mRNA processing
- 3. 1 Introduction Many of the RNA molecules in bacteria and virtually all RNA molecules in eukaryotes are processed to some degree after synthesis
- 4. 1 Introduction • mRNAs are synthesized by either cellular or viral enzymes • RNA processing- a series of covalent modifications • Facilitate recognition of mRNAs by the protein synthesizing machinery • Most RNA-processing reactions were discovered in viral systems
- 5. Principles of Virology, ASM Press 1 Introduction
- 7. 2 5’Capping Principles of Virology, ASM Press
- 8. 2 5’Capping • Protects mRNAs from 5’ exonucleolytic attack • Essential for the efficient translation of most mRNAs • de novo synthesis by cellular enzymes, synthesis by viral enzymes and acquisition of preformed 5’ cap
- 9. 2 5’Capping Synthesis by cellular enzymes • Pre mRNA are substrates for cellular capping enzymes • RNA pol II • Cotranscriptional reaction that takes place when nascent RNA is only 20 to 30 nucleotides in length • Phosphorylation of paused RNA pol II at specific serines in the C-terminal domain of the largest subunit is the signal for binding of the capping enzyme and capping of nascent RNA
- 10. 2 5’Capping Principles of Virology, ASM Press
- 11. 2 5’Capping Acquisition of viral 5’Cap from cellular mRNA • 5’ caps of orthomyxoviral and bunyaviral mRNAs are produced by cellular capping enzymes • Viral cap dependent endonucleases cleave cellular transcripts to produce the primers needed for viral mRNA synthesis- cap snatching • The 5’ terminal segments and caps of influenza virus mRNAs are obtained from cellular pre-mRNA in nucleus • Bunyaviral mRNA synthesis is primed with 5’ terminal fragments cleaved from mature cellular mRNAs in cytoplasm
- 12. 3 Polyadenylation Addition of poly(A) tail to 3’ end
- 13. 3 Polyadenylation History Principles of Virology, ASM Press
- 14. 3 Polyadenylation • Stability and increases the efficiency of translation • 3’ terminal stemloop structures instead poly(A) tail in mRNAs that encode histones and also in case of reoviral and arenaviral mRNAs • Carried out by either cellular or viral enzymes
- 15. 3 Polyadenylation By cellular Enzymes • Transcription of a gene proceeds beyond the site at which poly(A) tail is added • The 3’ end of the mRNA is determined by endonucleolytic cleavage of its pre-mRNA • Poly(A) is then added to the new 3’ terminus • Termination of transcription • Degradation of RNA downstream to the cleavage site
- 16. 3 Polyadenylation Principles of Virology, ASM PressLehninger principles of Biochemistry, WH Freeman
- 17. 3 Polyadenylation By Viral Enzymes • Either posttranscriptional or during viral mRNA synthesis • Vaccinia virus- 2 processes with one enzyme • Other viruses- during mRNA synthesis • Poly(U) sequence present at the 5’ end of the (-) strand RNA template Principles of Virology, ASM Press
- 18. 4 Splicing Non-coding regions are removed and coding regions are joined together
- 19. 4 Splicing Discovery • Nuclear precursors of mRNAs are larger than mRNAs transported in cytoplasm and heterogenous in size • Both ends of hnRNA retained • 1993- Phillip Sharp and Richard Roberts • Adenoviral late mRNAs
- 20. 4 Splicing Principles of Virology, ASM Press
- 21. 4 Splicing 4 classes of introns • Group I: Found in some nuclear, mitochondrial and chloroplast genes that code for rRNAs, mRNAs and tRNAs • Group II: Found in the primary transcripts of mitochondrial or chloroplast mRNAs in fungi,algae and plants • Rare examples of introns found in bacteria
- 22. 4 Splicing Lehninger principles of Biochemistry, WH Freeman
- 23. 4 Splicing Group III Introns • Largest class • Found in nuclear mRNA primary transcripts • Spliceosomal introns • Splicing by lariat mechanism • Spliceosome is made up of specialized RNA-protein complexes: snRNPs
- 24. 4 Splicing Lehninger principles of Biochemistry, WH Freeman
- 25. 4 Splicing Lehninger principles of Biochemistry, WH Freeman
- 26. 4 Splicing Differential RNA processing Lehninger principles of Biochemistry, WH Freeman
- 27. 4 Splicing Principles of Virology, ASM Press
- 28. 5 Conclusion
- 29. 5 Conclusion • Eukaryotic mRNAs are modified by addition of a 7- methylguanosine residue at the 5’ end and by cleavage and polyadenylation at 3’ end • Introns are removed by splicing • Group I introns require guanosine cofactor • Some group I and group II introns are capable of self splicing • Group III introns are spliced with the aid of RNA-protein complexes called snRNPs • Complex transcripts can have either more than one site for cleavage and polyadenylation or both
- 30. 6 Reference •Flint, Racaniello, 2015, Principles of virology, 4th edition •David L Nelson, M Cox, 2013, Lehninger Principles of Biochemistry, sixth edition, W H Freeman and Company
- 31. THANK YOU!