1. ©2000 Timothy G. Standish 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. ©2000 Timothy G. Standish Nuclear Splicing Timothy G. Standish, Ph. D.

3. mRNA Transcription Introduction The Central Dogma of Molecular Biology Cell Polypeptide (protein) Translation Ribosome Reverse transcription DNA

4. ©2000 Timothy G. Standish DNA Cytoplasm Nucleus Eukaryotic Transcription Export G AAAAAA RNA Transcription Nuclear pores G AAAAAA RNA Processing mRNA

5. ©2000 Timothy G. Standish A “Simple” Eukaryotic Gene Terminator Sequence Promoter/ Control Region Transcription Start Site RNA Transcript 5’ Untranslated Region 3’ Untranslated Region Exons Introns 3’5’ Exon 2Exon 3 Int. 2 Exon 1 Int. 1 3’5’ Exon 2Exon 3Exon 1 Int. 2Int. 1

6. ©2000 Timothy G. Standish 3’5’ Exon 2Exon 3 Int. 2 Exon 1 Int. 1 Processing Eukaryotic mRNA Protein Coding Region 3’ Untranslated Region5’ Untranslated Region 3’ AAAAA 3’ Poly A Tail 5’ G 5’ Cap Exon 2 Exon 3Exon 1 Int. 2Int. 1 RNA processing achieves three things:  Removal of introns  Addition of a 5’ cap  Addition of a 3’ tail l This signals the mRNA is ready to move out of the nucleus and may control its lifespan in the cytoplasm

7. ©2000 Timothy G. StandishIntrons 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: 1 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 2 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 3 Nuclear introns - Found in eukaryotic nuclear genes -Require proteins and other RNAs for splicing

8. ©2000 Timothy G. Standish Nuclear Intron Splicing Exon/intron junctions have short but well- conserved consensus sequences The generic sequence of an intron is: GT... AG in DNA or GU... AG 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. ©2000 Timothy G. Standish Mutation in GU to UU GU 3’ AG Ex 2Ex 3In 2 AG5’UU Ex 1In 1 Mutation in AG to AA Nuclear Intron Splicing 3’ Ex 2Ex 3 AG5’UU Ex 1In 1 3’ 5’ Ex 2Ex 3Ex 1 AGGU 3’ 5’AGGU Ex 2Ex 3In 2Ex 1In 1 3’ 5’ Ex 3Ex 1 AAGU 3’ 5’AGGU Ex 2Ex 3In 2Ex 1In 1

10. ©2000 Timothy G. Standish Splicing Order Some gene transcripts have been shown to lose 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. ©2000 Timothy G. Standish Common Splicing Mechanism Exon 2Exon 1 Intron AGAGU 3’5’ 18-40 BP Branch site Left (donor) 5’ splice site Right (acceptor) 3’ splice site Py 80 NPy 80 Py 87 Pu 75 A Py 95 (Animal-Subscripts indicate percent frequency) U A C U A A C (Yeast) The branch sequence allows identification of the 3’ splice site

12. ©2000 Timothy G. Standish AGA G U 3’ 5’ Common Splicing Mechanism Folding U OH O O O O P G O O O O O P O O O P N O O O O P A O HO O O OP O OH O P Exon 1

13. ©2000 Timothy G. Standish Exon 1 Common Splicing Mechanism G OHO O O O OP O O O P A O HO O O OP O OH O P N O O O O P U O O O O P + + - - - - - Lariat Formation Transesterification reaction between 2’hydroxyl group on adenine in the branch site and phosphate connecting intron with exon 1

14. ©2000 Timothy G. Standish Common Splicing Mechanism G OHO O O O OP O O O P A O HO O O OP O OH O P U O O O O P N O O O HO P O Lariat Formation Exon 1

15. ©2000 Timothy G. Standish Common Splicing Mechanism Lariat Formation Exon 2 AAG G U 3’ 5’ 3’ Exon 1 Intron Lariat Yee ha! Lariat

16. ©2000 Timothy G. Standish + + Common Splicing Mechanism Lariat Removal Exon 1 N OH O O O HO P O - - - - - 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 Exon 2 A O HO O O O P G O O O P N O O O P O O P OH Intron

17. ©2000 Timothy G. Standish Common Splicing Mechanism Lariat Removal 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 Exon 2 O N O HO O O P N O O O P O O P OH Exon 1

18. ©2000 Timothy G. Standish Common Splicing Mechanism 3’ Exon 2Exon 1 5’ A AG G U Intron lariat Following excision, the lariat is rapidly degraded

19. ©2000 Timothy G. Standish Common Splicing Mechanism 3’ Exon 2Exon 1 5’ Following excision, the lariat is rapidly degraded

20. ©2000 Timothy G. Standish 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. ©2000 Timothy G. Standish