Splicing: A basal process where all introns are removed, and exons are joined linearly to produce a single mRNAand protein isoform per gene.
Alternative Splicing: A regulated process generating multiple mRNAand protein isoforms from a single gene. This involves selective inclusion or exclusion of exons, or intron retention, increasing proteomic diversity. Alternative splicing is often tissue-specific.

Regulatory Significance of Splicing

Splicing is not merely RNA processing but a key regulatory step influencing:

mRNA Stability: Splicing can affect mRNAturnover rates.
Coding Sequence Determination: Alternative splicing dictates the final protein-coding sequence.
Protein Diversity: Generates multiple protein isoforms with varied localization and functions, contributing to tissue-specific protein expression and functional diversity.

Splicing Defects and Human Diseases

Aberrant RNA splicing is implicated in various human diseases, particularly neurological disorders, due to the production of non-functional or dysfunctional proteins.

Splicing Pathologies

Splicing defects are increasingly recognized in disease etiology, especially in neurological conditions, reflecting the nervous system’s reliance on precise gene expression.

Disease Examples

Several pathologies are linked to splicing errors:

Ataxia-Telangiectasia (A-T)

Ataxia-Telangiectasia (A-T) is characterized by neurological deficits, immune deficiency, and cancer predisposition. Aberrant splicing of the ATM kinase gene is central to A-T. ATM kinase is critical for DNArepair and cell cycle control. Splicing defects lead to reduced ATM function, causing cerebellar ataxia and increased cancer susceptibility.

Spinal Muscular Atrophy (SMA)

Spinal Muscular Atrophy (SMA) is a severe neuromuscular disease and a major cause of infant mortality. It results from defects in the SMN1 (Survival Motor Neuron 1) gene. Splicing aberrations in SMN1 reduce functional SMN protein, essential for motor neuron survival. SMN deficiency leads to motor neuron degeneration and muscle atrophy, causing paralysis.

Intron-Exon Structure and Splicing Signals

Understanding gene organization and splice signals is crucial for comprehending splicing mechanisms.

Exon-Intron Arrangement

Eukaryotic genes feature alternating exons and introns. Human genes are composed of $\sim$95% intronic DNA and $\sim$5% exonic coding sequences. A typical human gene spans 27 kb, with only 1.3 kb coding. Genes average 7-8 exons. Introns, though non-coding, are functionally significant.

Splice Site Boundaries

Introns are defined by conserved splice sites:

5’ Splice Site (Donor Site): At the 5’ intron end.
3’ Splice Site (Acceptor Site): At the 3’ intron end.

Intron Size and Regulatory Elements

Intron size varies widely in humans ( $<$ 100 bp to $>$ 100 kb). Introns contain regulatory sequences:

miRNA Genes: Some introns encode microRNAs.
Enhancers and Insulators: Regulatory DNAsequences within introns modulate gene expression.

Increased intron size and number during evolution correlate with expanded regulatory capacity in higher eukaryotes.

Exon Size Conservation

Exon size is evolutionarily conserved (50-300 nucleotides), suggesting functional constraints, possibly related to protein structure or splicing efficiency.

Intron Classification

Introns are classified into five main types based on splicing mechanisms. We focus on self-splicing and spliceosomal introns.

Self-Splicing Introns

Self-splicing introns are catalytic RNA(ribozymes) that excise themselves without protein enzymes.

Group I Introns: Found in rRNA, tRNA, and mRNA in lower eukaryotes, bacteria, and viruses. Splicing is intermolecular, initiated by a free guanosine.
Group II Introns: Primarily in organelles of fungi, plants, algae, and some bacteria. Splicing is intramolecular, initiated by an internal adenosine.

Spliceosomal Introns

Spliceosomal introns are the major type in eukaryotic protein-coding genes. Their removal is catalyzed by the spliceosome, a ribonucleoprotein complex. Spliceosomal introns are evolutionarily derived from self-splicing introns, utilizing protein and components.

Splicing Mechanisms: Transesterification

Intron removal involves two sequential transesterification reactions for all intron types.

