miRNA’s and RNA interference

Lecture Overview

  • The lecture covers gene expression, specifically focusing on microRNA (miRNA) and RNA interference, presented by Dr. Jerome Korzelius at the University of Kent.

Gene Expression: Central Dogma

  • Central dogma of molecular biology outlines the flow of genetic information:

    • Replication: DNA is copied to ensure genetic continuity.

    • Transcription: DNA is transcribed to produce RNA.

    • Translation: mRNA is translated to synthesize proteins.

Discrepancy Between Protein and mRNA Levels

  • Key Point: Protein and mRNA levels may not always correlate.

    • Factors influencing correlation include:

      • Environmental stress (heat stress, infection).

      • Developmental stages.

  • Examples:

    • In bacteria:

      • Environmental changes lead to gene-specific alterations in mRNA decay rates.

      • Rapidly growing bacteria exhibit transcriptome-wide rapid decay, while slowly growing bacteria experience slow decay.

Age and Protein Expression

  • Research by Kelmer-Sacramento et al. (2020) indicates:

    • Reduced proteasome activity in aging brains results in loss of ribosome stoichiometry and protein aggregation.

    • The association between mRNA and protein levels decreases with age.

Regulation of mRNA and Protein Levels

  • Regulation Mechanisms:

    • Qualitative Control: Multiple protein forms can arise from one mRNA due to splicing.

    • Quantitative Control: Control over mRNA levels impacts translation efficiency.

      • Mechanisms include mRNA decapping, deadenylation, and small RNA regulation.

Focus of Lecture: Control of mRNA By Small RNAs

  • Exploration of:

    • Discovery and role of small non-coding RNAs in gene silencing.

    • RNA Interference (RNAi) and microRNAs (miRNAs):

    • Mechanisms of siRNA and miRNA function and processing.

    • Impact of miRNA abundance on gene expression in eukaryotes.

    • RNAi as a tool for gene function study.

Types of Non-Coding RNAs

  • Two Classes Based on Size:

    • Long (>200 bp): Involves transcriptional silencing in the nucleus.

    • Short (20-30 bp): Engages in post-transcriptional gene silencing in the cytoplasm.

Discovery of Small Non-Coding RNAs

  • Serendipitous discovery leading researchers to evaluate small RNAs and gene regulation.

Transgene Induced Gene Silencing (TIGS)

  • Example of CHS gene resulting in unexpected white flowers due to silencing of the endogenous gene by the addition of a transgene.

    • Impact can be hereditary, spreading effect observed.

The Role of C. elegans lin-4 Gene

  • Function: lin-4 gene encodes a non-coding RNA that regulates transcription factor lin-14 by binding to its 3’ UTR, thus controlling mRNA levels through degradation mechanisms.

Discovery of RNA Interference (RNAi)

  • Key Findings: RNAi is triggered by double-stranded RNA (dsRNA), leading to specific silencing of homologous mRNA.

    • The mechanism involves degradation of the targeted mRNA without affecting others.

  • Notable example with C. elegans unc-22 gene and its mutant phenotype (twitching) illustrating RNAi effects.

Nobel Prize in RNAi

  • The significance of RNA interference was recognized with a Nobel Prize in Physiology or Medicine in 2006.

Definitions

  • RNA Interference (RNAi): Process where short interfering RNAs (siRNA) modulate mRNA expression through inhibition or degradation.

  • MicroRNAs (miRNA): Endogenous short interfering RNAs processed from longer precursors that regulate mRNA expression.

Steps in siRNA/miRNA-Mediated Gene Silencing

  1. Processing by Drosha: Pri-miRNAs processed to pre-miRNAs.

  2. Dicer Processing: Precursor RNAs cleaved to 21-23 nucleotide fragments.

  3. Incorporation into RISC: RISC complex formed to target mRNA.

  4. mRNA Inactivation: Binding leads to degradation or translational repression of mRNA.

Mechanisms of RISC Function

  • RISC binding often destabilizes mRNA, inhibiting translation.

  • In mammalian cells, different Ago isoforms dictate the efficiency and type of RNAi.

Differences between siRNA and miRNA

  • miRNAs: Often bind partially, leading to translational repression.

  • siRNAs: Typically bind fully to their target's 3’ UTR, resulting in degradation.

Widespread Role of MicroRNAs

  • Eukaryotic genomes can encode hundreds of miRNAs, affecting vast numbers of target mRNAs across developmental stages.

Applications of RNAi in Research

  • Utilized in gene function studies through RNAi knockdown in mammalian systems and efficient protocols in C. elegans.

Summary Points

  • siRNAs and miRNAs regulate mRNA levels by binding to the 3’ UTR and inhibiting translation or degradation.

  • Processing involves Drosha and Dicer with RISC complex playing a central role.

  • siRNA/shRNA technology serves as a powerful tool for gene function studies.

Suggested Readings

  • Essential Genes, 4th Edition, Chapter 28: Regulatory RNA

  • Genes XII, Chapter 30: Regulatory RNA

  • Youtube Lecture by David Bartel: Introduction to miRNAs

  • Nature Overview on Antisense RNA Research: Historical insights into discoveries in RNA interference and miRNAs.