Questions with Answers on “MicroRNAs: Role in Post-Transcriptional Regulation”

1. What are microRNAs, and how do they function in post-transcriptional gene regulation?

Answer:
Definition: MicroRNAs (miRNAs) are small, non-coding RNAs, typically 20-24 nucleotides long, that regulate gene expression post-transcriptionally.

Function:

1. Transcription and Processing:
   • miRNAs are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II or III.
   • Pri-miRNAs are processed in the nucleus by Drosha and DGCR8 into precursor miRNAs (pre-miRNAs).
2. Export and Maturation:
   • Pre-miRNAs are exported to the cytoplasm by Exportin-5 and further processed by Dicer into mature miRNA duplexes.
3. Target Regulation:
   • Mature miRNAs are incorporated into the RNA-induced silencing complex (RISC), where they guide the complex to complementary target mRNAs, leading to either degradation or translational repression.

2. Discuss the biogenesis pathway of microRNAs.

Answer:
Biogenesis Pathway:

1. Transcription:
   • miRNAs are transcribed from miRNA genes as pri-miRNAs by RNA polymerase II or III.
2. Nuclear Processing:
   • Pri-miRNAs are cleaved by the Drosha-DGCR8 complex to form pre-miRNAs (~70 nucleotides long).
3. Export to Cytoplasm:
   • Pre-miRNAs are exported via Exportin-5 and Ran-GTP.
4. Cytoplasmic Processing:
   • Dicer cleaves pre-miRNAs into mature miRNA duplexes (~22 nucleotides).
5. RISC Loading:
   • The guide strand of the duplex is incorporated into RISC, where it directs the complex to target mRNAs.

3. Explain the role of the Drosha-DGCR8 complex in miRNA processing.

Answer:

• Location: Found in the nucleus.
• Function: Processes primary miRNAs (pri-miRNAs) into precursor miRNAs (pre-miRNAs).
• Mechanism:
  • Drosha, an RNase III enzyme, cleaves the pri-miRNA at specific sites.
  • DGCR8 acts as a cofactor, recognizing the hairpin structure of pri-miRNAs.
    This step is essential for generating pre-miRNAs for further cytoplasmic processing.

4. What is the significance of Dicer in miRNA maturation?

Answer:

• Role: Dicer is a cytoplasmic RNase III enzyme crucial for converting pre-miRNAs into mature miRNA duplexes.
• Mechanism:
  • It recognizes the loop structure of pre-miRNAs and cleaves them into ~22-nucleotide duplexes.
  • One strand (the guide strand) is incorporated into RISC, while the passenger strand is degraded.

5. How do miRNAs identify their target mRNAs?

Answer:

• Mechanism: miRNAs recognize target mRNAs through sequence complementarity between their seed region (nucleotides 2-8 of the miRNA) and the target’s 3′ untranslated region (3′ UTR).
• Outcome: Depending on the degree of complementarity, miRNAs either:
  • Induce mRNA degradation (perfect match).
  • Suppress translation (partial match).

6. Discuss the role of Argonaute proteins in miRNA function.

Answer:

• Role in RISC: Argonaute proteins are the core components of the RNA-induced silencing complex (RISC).
• Functions:
  1. Bind to the guide strand of miRNA.
  2. Facilitate target mRNA recognition and cleavage (for perfect matches).
  3. Inhibit translation by stalling ribosomes (for imperfect matches).

7. Describe the dual regulatory role of miRNAs in cancer.

Answer:

• Tumor Suppressors:
  • miRNAs downregulate oncogenes. For example, let-7 targets RAS oncogene mRNA.
• Oncogenes:
  • miRNAs, like miR-21, suppress tumor suppressor genes, leading to cancer progression.
• Therapeutic Implications: Understanding miRNA dysregulation offers opportunities for targeted cancer therapies.

8. Explain the seed region of miRNAs and its significance in target recognition.

Answer:

• Definition: The seed region is a conserved 7-8 nucleotide sequence at the 5′ end of miRNAs.
• Significance:
  • Critical for binding target mRNAs.
  • Determines specificity of target recognition.
  • Mutations in the seed region can alter miRNA function and lead to diseases.

9. How do miRNAs regulate gene expression in plants and animals?

Answer:

• In Animals:
  • Bind to 3′ UTR of mRNAs, leading to translational repression or mRNA degradation.
• In Plants:
  • Bind with near-perfect complementarity to mRNA coding regions, causing direct cleavage of target mRNAs.

10. Discuss the clinical significance of miRNAs in neurodegenerative diseases.

Answer:

• Roles:
  • miRNA dysregulation contributes to Alzheimer’s, Parkinson’s, and Huntington’s diseases.
  • For example, miR-29 is implicated in preventing amyloid-beta plaque formation in Alzheimer’s.
• Potential Therapies: miRNA-based treatments could restore normal gene expression.

11. How are miRNAs involved in developmental biology?

Answer:

• Functions:
  • Regulate genes involved in cell differentiation, proliferation, and apoptosis.
  • For example, miR-430 in zebrafish clears maternal mRNAs during embryogenesis.

12. What are the therapeutic applications of miRNAs?

Answer:

• Applications:
  • miRNA Mimics: Restore lost miRNA function in diseases.
  • miRNA Inhibitors: Block overactive miRNAs.
  • Cancer Therapy: Target oncogenic miRNAs.

13. Describe the methods used to study miRNA functions.

Answer:

• Techniques:
  1. Microarray and RNA sequencing to identify miRNA expression profiles.
  2. Luciferase reporter assays to validate miRNA-mRNA interactions.
  3. CRISPR/Cas9 to knock out miRNA genes.

14. What is the role of miRNAs in stem cell differentiation?

Answer:

• miRNAs, like miR-145, regulate the balance between self-renewal and differentiation in stem cells by targeting pluripotency genes like OCT4 and SOX2.

15. How does Exportin-5 facilitate miRNA processing?

Answer:

• Exportin-5 recognizes pre-miRNAs in the nucleus and transports them to the cytoplasm using Ran-GTP as an energy source.

16. Discuss the relationship between miRNAs and metabolic disorders.

Answer:

• Dysregulated miRNAs, like miR-33, affect lipid metabolism, insulin sensitivity, and obesity, contributing to metabolic diseases.

17. What are circular RNAs (circRNAs), and how do they interact with miRNAs?

Answer:

• CircRNAs act as miRNA sponges, sequestering miRNAs and preventing them from binding to their target mRNAs.

18. Explain the concept of miRNA sponges in gene regulation.

Answer:

• miRNA sponges are RNA molecules that bind miRNAs, reducing their availability to target mRNAs, thereby modulating gene expression.

19. What challenges are associated with miRNA-based therapies?

Answer:

• Challenges:
  1. Delivery of miRNAs to specific tissues.
  2. Off-target effects.
  3. Stability of miRNAs in vivo.

20. How does miRNA dysregulation lead to immune system disorders?

Answer:

• Dysregulated miRNAs, such as miR-155, alter cytokine production and immune cell differentiation, leading to autoimmune diseases like lupus.