Group I Splicing (Intermolecular)

Group I splicing is intermolecular, initiated by a free guanosine (G, GTP, GDP).

1st Transesterification: 3’-OH of guanosine attacks the 5’ splice site, cleaving the RNAbackbone and attaching guanosine to the 5’ intron end.
2nd Transesterification: 3’-OH of the 5’ exon attacks the 3’ splice site, joining exons and releasing the linear intron.

Group II Splicing (Intramolecular)

Group II splicing is intramolecular, initiated by an adenosine at the branch site within the intron.

1st Transesterification: 2’-OH of the branch site adenosine attacks the 5’ splice site, forming a lariat and cleaving the 5’ exon.
2nd Transesterification: 3’-OH of the 5’ exon attacks the 3’ splice site, joining exons and releasing the lariat intron.

Spliceosomal Splicing

Spliceosomal splicing, while similar to Group II, is catalyzed by the spliceosome, a complex of $\sim$150 proteins and small nuclear ribonucleoproteins (snRNPs). The spliceosome positions reactants and coordinates two transesterification reactions, lacking intrinsic catalytic activity itself.

The Spliceosome and snRNPs

The spliceosome is the machinery for spliceosomal intron removal, composed of snRNPs and associated proteins.

snRNP Composition and Function

snRNPs are core spliceosome components, comprising small nuclear RNA() and proteins. Key snRNPs are U1, U2, U4, U5, and U6.

snRNP Roles

U1 snRNP: (U1 snRNA + proteins) - Recognizes and binds the 5’ splice site.
U2 snRNP: (U2 snRNA + proteins) - Binds the branch site.
U4/U6 and U5 snRNPs: Function in later assembly and catalysis. U5 interacts with both exons; U4/U6 regulate catalytic activity.

snRNPs recognize splice sites and catalyze splicing via -mRNAand -base pairing.

snRNA-Mediated Splice Site Recognition

within snRNPs recognize mRNAsplice signals through base pairing. For example, U1 snRNA targets the 5’ splice site consensus, and U2 snRNA targets the branch site, guiding spliceosome assembly and action.

Splice Site Consensus Sequences

Human introns have specific consensus sequences for spliceosome recognition.

5’ Splice Site: GU

The 5’ splice site consensus includes the GU dinucleotide at the 5’ intron end. This GU is highly conserved and crucial for U1 snRNP binding.

3’ Splice Site: AG and Polypyrimidine Tract

The 3’ splice site consensus features the AG dinucleotide at the 3’ intron end and an upstream polypyrimidine tract (U/C-rich region). The AG is conserved; the polypyrimidine tract is recognized by factors like U2AF.

Mutations in Splice Sites

Mutations in splice site consensus sequences disrupt splicing, causing aberrant mRNAand disease, leading to:

Exon Skipping
Intron Retention
Cryptic Splice Site Activation: Activation of pseudo splice sites.

These defects result in non-functional or altered proteins, contributing to genetic disorders.

Therapeutic Splice Site Correction

Therapeutic strategies aim to correct aberrant splicing using antisense oligonucleotides (AONs) to:

Block Aberrant Splice Sites
Promote Exon Inclusion
Modulate Alternative Splicing

These AON-based therapies aim to restore normal splicing and protein function in splicing-related diseases.

Spliceosome Regulation and Kinetics

Spliceosome assembly and splicing kinetics are regulated, involving multiple factors and steps.

Early Complex Formation: Complex E

Initial spliceosome assembly involves forming the Commitment Complex (Complex E), or presplicing complex, through 5’ and 3’ splice site recognition by splicing factors.

Splicing Activator Proteins

Splicing activators are essential for early spliceosome assembly, defining 5’ and 3’ splice site pairs and promoting Complex E formation.

ASF/SF2 and U2AF65/U2AF35

ASF/SF2: Binds the 5’ splice site, enhancing U1 snRNP recognition.
U2AF65/U2AF35: Recognizes the polypyrimidine tract and 3’ splice site. U2AF65 binds the polypyrimidine tract, and U2AF35 interacts with the 3’ splice site AG.

