BIOL21101_Module13_organelles_3_

Genome Maintenance & Regulation

Instructor: Pawel GrzechnikFocus: Detailed analysis of the regulation of gene expression specifically in mitochondria and chloroplasts.

Learning Outcomes

By the end of this course, students will:

• Comprehend the distinct properties of DNA and RNA present in organelles, focusing on their evolutionary origins, genome structure, and gene content.

• Understand the intricate processes of transcription and RNA turnover, including how they differ in organelles compared to the nucleus.

• Gain insights into replication mechanisms, heteroplasmy, and mutation rates in mitochondrial and chloroplast DNA (cpDNA).

• Recognize the significance of coordination between the expression of nuclear encoded genes and those of organelles for proper cellular function.

• Develop a foundational knowledge of mitochondrial diseases and explore various therapeutic approaches, including advances in gene therapy.

Eukaryotic DNA

Eukaryotic cells contain additional DNA located outside the nuclear genome in organelles such as:

• Mitochondria

• Chloroplasts

Diagram:

A simplified map showcasing the composition and location of organellar DNA, including mitochondrial DNA (mtDNA) and chloroplast DNA (cpDNA).

Endosymbiotic Theory

The endosymbiotic theory posits that mitochondria and chloroplasts originated from prokaryotic cells that were engulfed by ancestral eukaryotic cells.

• Engulfing Events:

  • Cyanobacterium: The precursor to chloroplasts, responsible for photosynthesis.

  • Aerobic Prokaryote: The precursor to mitochondria, essential for aerobic respiration.

• Result: Modern eukaryotic cells exhibit dual ancestry, comprising both prokaryotic and eukaryotic lineage.

DNA Accumulation in Organelles

Over evolutionary time, selective forces have minimized excessive DNA accumulation in mitochondria and chloroplasts.

• A significant evolutionary trend includes the transfer of many organelle genes to the nuclear genome, leading to more efficient regulation from the nucleus.

• As a result, organelle genomes are generally compact and exhibit reduced sizes compared to nuclear genomes.

Importance of Gene Expression

The expression of genes encoded by organellar DNA is crucial for the efficient functioning of mitochondria and chloroplasts.

Diagram:

Illustration depicting the functional roles of mtDNA and cpDNA and the products that result from their expression in respective organelles.

Mitochondria Visualization

Techniques such as fluorescent staining in HeLa cells allow visualization of mitochondria and nuclei.

• Red emission indicates the presence of mitochondria (utilizing MitoTracker).

• Blue emission represents nuclei (using DAPI staining).

Mitochondrial DNA (mtDNA)

The characteristics of mtDNA include:

• Inheritance: Exclusively maternally inherited, resulting in a matrilineal lineage of mtDNA.

• Structure: Unique circular genome that codes for essential polypeptides involved in the respiratory electron transport chain.

• Distinction: The structure diverges significantly from that of the nuclear genome.

mtDNA Replication

The enzyme responsible for mtDNA replication is DNA Polymerase Gamma, which has the following characteristics:

• Replication Process: Features bidirectional replication, meaning it unwinds and synthesizes DNA in both directions from the replication origin.

• Leading Strand Synthesis: Initiates at a particular site known as the D-loop, where replication begins and is regulated.

mtDNA Structure

The human mitochondrion's DNA consists of approximately 16.6 kbp, with the following components:

• Composition:

  • 2 rRNA genes

  • 13 protein-coding genes essential for electron transport

  • 22 tRNA genes that utilize a non-standard genetic code

• Transcription Mechanism: MtRNA transcription is conducted by a single-subunit RNA polymerase that functions similarly to T7 RNA polymerase.

tRNA's Role in mtRNA Processing

In mtRNA processing, tRNAs function as 'punctuation marks' that delineate coding sequences:

• Transcription Initiation: Starts from the D-loop, producing mRNAs that are transcribed as polycistronic precursors.

• Processing Model: This process follows the tRNA punctuation model, where 22 interspersed tRNAs mark boundaries for individual coding regions.

tRNA Processing Enzymes

Key enzymes involved in the maturation of tRNA include:

• RNase P: Functions as an endonuclease that processes tRNA at the 5′ end; it is universally present across all domains of life.

