Non-coding RNA Regulation of Gene Expression: RNAi, microRNAs, and lncRNAs in Genetics - Nov 19

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Non-coding RNA Regulation of Gene Expression

Introduction to Non-coding RNAs

Non-coding RNAs (ncRNAs) are RNA molecules that do not encode proteins but play crucial roles in regulating gene expression. They include small interfering RNAs (siRNAs), microRNAs (miRNAs), and long non-coding RNAs (lncRNAs), each with distinct mechanisms and biological functions.

siRNAs and miRNAs are short, double-stranded RNAs involved in post-transcriptional gene silencing.
lncRNAs are longer RNA molecules (>200 nucleotides) that regulate gene expression at multiple levels, including chromatin modification and transcriptional control.
Other non-coding elements include transposons and repetitive DNA sequences.

Genomic Context of Non-coding DNA

The majority of eukaryotic genomes consist of non-coding DNA, which includes regulatory sequences, repetitive elements, and unique noncoding regions.

Category	Percentage of Genome	Description
Repetitive DNA (transposons & related)	44%	Includes transposable elements and related sequences
Introns & regulatory sequences	24%	Non-coding regions within genes and regulatory elements
Unique noncoding DNA	15%	Non-repetitive, noncoding regions
Repetitive DNA (unrelated to transposons)	15%	Other repetitive sequences
Exons (coding for protein/rRNA/tRNA)	1.5%	Protein-coding and functional RNA genes

RNA Interference (RNAi) and Gene Silencing

Discovery and Mechanism of RNAi

RNA interference (RNAi) is a potent and specific mechanism for gene silencing, first characterized in Caenorhabditis elegans. The process involves double-stranded RNA (dsRNA) triggering the degradation of complementary mRNA, thereby inhibiting gene expression.

Key Experiment: Injection of dsRNA into C. elegans led to gene-specific silencing, more effective than single-stranded RNA.
Hypothesis: dsRNA acts via an antisense mechanism to silence genes.
Findings: dsRNA is required for gene regulation; single-stranded sense or antisense RNA is not sufficient.
Implications: RNAi provides a tool for targeted gene knockdown and functional genomics.

Experimental Evidence: unc-22 Gene in C. elegans

The unc-22 gene is used as a model to study RNAi. Loss-of-function mutants exhibit a twitching phenotype proportional to unc-22 levels.

Experiment: Injection of different RNA types (sense, antisense, dsRNA) and assessment of twitching.
Result: Only dsRNA causes twitching, indicating gene silencing is specific to dsRNA.
Specificity: The effect is gene-specific but can be observed in multiple genes.

Gene	RNA Type Injected	Phenotype
unc-22	dsRNA	Strong twitching (KO)
unc-22	sense/antisense	No twitching
Other genes	dsRNA	Gene-specific silencing

Effect on mRNA Levels

RNAi leads to a reduction in target mRNA levels, as shown by probe-based detection in embryos. dsRNA injection results in decreased mRNA, while sense or antisense RNA does not.

Conclusion: dsRNA specifically degrades complementary mRNA, confirming the mechanism of RNAi.

Types and Functions of Regulatory RNAs

siRNA vs. microRNA (miRNA)

Both siRNAs and miRNAs are short, double-stranded RNAs that mediate gene silencing, but they differ in origin and function.

siRNA: Derived from exogenous sources (e.g., viruses, transposons, synthetic RNA); perfect complementarity to target mRNA.
miRNA: Encoded by endogenous genes; imperfect complementarity, often regulates multiple targets.
Key protein: Dicer is essential for processing both siRNAs and miRNAs.

Mechanism of Action

Both siRNAs and miRNAs are processed by Dicer and incorporated into the RNA-induced silencing complex (RISC) or RNA-induced initiation of transcription (RITS).

RISC: Mediates mRNA degradation or translation inhibition.
RITS: Involved in chromatin remodeling and transcriptional silencing.

Seed sequence: miRNAs typically bind to target mRNAs via a 6-8 nucleotide seed sequence.

Functions of RNAi Pathways

Transposon silencing: Prevents mobilization of transposable elements.
Viral defense: Protects cells from viral infection by degrading viral RNA.
Gene regulation: Fine-tunes gene expression during development and cellular responses.

Long Non-coding RNAs (lncRNAs) and Dosage Compensation

Role of lncRNAs

lncRNAs are involved in diverse regulatory processes, including X-chromosome inactivation and dosage compensation.

X-inactivation: lncRNAs such as Xist coat the X chromosome, leading to its silencing in female mammals.
Dosage compensation: Ensures equal expression of X-linked genes between males and females.

Experimental Identification of Non-coding RNAs

Genetic Screens and Epistasis Analysis

Forward genetic screens in C. elegans have identified regulatory RNAs involved in developmental timing (heterochrony). The Lin-4 and Lin-14 genes are key examples:

Lin-4 mutants: Fail to initiate neuronal structures.
Lin-14: Codes for a protein involved in neuron development; Lin-14 mutations are epistatic to Lin-4.
miRNA identification: Lin-4 mapped to a region without protein-coding genes, but small RNAs (~21 bp) were found, complementary to Lin-14 mRNA.

Experimental Approaches to Validate miRNAs

Knockout (KO) Dicer: Loss of Dicer disrupts miRNA processing.
CRISPR/Cas9 mutation: Targeted mutation in miRNA sequence.
KO Drosha: Disrupts miRNA maturation.
Injecting dsRNA: Not a good method for validating endogenous miRNAs.

Summary Table: Comparison of siRNA, miRNA, and lncRNA

Type	Length	Origin	Function	Key Protein
siRNA	~21-23 nt	Exogenous (virus, transposon, synthetic)	mRNA degradation, gene silencing	Dicer
miRNA	~21-23 nt	Endogenous (genomic)	Translational repression, mRNA degradation	Dicer
lncRNA	>200 nt	Endogenous	Chromatin modification, transcriptional regulation	Varies (e.g., Xist)

Key Equations and Concepts

Gene silencing by RNAi:

Seed sequence binding: miRNA binds target mRNA via 6-8 bp seed region.

Additional info:

RNAi and non-coding RNAs are central to modern genetic research, providing tools for gene function analysis and therapeutic applications.
Epistasis experiments help determine genetic pathways and regulatory hierarchies.