These activators initiate splice site recognition and pre-mRNAcommitment to splicing.

Tissue-Specific Splicing Factors

Tissue-specific expression of splicing factors (activators and repressors) regulates alternative splicing, generating tissue-specific protein isoforms. Varying levels of these factors in different tissues lead to diverse splicing patterns.

Energetic Cost and Efficiency

Spliceosomal splicing is energy-intensive, requiring ATP and GTP hydrolysis. Despite this cost, it is highly efficient and precise, ensuring accurate intron removal and exon joining, highlighting its critical role in eukaryotic gene expression.

Conclusion

This lecture explored RNA metabolism, covering degradation, translational control, and splicing. We discussed:

RNA Degradation: Essential for regulating RNA lifespan and protein expression, with distinct mechanisms in prokaryotes (degradosome) and eukaryotes (5’-3’ and 3’-5’ pathways, NMD).
RNA Silencing and Translational Control: Mechanisms for temporal and spatial gene expression regulation through mRNAstorage, transport, and localized translation, crucial for development and cellular functions.
RNA Splicing: A fundamental eukaryotic process for mRNAmaturation, involving constitutive and alternative splicing, catalyzed by the spliceosome, and critical for proteomic diversity. Splicing defects are linked to various human diseases.

Understanding these RNA processes is crucial for comprehending gene regulation, cellular biology, and the molecular basis of diseases. Further research into RNA biology promises advancements in biotechnology and therapeutic interventions.

