BIOS5010_Lecture 9 miRNA and RNAi

Gene Expression and its Control

Lecture Title: Gene Expression and its Control - BIOS5010 Lecture 9: miRNA’s and RNA Interference

Presented by: Dr. Jerome KorzeliusEmail: J.Korzelius@kent.ac.ukAffiliation: University of Kent

Gene Expression: Central Dogma

The Central Dogma of Molecular Biology outlines the fundamental processes of genetic information flow within biological systems. This framework can be summarized as follows:

Replication: The process by which DNA is copied to produce identical DNA molecules, crucial for cell division and inheritance.
Transcription: The synthesis of RNA from a DNA template, where the information coded in DNA is transcribed to messenger RNA (mRNA).
Reverse Transcription: A process where RNA is converted back into DNA, a mechanism utilized by retroviruses like HIV to integrate into the host genome.
Translation: The process where ribosomes synthesize proteins based on the sequence of the mRNA, ultimately determining the function and role of proteins in cellular processes.

Correlation Between Protein and mRNA Levels

It is essential to understand that protein levels do not consistently correlate with mRNA levels due to various influencing factors:
- Environmental Conditions: Different stressors (e.g., heat shock, infection) can alter translation efficiency and mRNA stability, leading to discrepancies between the amount of mRNA present and the corresponding protein.
- Developmental Stages: As organisms develop, the regulation of gene expression can change, affecting how much protein is ultimately produced.
Example: In bacteria, growth rates significantly influence mRNA decay rates:
- Rapidly Growing Bacteria: Exhibit a high transcriptome-wide rapid decay rate for mRNA.
- Slowly Growing Bacteria: Demonstrate a slower decay rate, influencing the protein synthesis dynamics within the cell.

Age and Gene Expression

Recent studies (Kelmer-Sacramento et al., 2020) indicate that:

The association between mRNA and protein levels tends to decrease with age, which suggests that the efficiency and regulation of gene expression might decline over time.

Mechanisms Regulating mRNA to Protein Conversion

Regulatory Mechanisms:

Qualitative Control: This mechanism allows for the production of different protein isoforms from a single mRNA transcript through alternative splicing, contributing to protein diversity in eukaryotes.
Quantitative Control: Involves the regulation of mRNA abundance, which directly impacts the efficiency of translation into proteins. This can include processes such as mRNA decapping, degradation via exosomes, and deadenylation.

This week’s focus revolves around the role of small non-coding RNAs (such as miRNAs) in regulating mRNA stability and gene expression.

Lecture Focus: mRNA Control Through Small RNAs

Topics Discussed:

The discovery and functional importance of small non-coding RNAs in cellular processes.
Mechanisms of Gene Silencing: Focusing on RNA interference (RNAi) and microRNAs (miRNAs) which play pivotal roles in post-transcriptional regulation of gene expression.
Processing and Function of Small Interfering RNA (siRNA) and miRNA: Their biogenesis, structure, and mechanism of action as critical regulators of mRNA levels.

Types of Non-Coding RNAs

Classification by Size:
- Long Non-Coding RNAs (>200 bp): Typically involved in transcriptional silencing mechanisms in the nucleus, they can regulate the expression of nearby genes.
- Short Non-Coding RNAs (20-30 bp): These play a significant role in post-transcriptional gene silencing at the cytoplasmic level.

Historical Context of Small Non-Coding RNA Discovery

The discovery of small non-coding RNAs was somewhat serendipitous, illustrating how biological research can yield unexpected discoveries that significantly advance RNA biology.

Applications in Plant Genetics

Since the 1980s, researchers have applied traditional genetics and genetic transformation techniques to develop new plant varieties. A notable example being:
- The introduction of additional copies of the CHS gene in petunias, which resulted in more intense purple coloration, showcasing the relationship between gene copy number and phenotypic expression.

Transgene Induced Gene Silencing (TIGS)

Phenomenon: The addition of a transgene has the propensity to silence endogenous genes, causing a decrease in their mRNA transcripts. This silencing can propagate across generations and is heritable, as demonstrated in foundational work by Napoli et al. in 1990.

Case Study: C. elegans lin-4 Gene

Function: The lin-4 gene encodes a small non-coding RNA that regulates the transcription factor lin-14.
Mechanism of Action: lin-4 binds to conserved sequences located in the 3' UTR of lin-14 mRNA, resulting in the repression of lin-41 mRNA levels, illustrating how small RNAs can influence developmental transitions.

Discovery of RNA Interference (RNAi)

Mechanism: The phenomenon of RNA interference can be initiated by the injection of double-stranded RNA (dsRNA) that targets specific mRNA sequences for degradation.
Discovery: This process was first identified in the study of the C. elegans unc-22 gene where dsRNA led to observable phenotypic changes (twitching), helping to elucidate the mechanism of gene silencing.

Key Definitions

RNA Interference (RNAi): A biological process where small interfering RNAs (siRNAs) originating from long dsRNAs modulate gene expression through degradation of target mRNA or inhibition of its translation.
MicroRNAs (miRNAs): Endogenously expressed small RNA molecules, typically 21-23 nucleotides in length, derived from longer precursor sequences, that regulate mRNA stability and translation.

Steps in siRNA/miRNA-mediated Gene Silencing

MicroRNA Processing:
- The enzyme Drosha processes primary miRNAs (pri-miRNAs) into precursor miRNAs (pre-miRNAs) of approximately 70 nucleotides in length.
- The enzyme Dicer then cleaves pre-miRNAs, producing mature 21-23 nucleotide siRNAs.
siRNA Fragmentation:
- Dicer also cleaves longer dsRNA into short fragments suitable for regulatory function, allowing precise targeting of complementary mRNA.
RISC Incorporation:
- The RNA Induced Silencing Complex (RISC) integrates siRNAs, primarily incorporating Argonaute proteins, which are crucial for this process.
mRNA Inactivation:
- RISC binding to target mRNA results in either degradation of mRNA or inhibition of its translation, effectively silencing the gene.

Differences Between siRNA and miRNA-mediated Silencing

siRNA: Usually exhibits full complementarity with target mRNA, leading to complete degradation of the mRNA.
miRNA: Typically binds with partial complementarity, mainly promoting translational repression rather than degradation.

Role of miRNAs in Eukaryotes

Eukaryotic genomes contain hundreds of miRNAs that significantly influence gene regulation networks, particularly during crucial processes like development and tumor progression in cancer pathways.
Each miRNA can target multiple mRNAs, illustrating the intricate nature of gene regulatory mechanisms.

Tools for Studying Gene Function Using RNAi

RNA interference techniques enable comprehensive gene function screening through knockdown methodologies utilizing siRNA libraries. However, a concern remains regarding potential off-target effects associated with the use of short hairpin RNAs (shRNAs).

C. elegans as a Model for RNAi

Research Model: C. elegans is an extensively utilized model organism for RNAi studies due to its high efficiency with various delivery methods, including direct injection and feeding with bacteria expressing dsRNA.
Genetic variability among various C. elegans mutants also enhances sensitivity to RNAi, providing insights into the genetic regulation of RNAi responses.

Summary: Take-Home Points

Small non-coding RNAs, particularly siRNAs and miRNAs, are essential regulators of mRNA levels and gene expression.
Mechanisms involve binding to mRNA’s 3' UTR, leading to translational inhibition or degradation.
miRNAs undergo processing from larger precursors through the actions of Drosha and Dicer.
The RISC complex plays a critical role in the targeted mRNA silencing process, with significant implications for gene functional studies utilizing RNAi technology.