• RNase Z: Specifically matures the 3′ end of tRNA, completing the maturation process for functional tRNA molecules in mitochondria.

Gene Expression Machinery in Mitochondria

The distinction between mitochondrial RNA (mtRNA) and conventional mRNA includes:

• lacking Cap Structures: Unlike nuclear mRNA, mtRNA lacks a 7-methylguanylate cap and does not undergo splicing since it contains no introns.

• Polyadenylation: Occurs post-transcriptionally, a deviation from traditional eukaryotic mechanisms.

• Mitochondria possess their own ribosomes and specific gene expression machinery that influences the synthesis and maturation of proteins encoded by mtDNA.

mtRNA Degradation

The degradation of mtRNAs involves several mechanisms:

• Enzymes:

  • Polynucleotide phosphorylase (hPNPase): Dismantles RNA from the 3' end.

  • ATP-dependent helicase (hSuv3): Acts to unfold structured RNA, facilitating degradation.

• Process Overview: MtRNAs are consistently subject to degradation events that require specific mechanisms to preserve their integrity and functionality.

RNA Metabolism Overview

Key enzymes involved in mitochondrial RNA metabolism include:

• hSuv3: Responsible for unwinding secondary RNA structures to ensure proper processing and degradation.

• REXO2 exonuclease: Facilitates the removal of short RNA fragments that are not necessary for mitochondrial function.

• PNPases: Found in mitochondria, chloroplasts, and various bacterial systems, critical for exonucleolytic degradation and polyadenylation activities.

Key Takeaways on mtRNA Processing

In summary:

• Pre-RNAs undergo processing according to the tRNA punctuation model, necessitating tRNA maturation at specific ends via RNase P and RNase Z.

• The enzymes hSuv3 and PNPase play pivotal roles in the degradation processes of mtRNAs, essential for maintaining mitochondrial health by regulating RNA levels.

Mitochondrial Disorders Overview

Mitochondrial disorders are genetic conditions arising from dysfunctions in the mitochondria affecting cellular energy metabolism:

• Impact: Results in compromised oxidative phosphorylation and disturbances in the Krebs cycle, leading to energy deficits.

• Toxic Accumulation: Mutations can cause the accumulation of toxic by-products that can harm tissues and organ systems.

• Prevalence: These disorders manifest in approximately 1 in 5000 individuals, with symptoms being highly variable.

• Onset: Can occur either during childhood or adulthood, frequently influenced by nuclear-encoded mutations affecting mitochondrial function.

Clinical Implications of mtDNA Mutations

Mutations in mtDNA can lead to a wide spectrum of diseases ranging from mild to severe manifestations:

• Rare Disorders: Monogenic disorders often have profound effects on the nervous and muscular systems, highlighting the critical reliance on mitochondrial function for these tissues.

• Late-Onset Disorders: Mild mutations might correlate with disorders that emerge in late adulthood, emphasizing the cumulative effects of mitochondrial dysfunction.

• Healthy Individuals: Lower levels of mtDNA mutations (<1%) can typically be found without presenting any health issues, illustrating the variability in tolerance to these genetic changes.

Specific Mitochondrial Disorders

Notable mitochondrial disorders include:

• MELAS: Childhood-onset disorder featuring neurological and muscular symptoms, including seizures and muscle weakness.

• MERRF: Typically manifests in adolescence, characterized by muscle jerks, ataxia, and other neurological symptoms.

• LHON: Primarily affects adult males leading to vision loss, showing a gender bias linked to the disease’s inheritance pattern.

Mitochondrial DNA Characteristics

Mitochondrial DNA is known for its high mutation rates compared to nuclear DNA:

• Replication Errors: The high replication error rate (10^3 to 10^4) contributes to increased mutations.

• Heteroplasmy: Continuous replication leads to heteroplasmy, where cells may carry a mixture of normal and mutated mtDNA, with significant implications for energy production and disease.

• Mutation Rate: It is estimated that the mutation rate is approximately 30-fold higher than that of nuclear DNA, contributing to the diverse clinical presentations of mitochondrial pathologies.