--- title: "Lecture Notes: RNA Degradation, Silencing, and Splicing" author: "Your Name" date: "2025-02-05" format: html: toc: true # Table of Contents toc-depth: 2 code-tools: true theme: cosmo # Or "journal" for Distill-like minimalism --- # Introduction In this lecture, we will explore RNA metabolism, focusing on RNA degradation, translational control, and splicing. We will examine: - Mechanisms of RNA degradation in prokaryotes and eukaryotes, including key enzymes and regulatory sequences. - RNA silencing and translational control, and their impact on gene expression and cellular functions. - The process of RNA splicing, including constitutive and alternative splicing, the spliceosome machinery, and associated human pathologies. Understanding these processes is crucial for comprehending gene regulation and cellular biology. # RNA Degradation Mechanisms RNA degradation is a crucial regulatory process that controls RNAlifespan and protein expression, essential for cellular homeostasis and responses to environmental changes. ## Prokaryotic RNA Degradation: The Degradosome In prokaryotes, RNAdegradation is rapid and often co-transcriptional, primarily mediated by the **degradosome**, a multi-enzyme complex. - **Degradosome Components:** - **Ribonuclease E (RNase E):** An endoribonuclease initiating degradation with non-specific internal cleavages. - **Polynucleotide Phosphorylase (PNPase):** A 3' to 5' exoribonuclease degrading RNA fragments. - **RNA Helicases:** Unwind RNA secondary structures to facilitate ribonuclease access. - **Mechanism:** Degradation is initiated when ribosome-free RNAregions become accessible. RNase E introduces cuts, followed by 3' to 5' degradation by PNPase, resulting in short mRNAhalf-lives in prokaryotes. - **Regulation:** Prokaryotic RNAdegradation is generally non-specific and not tightly regulated. ## Eukaryotic RNA Degradation: Regulated Turnover Eukaryotic RNAdegradation is a regulated process critical for controlling protein expression. mRNAhalf-life is a key regulatory parameter, influenced by specific RNAsequences. ### Destabilizing Sequences: AU-Rich Elements (AREs) **AU-Rich Elements (AREs)** are -rich sequences in the 3' UTR of mRNAthat signal for rapid degradation by recruiting destabilizing enzymes. ### Deadenylation-Dependent Decay The primary eukaryotic mRNAdegradation pathway is deadenylation-dependent decay, initiated by shortening of the 3' poly(A) tail by deadenylases like the CCR4-NOT complex (including CCR4 and PARN). Deadenylation destabilizes mRNAby disrupting the protective loop formed by the poly(A) tail and 5' cap. ### Decapping and 5'-3' Degradation Following deadenylation, the 5' cap is removed by the decapping enzyme DCP1. The uncapped mRNAis then degraded 5' to 3' by the exoribonuclease XRN1, the primary enzyme in this pathway. ## Nonsense-Mediated Decay (NMD) **Nonsense-Mediated Decay (NMD)** is a surveillance pathway in eukaryotes that specifically degrades mRNAcontaining premature termination codons (PTCs) or nonsense codons (UAA, UGA, UAG). NMDprevents the production of truncated proteins from aberrant mRNA. ### Mechanism of NMD NMDis triggered during translation when a ribosome encounters a PTC. ### Role of Exon Junction Complexes (EJCs) in NMD **Exon Junction Complexes (EJCs)**, deposited at exon-exon junctions during splicing, are crucial for NMD. 1. During the first translation round, ribosomes typically displace EJCs. 2. If a PTC is upstream of the last EJC, translation terminates prematurely, leaving EJCs on the mRNA. 3. These post-termination EJCs are recognized by UPF proteins (surveillance factors). 4. The mRNAis then targeted for degradation in P-bodys via the 5' to 3' pathway. This mechanism ensures degradation of mRNAwith PTCs, preventing synthesis of truncated proteins and conserving cellular energy. ## Major Eukaryotic mRNA Degradation Pathways Eukaryotes utilize two main mRNAdegradation pathways, distinguished by directionality and subcellular location: 5'-3' and 3'-5' degradation. ### 5'-3' Degradation Pathway The **5'-3' degradation pathway** is the primary route for normal mRNAturnover in eukaryotes, occurring mainly in cytoplasmic **P-bodies**. #### Cytoplasmic P-bodies **P-bodies** are cytoplasmic granules where mRNAdegradation and translational repression are concentrated. #### Pathway Steps 1. **Deadenylation:** Initial shortening of the poly(A) tail. 2. **Decapping:** Removal of the 5' cap by DCP1. 