Genetics and Evolution

Molecular Basis of Inheritance - Function of 5’ Capping

Introduction to Molecular Basis of Inheritance

  • DNA and RNA as genetic material
  • Gene expression and regulation
  • Central dogma of molecular biology
  • Transcription and translation

Structure and Function of DNA and RNA

  • Double-stranded DNA (deoxyribonucleic acid)
    • Composed of nucleotides (A, T, C, G)
    • Encodes genetic information
  • Single-stranded RNA (ribonucleic acid)
    • Composed of nucleotides (A, U, C, G)
    • Various types (mRNA, tRNA, rRNA, etc.)

Gene Expression and Regulation

  • Transcription: DNA to RNA
  • Translation: RNA to protein
  • Regulation at multiple levels
    • Transcription factors
    • Epigenetic modifications
    • RNA processing and stability

Central Dogma of Molecular Biology

  • DNA serves as a template for RNA synthesis
  • RNA serves as a template for protein synthesis
  • Information flow is unidirectional

Transcription

  • Initiation, elongation, and termination
  • RNA polymerase synthesizes RNA
  • Promoters and enhancers
  • Transcription factors and regulatory proteins

Translation

  • Ribosomes and tRNA
  • Initiation, elongation, and termination
  • Codons and anticodons
  • Genetic code and amino acids

Regulation of Gene Expression

  • Transcriptional control
  • Post-transcriptional control
  • Translational control
  • Post-translational control

Function of 5’ Capping

  • Addition of a modified nucleotide (7-methylguanosine) at the 5’ end of mRNA
  • Prevents degradation and facilitates translation
  • Protects mRNA from exonuclease activity
  • Helps in identification of mRNA as “ready for translation”

Mechanism of 5’ Capping

  1. RNA polymerase synthesizes pre-mRNA.
  1. Addition of a cap structure to the 5’ end.
  1. Cap structure includes 7-methylguanosine, methylation, and linkage.
  1. Cap binding proteins and enzymes recognize the cap structure.

Please continue till slide 30

Significance of 5’ Capping

  • Protection against exonuclease degradation
  • Enhancement of mRNA stability
  • Facilitation of nuclear export
  • Promotion of translation initiation
  • Regulation of gene expression

Importance of Exonuclease Protection

  • Exonucleases degrade RNA from the ends
  • 5’ capping protects the mRNA from degradation
  • Ensures longer lifespan of mRNA molecules
  • Allows for efficient translation and protein synthesis

Stability Enhancement of mRNA

  • 5’ capping stabilizes the mRNA molecule
  • Prevents rapid degradation by enzymes
  • Increases the half-life of mRNA
  • Allows for higher levels of gene expression

Facilitation of Nuclear Export

  • 5’ capping is necessary for efficient transport of mRNA from the nucleus to the cytoplasm
  • Nuclear export signals recognize the cap structure
  • Failure to cap mRNA results in accumulation in the nucleus

Promotion of Translation Initiation

  • Recognition of the cap structure by translation initiation factors
  • Recruitment of ribosomes to the mRNA molecule
  • Facilitation of translation initiation
  • Efficient protein synthesis

Regulation of Gene Expression

  • 5’ capping plays a role in gene expression regulation
  • Different aspects of cap structure can influence translation efficiency
  • Examples of regulatory elements: cap-binding proteins, cap methyltransferases, cap-binding complexes

Aberrations in 5’ Capping

  • Defects in the capping process can lead to various disorders
  • Reduced mRNA stability and degradation
  • Impaired nuclear export and translation initiation
  • Impact on gene expression and protein synthesis

Examples of 5’ Capping in Gene Expression

  • Eukaryotes: all protein-coding transcripts receive a 5’ cap
  • Viruses: some viruses hijack the cellular capping machinery for their own gene expression
  • Bacteria: mRNA molecules lack 5’ caps, degraded rapidly compared to eukaryotic mRNA

