Chapter 17: Non-Coding RNAs

Non-coding RNAs (ncRNAs) play various roles in cellular processes by binding to different types of molecules.
  - Functions of ncRNAs:
    - Scaffold: ncRNAs have multiple binding sites that allow them to form complexes with proteins.
    - Guide: They can direct a molecule (e.g., another RNA or protein) to a specific location within the cell.
    - Decoy: ncRNAs can sequester (bind and inactivate) microRNAs (miRNAs), preventing them from regulating their target mRNAs.

Small non-coding RNAs are double-stranded RNA (dsRNA) molecules that are typically 20-31 nucleotides in length and contain short 3′ overhangs.
RNA Interference (RNAi): A mechanism by which sncRNAs silence post-transcriptional expressions of mRNAs in a sequence-specific manner. The discovery of RNAi by Andrew Fire and Craig Mello in 1998 demonstrated its potential for genetic interference.

Experiment by Andrew Fire et al.:
  1. RNA Injection: Two types of RNAs were injected into the gonads of Caenorhabditis elegans:
     - (c) mex-3 antisense RNA
     - (d) a combination of mex-3 sense and antisense RNA.
  2. In situ Hybridization: A labeled probe complementary to the mex-3 mRNA was added to embryos. If the cells expressed mex-3, the mRNA would bind the probe and become labeled. The result was a significant reduction in mex-3 mRNA expression when treated with the double-stranded RNA compared to the antisense RNA alone.
Controls:
  a. Negative control
  b. Endogenous mex-3 RNA
  c. Injected mex-3 antisense RNA
  d. Injected sense:antisense dsRNA

Two primary types:
1. MicroRNAs (miRNAs): Endogenous small RNA molecules that are involved in gene regulation.
2. Small Interfering RNAs (siRNAs): Exogenous small RNA molecules that also play crucial roles in silencing genes.
Mechanism of Action: For both miRNAs and siRNAs, the general mechanism of mRNA silencing leads to a gene knockdown, resulting in reduced mRNA levels or translation inhibition.
  - miRNA Pathway:
    1. The miRNA gene is transcribed to produce primary-miRNA.
    2. Drosha enzyme processes this to form pre-miRNA.
    3. Dicer enzyme further processes it to form mature miRNA.
    4. The RNA-induced silencing complex (RISC) binds to the ds-miRNA; Argonaute/Slicer removes one strand, retaining the other to guide RISC to complementary mRNA.
  - Outcomes of miRNA Binding:
    - Exact binding leads to mRNA degradation by RISC.
    - Partial binding leads to translation inhibition.
  - siRNA Pathway: Similar to the miRNA pathway but begins from the Dicer step.

Long non-coding RNAs are dsRNA molecules longer than 200 nucleotides.
Many functioning as decoy ncRNAs: for example, linc-MD1 acts as a sponge for miR-133 and miR-135 during muscle differentiation, allowing muscle regulatory factors (RTFs) to activate their target genes. In diseases like Duchenne muscular dystrophy, reduced amounts of linc-MD1 lead to overactivity of these miRNAs, inhibiting important translational processes.

TABLE 17.1 Examples:
  - Telomerase RNA component (TERC):
    - Role: DNA replication
    - Type: IncRNA
  - X inactive-specific transcript (Xist RNA):
    - Role: Chromatin structure, transcription
    - Type: IncRNA
  - Hox transcript antisense intergenic RNA (HOTAIR):
    - Role: Chromatin structure, transcription
    - Type: IncRNA
  - COLDAIR:
    - Role: Chromatin structure, transcription
    - Type: IncRNA
  - RNaseP RNA:
    - Role: Processing of tRNA molecules
    - Type: IncRNA
  - Small nuclear RNA (snRNA):
    - Role: Splicing
    - Type: Small regulatory RNA
  - Transfer RNA (tRNA):
    - Role: Translation
    - Type: Small regulatory RNA
  - Ribosomal RNA (rRNA):
    - Role: Translation
    - Note: Variable regulatory functions
  - MicroRNA (miRNA) and small-interfering RNA (siRNA):
    - Role: mRNA degradation
    - Type: Small regulatory RNAs
  - Small nucleolar RNA (snoRNA):
    - Role: RNA modification
    - Type: Small regulatory RNAs
  - Signal recognition particle RNA (SRP RNA):
    - Role: Protein targeting and secretion
    - Type: IncRNA
  - CRISPR RNA (crRNA):
    - Role: Genome defense
    - Type: Small regulatory RNA
  - PIWI-interacting RNA (piRNA):
    - Role: Genome defense
    - Type: Small regulatory RNA

Defensive Strategies Against Bacteriophages:
- Bacteria utilize various mechanisms, including restriction enzymes and the CRISPR-Cas system.

Five critical genes: tracr, Cas9, Cas1, Cas2, and Crispr.
CRISPR stands for clustered, regularly interspaced, short, palindromic repeats; consists of DNA sequences from bacteriophage infections that provide a form of immune memory.
Cas refers to CRISPR-associated proteins involved in processing CRISPR sequences and mediating immune responses against foreign DNA.

Adaptation (Spacer Acquisition):
- A fragment (20-50 bp) of bacteriophage DNA is integrated into the CRISPR sequence.
- Spacers reflect past infections and are inherited by resulting daughter cells.
Expression:
   - Following re-exposure to phage DNA, expression of the CRISPR gene leads to the formation of:
     - Pre-crRNA (a type of lncRNA)
     - tracrRNA
     - Cas9 protein
   - Function of tracrRNAs: These guide ncRNAs help transport crRNAs to Cas9.
Interference:
- Each crRNA's spacer is complementary to a strand of the phage DNA, allowing it to guide the tracrRNA-crRNA-Cas9 complex to bind to the invasive DNA strand.
- Action of Cas9: Cas9 functions to degrade the phage DNA, inhibiting further phage replication and protecting the bacterial cell.