3. **5'-3' Exonucleolytic Degradation:** Degradation of the mRNAbody by XRN1. ### 3'-5' Degradation Pathway The **3'-5' degradation pathway** is active in the nucleus and cytoplasm, utilizing the **exosome complex**. #### Nuclear Exosome Complex The **exosome** is a multi-subunit complex with 3'-5' exoribonuclease activity, present in both the nucleus and cytoplasm. In the nucleus, it is crucial for degrading introns, rRNAs, and improperly processed mRNA. Decapping is not required for 3'-5' degradation. #### Roles of 3'-5' Degradation - **Intron Turnover:** Degradation of excised introns in the nucleus. - **rRNA Turnover:** Quality control and turnover of ribosomal RNA. - **Aberrant mRNA Degradation:** Nuclear degradation of improperly processed mRNA(e.g., unspliced mRNA) to prevent cytoplasmic translation of non-functional mRNA. # RNA Silencing and Translational Control Beyond degradation, cytoplasmic mRNAcan be translationally silenced and stored, providing sophisticated control over gene expression for temporal and spatial regulation of protein synthesis. ## Cytoplasmic Fate of mRNA: Translational Silencing and Storage Cytoplasmic mRNAis not always immediately translated or degraded. It can be stored in a translationally inactive form, a process known as **translational silencing**. This allows for: - **Temporally Controlled Translation:** mRNAcan be stockpiled and translated only when needed, crucial during development and in response to cellular signals. Storage in ribonucleoprotein complexes prevents ribosome binding. - **Spatial Control and Inheritance:** mRNAcan be localized to specific subcellular locations and even transmitted to daughter cells, influencing cell function and potentially heritable traits. ## mRNA Transport and Subcellular Localization mRNAlocalization ensures protein synthesis occurs at the correct place and time, often involving **ribonucleoprotein granules (RNPs)** and cytoskeletal motor proteins. ### Ribonucleoprotein Granules (RNPs) **RNPs** are mRNA-protein complexes that protect mRNAfrom degradation and facilitate transport and translational control. P-bodys are a type of RNP involved in degradation and silencing. ### Cytoskeletal Transport Mechanisms mRNAtransport utilizes the cytoskeleton and motor proteins: - **Actin-based transport:** Myosin motors on actin filaments mediate short-range transport, e.g., maternal RNAin yeast budding. - **Microtubule-based transport:** Dynein and kinesin motors on microtubules facilitate long-range transport, e.g., dynein for maternal RNAin *Drosophila* oocytes. ### Localized Protein Synthesis and Cellular Functions Localized mRNAtranslation enables precise protein synthesis where needed, impacting: - **Cell Migration:** Localized beta-actin mRNAtranslation at pseudopods in fibroblasts. - **Cell Polarity and Asymmetric Division:** Establishment of cell polarity and asymmetric divisions. ## Maternal mRNA and Embryonic Asymmetry **Maternal mRNA** deposited in oocytes are critical for early embryonic development, establishing initial asymmetries in the zygote. ### Maternal mRNA Deposition Oocytes accumulate maternal mRNAduring oogenesis, often translationally silenced until fertilization. ### Drosophila Model: Bicoid and Oscar In *Drosophila*, maternal mRNAlike **Bicoid** and **Oscar** establish the anterior-posterior axis: - **Bicoid mRNA:** Localized anteriorly, encodes a transcription factor for anterior development. - **Oscar mRNA:** Localized posteriorly, essential for germ cell and posterior axis formation. Nurse cells synthesize these mRNAand transport them to the oocyte. Dynein-mediated transport along microtubules positions Bicoid and Oscar mRNAat opposite poles, initiating embryonic asymmetry. ### 3' UTRs as Localization Signals **3'** in mRNAoften contain cis-regulatory elements that act as localization signals, recognized by RNA-binding proteins (RBPs). These RBPs mediate transport via interactions with cytoskeletal motors, determining mRNAdestination within the cell. # RNA Splicing: Intron Removal and Exon Ligation **RNA splicing** is a fundamental eukaryotic process for gene expression, removing non-coding **introns** from precursor mRNA(hnRNA) and ligating coding **exons** to form mature mRNA. ## Introduction to Splicing ### hnRNA Maturation Splicing is essential for the maturation of **heterogeneous nuclear RNA** (hnRNA), the primary transcript, into functional mRNA. hnRNAcontains both introns and exons; splicing excises introns, yielding translatable mRNA. ### Constitutive and Alternative Splicing Splicing occurs in two main forms: - **Constitutive Splicing:** A basal process where all introns are removed, and exons are joined linearly to produce a single mRNAand protein isoform per gene. - **Alternative Splicing:** A regulated process generating multiple mRNAand protein isoforms from a single gene. This involves selective inclusion or exclusion of exons, or intron retention, increasing proteomic diversity. Alternative splicing is often tissue-specific. ### Regulatory Significance of Splicing Splicing is not merely RNA processing but a key regulatory step influencing: - **mRNA Stability:** Splicing can affect mRNAturnover