Questions with Answers on “RNA Splicing and Alternative Splicing Mechanisms”

1. What is RNA splicing, and why is it essential in eukaryotic gene expression?

Answer:
RNA splicing is the process by which non-coding sequences (introns) are removed from pre-mRNA, and coding sequences (exons) are joined together to form a mature mRNA molecule. This step occurs during the post-transcriptional modification of RNA.

Importance:

1. Formation of Functional mRNA: Splicing ensures the formation of mature mRNA capable of being translated into functional proteins.
2. Gene Expression Regulation: Proper splicing is essential for accurate gene expression and cellular function.
3. Error Prevention: Defects in splicing can lead to diseases such as cancer and neurodegenerative disorders.

2. Describe the structure and function of the spliceosome in RNA splicing.

Answer:
Structure:
The spliceosome is a large ribonucleoprotein complex composed of five small nuclear ribonucleoproteins (snRNPs), namely U1, U2, U4, U5, and U6, along with associated proteins.

Function:

1. Recognizes conserved splice sites at the 5′ and 3′ ends of introns.
2. Catalyzes the removal of introns via two transesterification reactions.
3. Joins adjacent exons to create a continuous coding sequence.

3. Explain the steps involved in the RNA splicing process.

Answer:

1. Recognition of Splice Sites: The 5′ splice site, 3′ splice site, and branch point are recognized by snRNPs.
2. Assembly of the Spliceosome: U1 binds the 5′ splice site, and U2 binds the branch point. U4/U6 and U5 join to form the active spliceosome.
3. First Transesterification Reaction: The 5′ splice site is cleaved, and the 5′ end of the intron forms a lariat structure by attaching to the branch point.
4. Second Transesterification Reaction: The 3′ splice site is cleaved, and exons are ligated together.

4. What are the conserved sequences required for RNA splicing?

Answer:

1. 5′ Splice Site: A conserved GU sequence at the intron’s 5′ end.
2. 3′ Splice Site: A conserved AG sequence at the intron’s 3′ end.
3. Branch Point: An adenine (A) residue located upstream of the 3′ splice site.
4. Polypyrimidine Tract: A stretch of pyrimidines between the branch point and the 3′ splice site.

These sequences are recognized by the spliceosome during the splicing process.

5. Define alternative splicing and its significance in gene expression.

Answer:
Definition:
Alternative splicing is a process that allows a single pre-mRNA transcript to be spliced in multiple ways, producing diverse mRNA isoforms and protein variants.

Significance:

1. Protein Diversity: Increases the complexity of the proteome without increasing the genome size.
2. Tissue-Specific Expression: Enables the generation of tissue-specific proteins.
3. Regulation of Gene Function: Controls protein function and activity in response to environmental signals.

6. What are the different types of alternative splicing?

Answer:

1. Exon Skipping: An exon is skipped in some mRNA isoforms.
2. Mutually Exclusive Exons: One of two exons is included, but not both.
3. Alternative 5′ Splice Site: Different 5′ splice sites are selected.
4. Alternative 3′ Splice Site: Different 3′ splice sites are selected.
5. Intron Retention: An intron is retained in the mRNA.

7. How is splicing regulated in cells?

Answer:

1. Splicing Factors: Proteins like SR proteins and hnRNPs bind to enhancer or silencer sequences to regulate splicing.
2. Enhancers and Silencers: Cis-regulatory elements in the RNA, such as exonic splicing enhancers (ESEs) and intronic splicing silencers (ISSs).
3. Signal Transduction: Cellular signaling pathways can influence splicing factor activity.

8. Discuss the role of introns in alternative splicing.

Answer:

1. Regulation: Introns contain regulatory elements like enhancers and silencers.
2. Evolutionary Advantage: Introns provide a platform for the evolution of new protein-coding sequences.
3. mRNA Stability: Introns can affect the stability and localization of mRNA.

9. What is exon skipping, and how does it affect gene expression?

Answer:
Definition:
Exon skipping occurs when specific exons are excluded from the mature mRNA.

Impact:

1. Protein Function: Produces protein isoforms with altered structures and functions.
2. Disease Association: Errors in exon skipping can lead to diseases like Duchenne muscular dystrophy.

10. Describe the consequences of splicing errors.

Answer:

1. Disease: Mutations in splicing sites or factors can lead to diseases like cancer, spinal muscular atrophy, and retinitis pigmentosa.
2. Protein Dysfunction: Mis-spliced mRNA may produce non-functional or harmful proteins.
3. Gene Expression Alteration: Splicing errors can disrupt the normal regulation of gene expression.

11. What is the role of SR proteins in splicing?

Answer:
SR proteins are splicing factors that bind to exonic splicing enhancers (ESEs) to promote exon inclusion. They help recruit the spliceosome and enhance splicing fidelity.

12. Explain intron retention and its functional significance.

Answer:
Definition:
Intron retention occurs when introns are retained in the mature mRNA.

Significance:

1. Regulatory Role: May regulate mRNA localization, stability, or translation.
2. Functional Proteins: In some cases, retained introns can encode functional protein domains.

13. How do mutations in splice sites affect gene expression?

Answer:

1. Skipping of Exons: Leads to loss of critical protein domains.
2. Activation of Cryptic Sites: Can introduce new, aberrant splice sites.
3. Loss of Function: Results in non-functional proteins or mRNA degradation.

14. What is trans-splicing, and where does it occur?

Answer:
Trans-splicing is the process by which exons from two different RNA molecules are joined together. It is observed in some lower eukaryotes, such as trypanosomes.

15. How is splicing studied experimentally?

Answer:

1. In vitro Splicing Assays: Using purified spliceosomes and RNA substrates.
2. Mutagenesis Studies: Analyzing effects of mutations in splice sites or regulatory elements.
3. Bioinformatics Tools: Predicting splicing patterns and regulatory sequences.

16. What is the significance of the branch point in splicing?

Answer:
The branch point is critical for the formation of the lariat intermediate, which is necessary for intron removal.

17. Discuss the evolution of alternative splicing.

Answer:
Alternative splicing has evolved to increase proteome complexity without enlarging the genome. It provides a mechanism for functional innovation and adaptability.

18. How do external factors influence alternative splicing?

Answer:

1. Stress: Alters splicing factor activity.
2. Temperature: Can affect splice site recognition.
3. Drugs: Certain drugs modulate splicing in diseases.

19. What are cryptic splice sites?

Answer:
Cryptic splice sites are non-canonical sequences that can be used for splicing due to mutations or misrecognition by the spliceosome.

20. How is splicing linked to therapeutic applications?

Answer:

1. Gene Therapy: Correcting splicing defects using antisense oligonucleotides.
2. Cancer Treatment: Targeting splicing regulators in cancer therapy.
3. Rare Diseases: Treating splicing-related genetic disorders.