DIAGNOSTIC GENOMICS Lecture 6: Expression of the Genome – miRNAs and RNA Interference

DIAGNOSTIC GENOMICS Lecture 6: Expression of the Genome – miRNAs and RNA Interference

Learning Objectives

  • Compare Functional vs Regulatory RNAs

  • Summarize RNA-mediated repression of gene expression – RNAi

  • Describe RNAi molecules

  • Define microRNA (miRNA) and describe their structure/characteristics

  • Outline biological synthesis of miRNA

  • Describe the molecular activities of miRNAs

  • Discuss examples of dysregulated miRNA activity and disease

Functional and Regulatory RNAs

  • Both functional and regulatory RNAs are expressed in the genome.
      - Functional RNAs:
        - Directly involved in biochemical activities.
        - Types include:
          - mRNA (messenger RNA): Codes for proteins.
          - tRNA (transfer RNA): Required for codon recognition by ribosome; delivers the correct amino acid during translation.
          - rRNA (ribosomal RNA): Interacts with mRNA and tRNA; catalyzes peptide bond formation during translation.
      - Regulatory RNAs:
        - Direct or coordinate the activities of other biomolecules.
        - Types include:
          - lncRNA (long non-coding RNA): Acts as molecular scaffolds that interact with RNA-binding proteins to regulate activity.
          - miRNA (microRNA): A type of regulatory RNA involved in gene regulation.
          - siRNA (short interfering RNA): Involved in RNA interference.
          - piRNA (piwi-interaction RNA): Related to transposon silencing and germline development.

Regulatory RNAs

  • Approximately 30% to 70% of genes in higher eukaryotes are regulated by RNA activities.
      - Control the fate of other molecules, compared to a conductor leading an orchestra.
      - Functions include:
        - Development
        - Cellular homeostasis
        - Protection from viruses and transposons.

  • Characteristics:
      - Length (long vs. short)
      - Origin/Mechanism of generation
      - Regulatory mechanism employed to target(s).

Small RNAs

  • Small RNAs repress or "silence" expression of homologous genes through various mechanisms:
      - Translation Inhibition
      - mRNA Turnover
      - Transcriptional Silencing of target promoters.
      - Involvement in RNA interference (RNAi).

Types of Small RNAs

  • siRNA (short interfering RNA):
      - Length: 21-23 nucleotides (nt).
      - Prepared artificially, mainly used as research tools (potential therapeutics).

  • miRNA (microRNA):
      - Length: Usually 21-22 nt.
      - Derived from longer precursor RNAs; regulates gene expression.

miRNA Production Overview

  • miRNA Synthesis Process:
      - Begins with a long primary transcript known as pri-miRNA.
      - Cleavage occurs in two steps:
        1. First Cleavage: Occurs in the nucleus via Drosha, producing pre-miRNA.
        2. Second Cleavage: Occurs in the cytoplasm via Dicer, resulting in mature miRNA of ~22 nt.

Characteristics of pri-miRNA

  • Pri-miRNA Transcripts:
      - Can be transcribed by RNA polymerase II (Pol II) or RNA polymerase III (Pol III).
      - Capped and polyadenylated; often contains introns but typically does not code for proteins (non-coding RNA).
      - Subject to similar transcriptional and post-transcriptional mechanisms of regulation as mRNA.

Structure of pri-miRNA

  • May contain pre-miRNA(s) at various locations along its length:
      - Possible locations:
        - Coding (exonic) regions
        - Non-coding regions
        - Intronic regions
        - 3' UTR (untranslated regions).

Production of Multiple miRNAs

  • A single pri-miRNA can give rise to multiple miRNAs.
      - Example from Caenorhabditis elegans showing seven different miRNAs (miR-35, miR-36, miR-37, miR-38, miR-39, miR-40, miR-41) generated from a single precursor transcript.

Small RNAs and Target Gene Expression Inhibition

  • Small RNAs inhibit target gene expression via the RNA-Induced Silencing Complex (RISC).

  • Three modes of action for gene inhibition:
      - Trigger Destruction of mRNA Target: Shortens its half-life.
      - Inhibit Translation of mRNA.
      - Induce Chromatin Modifications: Silencing transcription of target genes.

RISC Complex

  • RISC Structure:
      - Complex of RNA and protein molecules that repress the expression of specific targets.
      - Single-stranded miRNA acts as a complementary guide to its target sequence.

  • Significant components include members of the Argonaute family.

  • Functionality:
      - If target is highly homologous, as is common with siRNA, RNA target is degraded.
      - Argonaute serves as the catalytic unit to perform "mRNA slicing." If no mRNA remains, no protein is produced.
      - For targets with less perfect complementarity (more common with miRNAs), the translation is inhibited, which may lead to sequestration of the mRNA targets into P-bodies within the cytoplasm.

RISC Effects on Chromatin

  • RISC can recruit chromatin remodeling proteins to target promoters, resulting in repression via reduced histone acetylation.

  • Localization is mediated via the guide sequence, targeting complementary regions to miRNA.

miRNA/Target Sequence Complementarity

  • Base pairing between miRNA and its target is the foundation for RNAi specificity.

  • Base pairing initiated at “seed” sequences within the miRNA:
      - The region of highest homology occurs at bases 2-9 of the 22 nt miRNA.
      - This complementarity can be employed to search for similar sequences in potential target genes.

Target Locations in mRNA

  • Predominantly located in the 3' UTRs of mRNAs.

  • miRNAs may target multiple binding sites within the same mRNA or engage different sites across different mRNAs.