rates. - **Coding Sequence Determination:** Alternative splicing dictates the final protein-coding sequence. - **Protein Diversity:** Generates multiple protein isoforms with varied localization and functions, contributing to tissue-specific protein expression and functional diversity. ## Splicing Defects and Human Diseases Aberrant RNA splicing is implicated in various human diseases, particularly neurological disorders, due to the production of non-functional or dysfunctional proteins. ### Splicing Pathologies Splicing defects are increasingly recognized in disease etiology, especially in neurological conditions, reflecting the nervous system's reliance on precise gene expression. ### Disease Examples Several pathologies are linked to splicing errors: #### Ataxia-Telangiectasia (A-T) ::: tcolorbox **Ataxia-Telangiectasia (A-T)** is characterized by neurological deficits, immune deficiency, and cancer predisposition. Aberrant splicing of the **ATM kinase** gene is central to A-T. ATM kinase is critical for DNArepair and cell cycle control. Splicing defects lead to reduced ATM function, causing cerebellar ataxia and increased cancer susceptibility. ::: #### Spinal Muscular Atrophy (SMA) ::: tcolorbox **Spinal Muscular Atrophy (SMA)** is a severe neuromuscular disease and a major cause of infant mortality. It results from defects in the **SMN1** (Survival Motor Neuron 1) gene. Splicing aberrations in *SMN1* reduce functional SMN protein, essential for motor neuron survival. SMN deficiency leads to motor neuron degeneration and muscle atrophy, causing paralysis. ::: ## Intron-Exon Structure and Splicing Signals Understanding gene organization and splice signals is crucial for comprehending splicing mechanisms. ### Exon-Intron Arrangement Eukaryotic genes feature alternating exons and introns. Human genes are composed of $\sim$``{=html}95% intronic DNA and $\sim$``{=html}5% exonic coding sequences. A typical human gene spans 27 kb, with only 1.3 kb coding. Genes average 7-8 exons. Introns, though non-coding, are functionally significant. ### Splice Site Boundaries Introns are defined by conserved **splice sites**: - **5' Splice Site (Donor Site):** At the 5' intron end. - **3' Splice Site (Acceptor Site):** At the 3' intron end. ### Intron Size and Regulatory Elements Intron size varies widely in humans ( $<$ 100 bp to $>$ 100 kb). Introns contain regulatory sequences: - **miRNA Genes:** Some introns encode microRNAs. - **Enhancers and Insulators:** Regulatory DNAsequences within introns modulate gene expression. Increased intron size and number during evolution correlate with expanded regulatory capacity in higher eukaryotes. ### Exon Size Conservation Exon size is evolutionarily conserved (50-300 nucleotides), suggesting functional constraints, possibly related to protein structure or splicing efficiency. ## Intron Classification Introns are classified into five main types based on splicing mechanisms. We focus on self-splicing and spliceosomal introns. ### Self-Splicing Introns **Self-splicing introns** are catalytic RNA(ribozymes) that excise themselves without protein enzymes. - **Group I Introns:** Found in rRNA, tRNA, and mRNA in lower eukaryotes, bacteria, and viruses. Splicing is intermolecular, initiated by a free guanosine. - **Group II Introns:** Primarily in organelles of fungi, plants, algae, and some bacteria. Splicing is intramolecular, initiated by an internal adenosine. ### Spliceosomal Introns **Spliceosomal introns** are the major type in eukaryotic protein-coding genes. Their removal is catalyzed by the **spliceosome**, a ribonucleoprotein complex. Spliceosomal introns are evolutionarily derived from self-splicing introns, utilizing protein and components. ## Splicing Mechanisms: Transesterification Intron removal involves two sequential **transesterification reactions** for all intron types. ### Group I Splicing (Intermolecular) Group I splicing is **intermolecular**, initiated by a free guanosine (G, GTP, GDP). 1. **1st Transesterification:** 3'-OH of guanosine attacks the 5' splice site, cleaving the RNAbackbone and attaching guanosine to the 5' intron end. 2. **2nd Transesterification:** 3'-OH of the 5' exon attacks the 3' splice site, joining exons and releasing the linear intron. ### Group II Splicing (Intramolecular) Group II splicing is **intramolecular**, initiated by an adenosine at the **branch site** within the intron. 