Understanding Heteroplasmy

Heteroplasmy refers to the presence of multiple mtDNA variants within a cell, leading to variable effects on cellular function:

• Tolerance Levels: Cells can tolerate high levels of pathogenic variants, but defects typically arise when levels exceed a threshold of approximately 80%.

Therapeutic Approaches

Therapeutic strategies targeting mitochondrial dysfunction include:

• Targeted Removal: Techniques aimed at removing mutated mtDNA to shift the proportions of mitochondrial DNA towards healthier variants, potentially ameliorating symptoms of mitochondrial disorders.

Strategies for mtDNA Elimination

Methods for eliminating mutated mtDNA involve:

• Mitochondrially Targeted Restriction Endonucleases (mitoRE): These enzymes are designed to preferentially target and cut pathogenic mtDNA sequences.

• Limitations: Safe application is hindered by the need for unique restriction sites that permit selective mutation targeting.

Advanced Gene Editing Techniques

Innovative approaches using gene editing technologies include:

• Programmable Nucleases: These can allow for precise targeting and editing of mtDNA, with various categories including:

  • Zinc-finger Nucleases (ZFNs): Engineered to recognize specific DNA sequences leading to targeted editing.

  • Transcription Activator-Like Effector Nucleases (TALENs): Another class of engineered nucleases that provide targeting capabilities based on specific DNA sequences.

Success in Correcting mtDNA Mutations

Emerging studies demonstrate successful correction of mtDNA mutations in cell cultures and animal models using advanced genetic techniques, providing hope for future therapeutic applications.

Summary on Mitochondrial Disorders

Recognizing key mitochondrial disorders, including MELAS, MERRF, and LHON, underscores the genetic underpinnings associated with mtDNA mutations:

• Heteroplasmy Effects: The concept of heteroplasmy highlights the coexistence of normal and mutant mtDNA, shaping both health outcomes and disease state.

• Mutation Correction Potential: Continued research aims at developing strategies for effective correction of mtDNA mutations, advancing the field of mitochondrial medicine.

Overview of Chloroplast DNA

Chloroplasts contain their own genome, primarily inherited maternally, encoding proteins that are essential for the photosynthesis process.

• This genetic information is crucial for energy acquisition and storage in plants, demonstrating the connection between chloroplast function and global carbon cycling.

Origin of Chloroplasts

All chloroplasts share a common evolutionary ancestor traceable back to cyanobacteria, establishing a significant evolutionary event that facilitated the establishment of photosynthetic capabilities in land plants and green algae, ultimately shaping terrestrial ecosystems.

cpDNA Replication

Chloroplast DNA replication encompasses:

• Mechanisms of Bidirectional Replication: Elongation initiated from multiple origins which allows efficient and thorough replication.

• D-loop Fusion: Represents significant replication events, critical in maintaining chloroplast function through genomic continuity.

Wheat Chloroplast DNA Composition

The chloroplast DNA of wheat exhibits:

• Structure: Comprising double-stranded DNA with an approximate size of 135 kb that contains:

  • 2 rRNA genes, 80 protein-coding genes, and 30 tRNA genes.

• The genome demonstrates high levels of conservation throughout evolutionary history, reflecting the importance of preservation in functions essential for photosynthesis.

Chloroplast Gene Expression

Distinct features of chloroplast RNA versus conventional mRNA include:

• Modification Mechanisms: Chloroplast RNA undergoes co-transcriptional capping and splicing, followed by polyadenylation which is essential for RNA stability and degradation efficiency.

Chloroplast RNA Metabolism

Chloroplast RNA degradation mechanisms resemble bacterial systems with:

• 3’ to 5’ degradation facilitated by PNPase or RNase II, ensuring efficient RNA turnover and regulation.

• Polyadenylation: Involves specific enzymes such as Ntr-PAP or PNPase contributing to RNA processing in a regulated manner.

Key Takeaways on Chloroplasts

In overview, key points include:

• The evolutionary roots of chloroplasts can be traced back to cyanobacteria, emphasizing their significant ancestral background.

• The metabolism and processing of RNA within chloroplasts show parallels to bacterial mechanisms, showcasing evolutionary conservation.

Comparison of mtDNA and cpDNA

Additional Topics

Forthcoming modules will explore non-coding RNAs, transposons, and various organelles, further expanding knowledge in cellular biology and genetics.