Week 5 - p2 - L3 -MicroRNA and siRNA: Mechanisms, Regulation, and Therapeutic Applications

MicroRNAs (miRNAs) identify and interact with specific messenger RNAs (mRNAs) primarily within the three prime untranslated region (3' UTR) of the mRNA, located after the end of the open reading frame.
The binding orientation between the miRNA and the mRNA is anti-parallel; the $5'$ end of the miRNA aligns with the target sequence in the mRNA.
The most critical determinant for target recognition is the seed region, which consists of nucleotides $2$ to $8$ of the miRNA.
The seed region typically demonstrates complete complementarity to its target mRNA sequence.
The bulge region follows the seed region; it is characterized by little to no complementarity. This structural bulge can manifest on either the miRNA side or the mRNA side.
The three prime complementary region of the miRNA has a highly variable amount of complementarity to the mRNA.
Predicting miRNA targets is challenging due to the low complexity of searching for matching sequences of only $7$ nucleotides across the genome.

Additional complementary regions outside the seed region play a vital role in defining the effectiveness of binding and the subsequent suppression of mRNA function.
A single miRNA can suppress multiple different genes or gene products because the seed sequence is found in multiple mRNAs.
Conversely, a single mRNA can possess multiple binding sites for different miRNA targets, allowing for highly coordinated gene expression.

The mature miRNA is bound by an Argonaute protein (Argo).
The Argonaute protein contains a cleft where the miRNA sits. It scans the entire transcriptome to find RNAs that can pair with the miRNA seed sequence.
Once a sufficiently strong binding is established, the target RNA is retained within the RNA-induced silencing complex (RISC).
The target RNA then undergoes translational regulation or degradation.
GW182 is a large scaffolding protein that binds directly to the Argonaute protein. It serves as a docking site for other proteins that execute the miRNA's effects on the target RNA.
The CCR4-NOT deadenylase complex is recruited to the site. Its functions include:
- Shortening the poly(A) tail of the mRNA.
- Destabilizing the poly(A) binding protein (PABP).
- Facilitating mRNA degradation.
- Inhibiting the formation of the closed-loop model of translation.

MicroRNAs can block translation through several distinct mechanisms:
- Initiation Block: This prevents the small ribosomal subunit from attaching to the mRNA and scanning to find the ATG start codon. Translation is stopped before it begins.
- Post-initiation/Elongation Block: The ribosome successfully forms and begins protein production, but elongation is halted. The ribosome then disassembles from the RNA, and any produced protein fragment is degraded.
Translational repression by miRNAs is reversible. If the miRNA is removed, the initiation block is released, and the mRNA can be translated again to produce protein.
Alternative to repression, the mRNA can be destroyed via decapping-mediated decay:
- Deadenylases attack the 3' end of the mRNA.
- Decapping enzymes attack the $5'$ cap.
- Once the cap is removed, the RNA is susceptible to $5'$ to $3'$ degradation.
- When the poly(A) tail is degraded, the RNA is susceptible to $3'$ to $5'$ degradation.

The biogenesis pathway involves:
1. Transcription of a miRNA gene to produce a primary miRNA (pri-miRNA) product.
2. Processing by the microprocessor complex (including Drosha).
3. Export from the nucleus via Exportin $5$ .
4. Processing by the Dicer complex to produce the mature miRNA.
MicroRNAs are critical for coordinating gene expression, particularly during development. Defects in this pathway are linked to cancer and other diseases.
Oncogenes (cancer-promoting genes) must be tightly regulated. If miRNA processing is defective (e.g., in transcription, processing, or export), lower levels of mature miRNA result in the overproduction of oncogenic proteins, driving tumorigenesis.
Tumor Suppressors act as brakes on cell proliferation. If miRNAs that target tumor suppressors are amplified or overproduced (due to genomic instability or efficient transcription factors/processing), the tumor suppressor protein levels drop, leading to over-proliferation of cells.

LET-7 is a highly conserved miRNA found in organisms ranging from worms to humans.
It plays an essential role in coordinating cellular proliferation levels.
In lung cancer studies, patients with tumors showing moderate expression of LET-7 have higher survival rates compared to those with low LET-7 levels.
Dysregulation of LET-7, whether through production or processing inhibition, results in more aggressive tumors and negative patient outcomes.
Small RNA sequencing using next-generation technologies shows that miRNA expression profiles can often serve as diagnostic tools for various cancers.

Small inhibitory RNAs (siRNAs) are a related class of small RNAs, but they are generally not encoded in the genome; they are often used experimentally.
A fundamental difference lies in complementarity:
- MicroRNAs have partial complementarity to their targets.
- siRNAs have complete ( $100\%$ ) complementarity to their targets.
The mechanism of action for siRNA involves the RISC complex cutting the mRNA in half (cleavage), leading to its destruction.
miRNAs are endogenous and processed via the microprocessor complex and Drosha, whereas siRNAs are typically exogenous, added to cells as long double-stranded RNA pieces that are then processed by Dicer.

The discovery of RNA interference (RNAi) using siRNAs provided an unbiased discovery tool for knocking out genes one at a time across species ( $C. elegans$ , flies, mice, humans) to observe phenotypes.
Biomedical Applications:
- Drug Discovery: Identifying proteins whose knockout leads to cell death in cancer cells, making them potential drug targets.
- Overcoming Drug Resistance: Identifying and targeting pathways that cancer cells use to survive initial therapies.
- Network Perspective: Knocking down multiple genes simultaneously to study signaling and biochemical pathways in combination.
FDA-approved siRNA drugs now exist which utilize complete complementarity to cleave target mRNAs and reduce protein production.
A major ongoing challenge in synthetic RNA therapy is the delivery of siRNAs into specific tissues of interest with high efficacy.

Antisense oligonucleotides (ASOs) are another type of small RNA used to modify RNA splicing rather than inducing cleavage.
Duchenne Muscular Dystrophy (DMD) Example:
- Patients often have a deletion of exon $52$ , which shifts the reading frame and results in non-functional protein.
- An ASO can be used to bind to exon $51$ in the primary transcript.
- This causes exon $51$ to be skipped during splicing.
- The resulting mature transcript lacks exons $51$ and $52$ but restores the reading frame for exons $53$ and $54$ .
- This allows the production of a functional protein, providing positive clinical effects for patients.