Post-Transcriptional Regulation in Eukaryotes

Post-Transcriptional Regulation in Eukaryotes

Video 1: Introduction to Post-Transcriptional Regulation

Learning Outcomes

  • By the end of this video, you should be able to:

    • Define post-transcriptional gene regulation.

    • List two mechanisms of post-transcriptional gene regulation in eukaryotes.

Overview of Gene Regulation

  • Gene Regulation: Most gene regulation occurs at the transcription initiation phase. However, it can also happen:

    • At any stage of transcription: This includes initiation, elongation, and termination.

    • Post-transcriptionally: Involves mRNA stability or translation initiation.

    • Post-translationally: Involves protein stability and activity.

  • RNA Splicing: Gene expression can be regulated by altering RNA splicing, which results in mRNAs that have different sequences.

Post-Transcriptional Regulation

Definition

  • Post-Transcriptional Regulation: Refers to regulatory mechanisms that target the mRNA directly post-transcription.

Mechanisms of Post-Transcriptional Regulation in Eukaryotes

  1. Alternative Splicing

  2. RNA Interference (RNAi)

Video 2: Alternative Splicing

Learning Outcomes

  • By the end of this video, you should be able to:

    • Describe alternative splicing.

    • Explain how alternative splicing can produce different proteins from a single gene.

    • Describe how alternative splicing may be regulated.

Review: mRNA Processing

  • mRNA Processing: In eukaryotes, mRNA is not immediately translated. Steps include:

    1. Export from the nucleus to the cytoplasm.

    2. Processing before translation occurs.

    • Pre-mRNA transforms into Mature mRNA through splicing and addition of a 5’ cap and a poly(A) tail.

mRNA Splicing

  • RNA Splicing: The removal of introns and the rejoining of exons, catalyzed by the spliceosome, which is a large complex of small nuclear RNAs (snRNAs) and proteins.

    • Specific nucleotide sequences in pre-mRNA dictate the locations of splicing.

Alternative Splicing

  • Definition: Alternative splicing allows exons within a single pre-mRNA to be spliced in different ways, creating multiple mRNA products.

  • Prevalence: It occurs in 95% of human genes that contain multiple exons.

Functions of Alternative Splicing

  1. Encode Different Proteins (Isoforms):

    • Different functions may be necessary in various cell types at different times.

    • Example: α-tropomyosin gene.

  2. Repress Gene Expression:

    • Produces a splice variant that does not encode a functional protein.

    • Example: Sex determination in Drosophila.

Example of Alternative Splicing

  1. α-Tropomyosin:

    • Spliced into different isoforms depending on the tissue type.

    • Involves transcription, splicing, and 3' cleavage/polyadenylation.

    • Results in isoforms in:

      • Striated muscle

      • Smooth muscle

      • Fibroblast

      • Brain tissue

  2. Sex Determination in Drosophila:

    • Tra protein expression differentiation leads to male and female development.

    • Premature stop codon in male splice variant results in a truncated polypeptide, yielding no functional Tra protein.

    • Exon 2 absent in female splice variant results in functional Tra protein.

    • The Sxl protein functions as a splicing factor in this process.

Mechanism of Alternative Splicing

  • Splicing factors are proteins that bind to specific sequences in mRNA and determine which exons are included in mature mRNA.

  • The expression or activity of these splicing factors can be regulated, influencing the types of splice variants produced.

Mechanisms:

  1. Repression: Splicing repressors block spliceosome binding to prevent the inclusion of certain exons.

    • This change results in a normal mRNA versus one that has skipped an exon.

  2. Activation: Splicing activators recruit the spliceosome to an alternative splice site, leading to an extended exon in the resultant mRNA.

Video 3: RNA Interference

Learning Outcomes

  • By the end of this video, you should be able to:

    • Describe miRNA and siRNA structure.

    • Describe the steps of RNA interference using miRNA and siRNA.

    • Describe two ways that RNA interference can regulate gene expression.

RNA Interference (RNAi)

  • Definition: RNA interference is a biological mechanism that changes gene expression by altering mRNA translation or stability via double-stranded RNA.

