SUNWAY UNIVERSITY - A CLASS ABOVE MONASH UNIVERSITY

School of MEDICAL & LIFE SCIENCES

Course: BIO1114 Cell Biology
Chapter 9: Control of Gene Expression II

Recap of Last Lecture

  • Core components discussed:

    • Histone Tail

    • Core Histone Proteins: H2A, H2B, H3, H4

  • Key terms:

    • TAF: TBP-associated factors involved in transcription.

    • Pol II: RNA Polymerase II, essential for transcription.

    • RTK: Receptor Tyrosine Kinases which are involved in signaling pathways.

  • Signaling pathways highlighted:

    • MAP Kinase Signaling

    • JAK/STAT Signaling

    • PI3K/AKT1 Signaling

  • Importance of Chromatin Modifications:

    • Regulate gene transcription initiation.

How are Genes Regulated?

  • Gene regulation in eukaryotes occurs at multiple stages:

    1. Chromatin Modifications

    2. Regulation of Transcription Initiation

    3. RNA Processing

    4. mRNA Stability

    5. Translation

    6. Protein Processing (post-translational modification)

Trans-acting Elements

  • Transcription Factors as trans-acting elements:

    • Bind specifically to DNA sequences (promoter, enhancer, silencer).

    • Interact with proteins to modulate transcriptional activity.

  • Structural domains identified:

    • DNA-binding domain: Facilitates the binding to DNA.

    • Regulatory Domain/Transcription-activator domain: Activates or represses transcription.

  • Functional Requirement:

    • Most transcription factors function as dimers (combinations of two molecules).

  • Example: Lac Repressor

Cis-acting Elements

  • Definition:

    • DNA sequences located on the same molecule as the gene they regulate.

    • Their function is to control gene expression by serving as binding sites for various transcription and regulatory proteins.

Histone Methylation

  • Definition:

    • Addition of methyl groups (-CH₃) to specific amino acids on histone tails.

  • Histone Methyltransferases (HMTs):

    • Enzymes that add one to three methyl groups to lysine or arginine residues on histones.

  • Effects of Histone Methylation:

    • Generally leads to reduced gene expression, but impact varies depending on the histone residue modified.

  • Visual representation:

    • Histone Tail with Methyl Groups displayed, indicating differential outcomes:

    • Heterochromatin: Generally more transcriptionally inactive.

    • Euchromatin: More transcriptionally active.

DNA Methylation

  • Two main mechanisms of transcriptional silencing via methylation:

    1. Blocking binding of transcription factors

    2. Inducing formation of heterochromatin

Implications of Mutations in Regulatory Elements

  • Inquiry:

    • How do mutations in cis-acting elements compare to those in trans-acting factors?

    • Which type of mutation causes more localized vs. global effects on gene expression?

Summary of Chromatin Modifications

  • Noteworthy Reference:

    • Flavahan, W. A., Gaskell, E., & Bernstein, B. E. (2017). Epigenetic plasticity and the hallmarks of cancer. Science, 357(6348), eaal2380. doi:10.1126/science.aal2380

Learning Objectives

  • Explain varying pathways regulating mRNA stability.

  • Describe how post-translational modifications influence gene expression.

Today's Lecture Focus

  • RNA Stability

  • RNA Interference

RNA Processing

  • Alternative Splicing:

    • Process to create mature mRNA involves excision of introns and ligation of exons.

    • Catalyzed and controlled by spliceosome:

    • Composed of 300+ proteins and five uracil-rich snRNPs (U1, U2, U4, U5, U6)

    • Produces variability in protein products from a single gene.

    • Involved in increasing transcriptome and proteome diversity (95% of protein-coding genes).

Various Types of Alternative Splicing

  • Basic splicing patterns include:

    1. Exon skipping

    2. Intron retention

    3. Mutually exclusive exons

    4. Alternative 5’ splice sites

    5. Alternative 3’ splice sites

mRNA Stability

  • mRNA characteristics:

    • Generally unstable.

    • Average half-life approximately 20 minutes.

    • Decay rate influenced by sequence, structure, and cellular environment.

  • Impact on protein expression:

    • Longer mRNA lifespan allows for more protein translation.

    • Rate of decay alterations can cause significant changes in protein levels.

Ribonucleases Role in mRNA Stability

  • Types of Ribonucleases:

    • Endoribonucleases: Cleave RNA at internal sites.

    • Exoribonucleases: Remove terminal ribonucleotides either from 5’ to 3’ or 3’ to 5’.

Regulation of mRNA Decay

  • RNA-binding Proteins (RBPs):

    • Key regulators of post-transcriptional events.

    • Bind to AU-rich elements (AREs) in the 3’ UTR of mRNA, affecting stability and decay rates.

  • RBP-RNA interactions can either stabilize or destabilize mRNA.

Mechanisms of mRNA Decay

  • Types of Decay Mechanisms:

    1. Deadenylation-dependent mRNA Decay

    2. Endonucleolytic cleavage-mediated Decay

    3. Nonsense-mediated Decay

    4. RNAi-dependent Pathway

Deadenylation-dependent mRNA Decay

  • Process:

    • 3’ poly-A tails are removed by deadenylase.

    • The 5’ cap is then removed, leading to degradation by exonucleases.

    • DcpS Protein: Scavenger decapping enzyme involved in mRNA decay.

Endonucleolytic Cleavage-mediated Decay

  • Mechanism:

    • Recognizes specific sequence elements for cleavage.

    • Generates exposed 3’ and 5’ ends for further decay by exonucleases.

Nonsense-mediated Decay

  • Definition:

    • Rapidly eliminates mRNAs with premature stop codons.

    • Function to remove faulty transcripts from mutated DNA sequences.

  • Mechanism Involving EJC: Exonic-junction complexes recruit core protein UPF2, which interacts with ribosomes when a premature stop codon is encountered, initiating decay.

RNAi-dependent Pathway

  • Significant Discovery:

    • Nobel Prize in Physiology or Medicine 2006 awarded to Andrew Z. Fire and Craig C. Mello for discovering RNA interference.

MicroRNAs

  • Characteristics:

    • Single-stranded RNA (20-25 nucleotides).

    • Endogenously produced and transcribed by RNA polymerase II.

  • MiRNA Processing:

    1. Transcription to primary miRNA (pri-miRNA).

    2. Processed into pre-miRNA and then mature miRNA by Dicer.

  • Functionality:

    • Can target multiple mRNAs or highly specific single targets to regulate gene expression and prevent translation.

Small Interfering RNAs (siRNAs)

  • Characteristics:

    • Double-stranded RNA (20-24 bp) with defined ends.

  • Processing:

    • Cleaved by Dicer to activate RNA-induced silencing complex (RISC).

    • RISC cleaves target mRNA, enhancing gene silencing.

Post-translational Regulation

  • Types of modifications:

    1. Proteolysis: Breakdown into smaller peptides or amino acids.

    2. Phosphorylation: Addition of phosphate groups for activation.

    3. Lipidation/Prenylation: Involves lipid additions.

    4. Ubiquitination: Marks proteins for degradation.

    5. Glycosylation: Adds carbohydrate groups, affecting protein folding and stability.

Ubiquitination Process

  • Definition:

    • Attachment of ubiquitin to a target protein via isopeptide bond to lysine.

  • Enzyme Process:

    • Involves E1 (activating), E2 (conjugating), and E3 (ligase) enzymes.

    • Polyubiquitination typically leads to proteasomal degradation of proteins.

Conclusion

  • This lecture encompasses the processes of gene expression control through various molecular mechanisms and highlights the significance of regulatory elements involved.

Closing Note

  • Happy Studying!

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