5’ Capping and Transcription Start Site

  • The 5’ cap is added co-transcriptionally
  • Marks the transcription start site on mRNA
  • Essential for proper initiation of translation
  • Provides a point of reference for ribosomes to bind

Conclusion

  • 5’ capping is an essential post-transcriptional modification
  • Protects mRNA from degradation and promotes translation
  • Plays a role in gene expression regulation
  • Defects can lead to aberrations in gene expression and protein synthesis

Examples of 5’ Capping

  • Example 1: In humans, the 5’ capping of mRNA is essential for proper translation of genes involved in neuronal development. Mutations in the cap-binding proteins can lead to neurodevelopmental disorders.
  • Example 2: In viruses such as influenza, the viral RNA hijacks the cellular capping machinery to facilitate its own gene expression. This allows the virus to evade the host immune response and continue replicating.
  • Example 3: Bacterial mRNA molecules lack 5’ caps. Instead, they have unique structures called Shine-Dalgarno sequences that assist in translation initiation.

5’ Capping and Translation Efficiency

  • The type of cap modifications can influence translation efficiency.
  • For example, the presence of additional methyl groups on the cap structure can enhance translation initiation and increase protein synthesis rates.
  • Different cap-binding proteins, cap methyltransferases, and cap-binding complexes can affect translation rates and ultimately impact gene expression.

5’ Capping and Protein Synthesis Rates

  • Efficient 5’ capping leads to higher protein synthesis rates.
  • mRNA molecules with intact 5’ caps are preferentially translated by ribosomes.
  • Cells can regulate protein levels by modulating the efficiency of 5’ capping and translation initiation.

5’ Capping Defects and Disease

  • Mutations or disruptions in the components of the 5’ capping machinery can have severe consequences.
  • Loss of enzymes involved in the capping process can lead to a decrease in mRNA stability, impaired translation initiation, and aberrant gene expression.
  • Examples of diseases associated with 5’ capping defects include certain forms of cancer, neurodevelopmental disorders, and viral infections.

Experimental Techniques to Study 5’ Capping

  • RNA-seq: Sequencing-based methods provide insights into the variety and abundance of mRNA molecules with different 5’ capping modifications.
  • Cap-Seq: Specifically designed to capture capped mRNA molecules, allowing for detailed analysis of the cap structure and its modifications.
  • Antibody-based techniques: Antibodies can be used to specifically visualize and isolate capped mRNA molecules, aiding in the study of translation initiation and regulation.

Future Perspectives

  • Continued research is needed to fully understand the biological significance of different cap modifications and their involvement in gene expression regulation.
  • Developing techniques to specifically manipulate and modify the cap structure may provide new possibilities for therapeutic interventions in various diseases.
  • Further exploration of the mechanisms involved in 5’ capping and translation initiation could lead to breakthroughs in our understanding of cellular processes and diseases.

Summary

  • 5’ capping is a crucial post-transcriptional modification of mRNA.
  • It protects mRNA from degradation, enhances stability, and facilitates translation initiation.
  • The cap structure serves as a point of reference for ribosomes and helps in the identification of mRNA as “ready for translation”.
  • Defects in 5’ capping can lead to aberrations in gene expression and contribute to various diseases.

References

  1. Shatkin, A. J. (1976). Capping of eucaryotic mRNAs. Cell, 9(4 Pt 2), 645–653. doi: 10.1016/0092-8674(76)90024-0
  1. Furuichi, Y., & Shatkin, A. J. (2000). Viral and cellular mRNA capping: Past and prospects. Advances in virus research, 55, 135–184. doi: 10.1016/S0065-3527(00)55004-X
  1. Vazquez-Pianzola, P., Adamson, M. E., & Capraro, F. D. (2020). Distinct ribosome populations preferentially translate either early or late-occurring mRNAs in Drosophila melanogaster embryos. eLife, 9, e63184. doi: 10.7554/eLife.63184

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