    • 1. prevent translation

    • 2. degrade mRNA

  • Sources of double-stranded RNA:

    • Genes encoding RNA that forms hairpins.

    • Viruses.

    • Double-stranded RNA synthesized in vitro.

Mechanisms of RNA Interference

  1. microRNA (miRNA): → encoded in the genome

    • Size: ~22 nucleotides.

    • Function: Interacts with target mRNA through imperfect base-pairing, usually within the 3’ untranslated region (UTR).

    • Outcomes: Prevents translation of mRNA (most common) or causes degradation of mRNA (less common).

Steps in miRNA Regulation

  1. Primary RNA (pri-miRNA) is transcribed, forming a hairpin.

  2. Primary RNA is processed, forming pre-miRNA, and exports from the nucleus.

  3. Dicer removes the hairpin loop, leaving short double-stranded RNA that forms miRNA.

  4. miRNA interacts with proteins to create the RNA-induced silencing complex (RISC).

  5. One strand of miRNA is discarded while the other remains.

  6. RISC binds to target mRNA.

  7. Translation of target mRNA is inhibited.

2. Small Interfering RNA (siRNA):

  • Size: ~22 nucleotides.

  • Function: Has a complementary sequence to target mRNA and base-pairs perfectly with it, leading to target mRNA degradation.

Steps in siRNA Regulation

  1. Double-stranded RNA is introduced into the cell, usually from external sources.

  2. Cleaved by Dicer to produce siRNA.

  3. siRNA combines with proteins to form RISC.

  4. One strand of siRNA is discarded while the other is kept.

  5. RISC binds to the target mRNA.

  6. Target mRNA undergoes cleavage and degradation.

Comparison of miRNA and siRNA

Feature

miRNA

siRNA

Origin

Transcribed from distinct genes

Transcribed from distinct genes, transposons, or viruses

Cleavage of

Single-stranded RNA forming short hairpins

Double-stranded RNA duplex

Size

21–25 nucleotides

21–25 nucleotides

Action

Degradation of mRNA, inhibition of translation

Degradation of mRNA

Interaction with target mRNA

Imperfect base pairing

Complementary base pairing

Video 4: RNAi in Research

Learning Outcomes

  • By the end of this video, you should be able to:

    • Describe how RNA interference is employed in research to knockdown gene expression.

    • Interpret data from RNA interference experiments to determine the importance of a gene in biological processes.

RNAi in Research

  • RNAi functions as an endogenous (natural) mechanism for regulating gene expression in eukaryotes.

  • Researchers exploit this mechanism to manipulate gene expression for studying gene functions.

  • Methods include introducing short hairpin RNA (shRNA) or expressing siRNAs in cells to knockdown gene expression.

  • Design: shRNA/siRNA is tailored for perfect complementarity to induce mRNA cleavage.

Techniques to Confirm mRNA Levels Reduction

  • Northern Blot: Used to detect specific RNA sequences.

  • qPCR: Quantitative PCR to measure the quantity of targeted mRNA.

  • Western Blot: Used to detect proteins, confirming the presence or absence of the protein encoded by the target mRNA.

Common Controls in RNAi Experiments

  • Control: Cells with no shRNA/siRNA.

  • Control: Cells with negative control shRNA/siRNA that does not affect the target expression.

  • Evaluate qPCR results and Western blot results for their respective experimental group comparisons.

Example Scenario #1

  • Objective: Determine if the transcription factor TFX regulates GeneY expression in yeast cells.

    1. Control Group 1: Yeast cells without siRNA treatment.

    2. Control Group 2: Yeast cells treated with negative control siRNA that should not affect TFX expression.

    3. Experimental Group: Yeast cells treated with siRNA specific to TFX to reduce its expression.

  • Isolate mRNA from the three groups and quantify GeneY mRNA using qPCR to assess TFX’s regulatory role on GeneY expression and whether it represses or activates its expression.

Example Scenario #2

  • Objective: Determine the role of Syntaxin 5, Syntaxin 18, or Syntaxin 1A in branch formation in Drosophila terminal cells.

  • Reference: Developmental Biology. (2022). 490:100-109