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Revelation 18:4

Revelation 18:4 4 And I heard another voice from heaven, saying, Come out of her, my people, that ye be not partakers of her sins, and that ye receive not of her plagues. RNA Processing And Nuclear Splicing. Timothy G. Standish, Ph. D. Introduction The Central Dogma of Molecular Biology.

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Revelation 18:4

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  1. Revelation 18:4 4And I heard another voice from heaven, saying, Come out of her, my people, that ye be not partakers of her sins, and that ye receive not of her plagues.

  2. RNA ProcessingAnd Nuclear Splicing Timothy G. Standish, Ph. D.

  3. IntroductionThe Central Dogma of Molecular Biology Reverse tanscription DNA Transcription Ribosome mRNA Translation Polypeptide (protein) Cell

  4. Eukaryotic Transcription Cytoplasm Nuclear pores AAAAAA AAAAAA DNA Transcription RNA RNA Processing G G mRNA Export Nucleus

  5. A “Simple” Eukaryotic Gene Transcription Start Site 3’ Untranslated Region 5’ Untranslated Region Introns 5’ 3’ Int. 1 Int. 2 Exon 1 Exon 2 Exon 3 Promoter/ Control Region Terminator Sequence Exons RNA Transcript 5’ 3’ Int. 1 Int. 2 Exon 1 Exon 2 Exon 3

  6. Processing Eukaryotic mRNA 5’ Untranslated Region 3’ Untranslated Region 5’ 3’ G AAAAA Exon 1 Exon 2 Exon 3 5’ Cap 3’ Poly A Tail 5’ 3’ Int. 1 Int. 2 Exon 1 Exon 2 Exon 3 Int. 1 Int. 2 Protein Coding Region • RNA processing achieves three things: • Removal of introns • Addition of a 5’ cap • Addition of a 3’ tail • This signals the mRNA is ready to move out of the nucleus and may control its life span in the cytoplasm

  7. Introns • Introns are intervening sequences that “interrupt” eukaryotic genes and must be removed before uninterrupted exons coding for proteins leave the nucleus as mRNA • Three types of intron are known: • Group I introns - Found in organelle and bacterial genes along with some lower eukaryotes nuclear genes • Can self splice without the aid of proteins • Require free GTP for splicing • Group II introns - Found in organelle and bacterial genes • Can self splice without the aid of proteins • Differ from Group I introns in sequence and mechanism • Nuclear introns - Found in eukaryotic nuclear genes • Require proteins and other RNAs for splicing

  8. Nuclear Intron Splicing • Exon/intron junctions have short but well conserved consensus sequences • The generic sequence of an intron is: • GTNN . . . NNAG in DNA or GUNN . . . NNAG in RNA • This sequence does not apply to the introns of organelles or yeast tRNA genes • Splice sites operate in pairs which are generic. Thus, if the end of one intron is mutated, that intron plus the following exon and next intron will be spliced out • The splicing apparatus is usually not tissue specific

  9. Nuclear Intron Splicing 3’ 3’ 5’ 5’ GU GU AG AA GU GU AG AG Ex 1 Ex 1 In 1 In 1 Ex 2 Ex 2 In 2 In 2 Ex 3 Ex 3 3’ 5’ Ex 1 Ex 2 Ex 3 Mutation in AG to AA 3’ 5’ Ex 1 Ex 3 Mutation in GU to UU 3’ 5’ UU AG GU AG Ex 1 In 1 Ex 2 In 2 Ex 3 3’ 5’ UU AG Ex 2 Ex 3 Ex 1 In 1

  10. Splicing Order • Some gene transcripts have been shown to loose their introns in a consistent order • The current model says that the hnRNA adopts different conformations after specific introns are removed thus making other introns available for removal • Thus, the removal of introns does not proceed sequentially along the transcript

  11. Common Splicing Mechanism Intron Exon 1 Exon 2 GU A AG 5’ 3’ 18-40 BP Left (donor) 5’ splice site Right (acceptor) 3’ splice site Branch site U A C U A A C (Yeast) Py80NPy80Py87Pu75APy95 (Animal-Subscripts indicate percent frequency) The branch sequence allows identification of the 3’ splice site

  12. Common Splicing Mechanism Folding O A HO P O A AG 3’ O G U O O O P O O OH P O O OH O OH P O O OH 5’ O N Exon 1 G O O O O P O OH P O O O U

  13. Common Splicing Mechanism Lariat Formation O A HO P O O O O - + O P O O OH - P O - + O OH O - OH - P O O OH O N Exon 1 G O O O O P O OH P O O O U Transesterification reaction between 2’hydroxyl group on adenine in the branch site and phosphate connecting intron with exon 1

  14. Common Splicing Mechanism Lariat Formation O A HO P O O O O O P OH O O P O O OH O P O O O HO OH G O O O O P O OH P O O N O U Exon 1

  15. Common Splicing Mechanism Lariat Formation Yee ha! Intron Exon 2 A AG 5’ 3’ 3’ G U Lariat Lariat l l Exon 1

  16. Common Splicing Mechanism Lariat Removal N G O A HO P O OH - O OH Intron O OH O O - - O P O O P O + P O - Exon 2 HO + HO - HO HO O OH O O N P O O Exon 1 A second nucleophilic transesterification reaction, this time between 3’ hydroxyl group on nucleotide 1 in exon 1 and the phosphate connecting intron 2 with exon 2

  17. Common Splicing Mechanism Lariat Removal N N OH O OH O Exon 1 O O P O O P O P O O Exon 2 HO HO HO A second nucleophilic transesterification reaction, this time between 3’ hydroxyl group on nucleotide 1 in exon 1 and the phosphate connecting intron 2 with exon 2

  18. Common Splicing Mechanism A AG G U Intron lariat Exon 1 Exon 2 5’ 3’ Following excision, the lariat is rapidly degraded

  19. Common Splicing Mechanism Exon 1 Exon 2 5’ 3’ Following excision, the lariat is rapidly degraded

  20. The Spliceosome • Spliceosomes are structures that form within the nucleus to remove introns from eukaryotic hnRNA • This structure is large, on the order of a ribosome subunit • Like the ribosome, spliceosomes are composed of both protein and RNA

  21. The End

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