POST-TRANSCRIPTIONAL GENE SILENCING PATHWAYS

Post-transcriptional gene silencing (PTGS) refers to the process by which gene expression is suppressed at the RNA level. This mechanism plays a crucial role in regulating gene expression and maintaining cellular homeostasis. PTGS pathways involve several key molecular processes, including RNA interference (RNAi), microRNA (miRNA) regulation, and small interfering RNA (siRNA) pathways.

1) RNA Interference (RNAi)

RNAi is a conserved biological process that regulates gene expression by targeting specific mRNAs for degradation or translational repression. The pathway involves the generation of small RNA molecules, typically 21-23 nucleotides in length, which guide the RNA-induced silencing complex (RISC) to complementary target mRNAs. Once bound, RISC cleaves the target mRNA or inhibits its translation, leading to gene silencing.

2) MicroRNA (miRNA) Regulation

MiRNAs are small non-coding RNAs that play a critical role in post-transcriptional gene regulation. These molecules are transcribed as primary miRNAs, which are processed into precursor miRNAs and eventually mature miRNAs. Mature miRNAs then associate with the RISC complex to target specific mRNAs for degradation or translational repression, thereby influencing gene expression.

3) Small Interfering RNA (siRNA) Pathway

The siRNA pathway shares similarities with the miRNA pathway but is triggered by exogenous double-stranded RNA (dsRNA) or endogenous hairpin RNAs. Dicer enzymes process long dsRNAs into siRNAs, which are then loaded onto the RISC complex to guide sequence-specific mRNA degradation or translational inhibition.

 

Generating Knockout Cell Lines

Knockout cell lines are valuable tools for studying gene function and understanding the molecular mechanisms underlying various biological processes. The generation of knockout cell lines involves disrupting specific genes within a cell’s genome, typically using techniques such as CRISPR/Cas9-mediated genome editing or homologous recombination.

1) CRISPR/Cas9-Mediated Genome Editing

CRISPR/Cas9 has revolutionized the field of genetic engineering by enabling precise and efficient genome editing. This technology utilizes a guide RNA (gRNA) to direct the Cas9 nuclease to a specific genomic locus, where it induces double-strand breaks. Subsequent repair processes can lead to gene knockout through non-homologous end joining (NHEJ) or targeted gene replacement via homology-directed repair (HDR).

2) Homologous Recombination

Homologous recombination is another method for generating knockout cell lines, particularly when precise gene targeting is required. This approach involves introducing a DNA construct containing homologous sequences to the target gene locus, promoting recombination events that result in gene disruption or replacement.

 

Signaling Pathways

Cellular signaling pathways orchestrate a wide range of biological processes by transmitting extracellular signals to intracellular effectors, ultimately regulating cellular responses such as growth, differentiation, and metabolism.

 

Subcellular Protein Relocalization

Subcellular protein relocalization refers to the dynamic movement of proteins within cells, often in response to various stimuli or cellular conditions. This process plays a critical role in modulating protein function and influencing diverse cellular functions, including signal transduction, vesicular trafficking, and organelle dynamics.