1. **1st Transesterification:** 2'-OH of the branch site adenosine attacks the 5' splice site, forming a lariat and cleaving the 5' exon. 2. **2nd Transesterification:** 3'-OH of the 5' exon attacks the 3' splice site, joining exons and releasing the lariat intron. ### Spliceosomal Splicing Spliceosomal splicing, while similar to Group II, is catalyzed by the **spliceosome**, a complex of $\sim$``{=html}150 proteins and **small nuclear ribonucleoproteins** (snRNPs). The spliceosome positions reactants and coordinates two transesterification reactions, lacking intrinsic catalytic activity itself. ## The Spliceosome and snRNPs The **spliceosome** is the machinery for spliceosomal intron removal, composed of snRNPs and associated proteins. ### snRNP Composition and Function **snRNPs** are core spliceosome components, comprising small nuclear RNA() and proteins. Key snRNPs are U1, U2, U4, U5, and U6. #### snRNP Roles - **U1 snRNP:** (U1 snRNA + proteins) - Recognizes and binds the 5' splice site. - **U2 snRNP:** (U2 snRNA + proteins) - Binds the branch site. - **U4/U6 and U5 snRNPs:** Function in later assembly and catalysis. U5 interacts with both exons; U4/U6 regulate catalytic activity. snRNPs recognize splice sites and catalyze splicing via -mRNAand -base pairing. ### snRNA-Mediated Splice Site Recognition within snRNPs recognize mRNAsplice signals through base pairing. For example, U1 snRNA targets the 5' splice site consensus, and U2 snRNA targets the branch site, guiding spliceosome assembly and action. ## Splice Site Consensus Sequences Human introns have specific **consensus sequences** for spliceosome recognition. ### 5' Splice Site: GU The **5' splice site** consensus includes the **GU** dinucleotide at the 5' intron end. This GU is highly conserved and crucial for U1 snRNP binding. ### 3' Splice Site: AG and Polypyrimidine Tract The **3' splice site** consensus features the **AG** dinucleotide at the 3' intron end and an upstream **polypyrimidine tract** (U/C-rich region). The AG is conserved; the polypyrimidine tract is recognized by factors like U2AF. ### Mutations in Splice Sites Mutations in splice site consensus sequences disrupt splicing, causing aberrant mRNAand disease, leading to: - **Exon Skipping** - **Intron Retention** - **Cryptic Splice Site Activation:** Activation of pseudo splice sites. These defects result in non-functional or altered proteins, contributing to genetic disorders. ### Therapeutic Splice Site Correction Therapeutic strategies aim to correct aberrant splicing using antisense oligonucleotides (AONs) to: - **Block Aberrant Splice Sites** - **Promote Exon Inclusion** - **Modulate Alternative Splicing** These AON-based therapies aim to restore normal splicing and protein function in splicing-related diseases. ## Spliceosome Regulation and Kinetics Spliceosome assembly and splicing kinetics are regulated, involving multiple factors and steps. ### Early Complex Formation: Complex E Initial spliceosome assembly involves forming the **Commitment Complex (Complex E)**, or presplicing complex, through 5' and 3' splice site recognition by splicing factors. ### Splicing Activator Proteins **Splicing activators** are essential for early spliceosome assembly, defining 5' and 3' splice site pairs and promoting Complex E formation. #### ASF/SF2 and U2AF65/U2AF35 - **ASF/SF2:** Binds the 5' splice site, enhancing U1 snRNP recognition. - **U2AF65/U2AF35:** Recognizes the polypyrimidine tract and 3' splice site. U2AF65 binds the polypyrimidine tract, and U2AF35 interacts with the 3' splice site AG. These activators initiate splice site recognition and pre-mRNAcommitment to splicing. ### Tissue-Specific Splicing Factors Tissue-specific expression of splicing factors (activators and repressors) regulates alternative splicing, generating tissue-specific protein isoforms. Varying levels of these factors in different tissues lead to diverse splicing patterns. ### Energetic Cost and Efficiency Spliceosomal splicing is energy-intensive, requiring ATP and GTP hydrolysis. Despite this cost, it is highly efficient and precise, ensuring accurate intron removal and exon joining, highlighting its critical role in eukaryotic gene expression. # Conclusion This lecture explored RNA metabolism, covering degradation, translational control, and splicing. We discussed: - **RNA Degradation:** Essential for regulating RNA lifespan and protein expression, with distinct mechanisms in prokaryotes (degradosome) and eukaryotes (5'-3' and 3'-5' pathways, NMD). - **RNA Silencing and Translational Control:** Mechanisms for temporal and spatial gene expression regulation through mRNAstorage, transport, and localized translation, crucial for development and cellular functions. - **RNA Splicing:** A fundamental eukaryotic process for mRNAmaturation, involving constitutive and alternative splicing, catalyzed by the spliceosome, and critical for proteomic diversity. Splicing defects are linked to various human diseases. Understanding these RNA processes is crucial for comprehending gene regulation, cellular biology, and the molecular basis of diseases. Further research into RNA biology promises advancements in biotechnology and therapeutic interventions.