Non-Coding RNAs (Ch. 17)

Introduction
  - Some genes do not encode polypeptides but are transcribed into non-coding RNAs (ncRNAs).
  - Estimates of the number of ncRNAs in humans range from several thousand to tens of thousands.
  - In most cell types, ncRNAs are more abundant than messenger RNAs (mRNAs).
  - In a typical human cell:
    - Approximately 20% of transcription involves the production of mRNAs.
    - Approximately 80% of transcription is associated with making ncRNAs.

Binding Interactions
- ncRNAs can bind to different types of molecules.
- RNA molecules can form stem-loop structures, which may bind to pockets on the surface of proteins.
Examples of Abundant ncRNAs
- The notes do not list specific examples but indicate a focus on those which are abundant in cellular contexts.

Scaffold
- A ncRNA binds a group of proteins, organizing them into a larger complex.
Guide
- A ncRNA binds to a protein and directs it to a specific site in the cell.
Alteration of Protein Function or Stability
   - A ncRNA can bind to a protein and alter:
     - The structure of the protein.
     - The ability of the protein to act as a catalyst.
     - The ability of the protein to bind to another molecule.
     - The stability of the protein.
Ribozyme
- RNA molecules that possess catalytic function.
Blocker
- An ncRNA physically prevents or blocks a cellular process from occurring.
Decoy
- An ncRNA recognizes another ncRNA and sequesters it, preventing its action.

MicroRNAs (miRNAs):
- miRNA binds to mRNA and inhibits translation.
- miRNA can bind to a decoy ncRNA, allowing translation of the mRNA to occur, demonstrating how decoy ncRNAs can modulate regulatory processes.

Categories by Length
  - Long non-coding RNAs (lncRNAs):
    - Longer than 200 nucleotides.
  - Small regulatory RNAs (short ncRNAs):
    - Shorter than 200 nucleotides, including microRNAs, typically 20-25 nucleotides long.

Emergence of Living Cells
  - Living cells may have originated from more primitive structures called protobionts.
  - Protobionts consisted of aggregates of molecules and macromolecules with a boundary (e.g., lipid bilayer).
  - They maintained a distinct internal chemical environment.
Characteristics of Protobionts
  - A boundary that separates internal contents from the external environment.
  - Polymers inside capable of information storage and having catalytic functions.
  - Candidates for self-replication.

Scientists suggest RNA was the first macromolecule in protobionts.
The RNA World period featured RNA molecules, but not DNA or proteins.
Functions of RNA included:
  1. Information storage via nucleotide base sequences.
  2. Self-replication as a ribozyme, using RNA templates for complementary RNA synthesis.
  3. Catalytic activity, synthesizing polypeptides and other organic molecules.
Evolution from RNA to DNA
  - RNA performed information storage and enzymatic functions.
  - DNA allowed RNA to bind cofactors, include modified bases, or bind peptides enhancing catalytic function.
  - DNA is predicted to be a more stable molecule for information storage.
  - Ancestral RNA molecules might have had the ability to synthesize DNA using RNA as a template.

The emergence of proteins as catalysts conferred significant advantages to early cells.
- Proteins can perform diverse functions, such as structural roles in cytoskeletal and membrane proteins.
Modern RNA's role in protein synthesis includes messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and even ribozymes, aligning with evolutionary history.

Definition:
- RNA interference is a regulatory mechanism found in most eukaryotes, mediated by ncRNAs, particularly microRNAs (miRNAs) and small interfering RNAs (siRNAs).
MicroRNAs (miRNAs):
   - These are transcribed from endogenous eukaryotic genes and regulate gene expression.
   - A single miRNA can inhibit translation of multiple mRNAs due to partial complementarity.
   - It’s estimated that up to 60% of human genes may be regulated by miRNAs.
Small Interfering RNAs (siRNAs):
- Typically derived from exogenous sources, such as viral infections or experimental treatments.

Transcription:
   - The synthesis of pri-miRNA, which folds into a hairpin recognized by Drosha and DGCR8.
   - Cleaved to form a pre-miRNA and exported from the nucleus.
Processing:
- The pre-miRNA is cut by dicer into 20 to 25 base pair products, miRNA or siRNA.
Formation of RISC (RNA-Induced Silencing Complex):
- Involves degradation of one strand of the double-stranded RNA.
- RISC recognizes specific mRNAs through complementary sequences.
Action Mechanism:
- siRNAs typically lead to complete degradation of their target mRNA, while miRNAs often inhibit translation without degrading mRNA.

CRISPR-Cas System:
  - A defensive mechanism found in prokaryotes against bacteriophages, plasmids, and transposons involving ncRNAs.
  - Type II system of CRISPR is discussed.
  - PIWI-interacting RNA (piRNA) interacts with PIWI proteins and inhibits transposable elements' movements.
Components of the CRISPR-Cas System:
- Found in prokaryotic chromosomes (CRISPR locus) first recognized in 1993.
- Contains repeated sequences and segments derived from bacteriophage DNA.
Phases of CRISPR-Cas Defense Mechanism:
  1. Adaptation Phase:
     - Involves acquisition of spacers from previous bacteriophage infections, integrating segments of bacteriophage DNA into CRISPR gene.
  2. Expression Phase:
     - After adaptation, the CRISPR, tracr, and Cas9 genes are expressed, creating pre-crRNA and tracrRNA that interact.
  3. Interference Phase:
     - The specific complementary interactions guide the Cas9 protein to cleave invading DNA, inhibiting phage proliferation.

Growing interest in CRISPR technology for gene editing within living cells.
The single guide RNA (sgRNA) links tracrRNA and crRNA, guiding the Cas9 protein to the target gene for editing.
Repair options after Cas9 cleavage:
1. Nonhomologous end joining (NHEJ): Typically results in small indels leading to gene inactivation.
2. Homologous recombination repair (HRR): Requires the presence of a donor DNA template carrying desired mutations.
Applications:
- CRISPR-Cas technology is utilized across various organisms including mice, human cell lines, and plants, showcasing its versatility and potential in genetic research and modification.