Revelation 18 4 4 And I heard another

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. © 2000 Timothy G. Standish

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

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

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

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

Processing Eukaryotic m. RNA 5’ Untranslated Region 3’ Untranslated Region Protein Coding Region 5’ 5’ G 3’ Int. 3 Exon 1 Int. 1 Exon 22 Exon 3 AAAAA 3’ 5’ Cap . nt I 1 . nt I 2 3’ Poly A Tail RNA processing achieves three things: 1 Removal of introns 2 Addition of a 5’ cap 3 Addition of a 3’ tail This signals the m. RNA is ready to move out of the nucleus and may control its lifespan in the cytoplasm © 2000 Timothy G. Standish

Introns are intervening sequences that “interrupt” eukaryotic genes and must be removed before uninterrupted exons coding for proteins leave the nucleus as m. RNA 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 © 2000 Timothy G. Standish

Nuclear Intron Splicing Exon/intron junctions have short but wellconserved 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 t. RNA 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 © 2000 Timothy G. Standish

Nuclear Intron Splicing 5’ Ex 1 GU AG In 1 5’ Mutation in AG to AA Ex 1 In 1 5’ GU Ex 1 Ex 2 AA Ex 2 5’ Mutation in GU to UU Ex 1 In 1 5’ UU AG Ex 2 5’ AG Ex 2 Ex 1 UU In 1 GU Ex 1 In 2 AG Ex 3 3’ 3’ Ex 3 GU Ex 3 AG In 2 Ex 3 3’ © 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 hn. RNA 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 © 2000 Timothy G. Standish

Common Splicing Mechanism Exon 1 5’ Intron GU A Left (donor) 5’ splice site Branch site 18 -40 BP AG Exon 2 3’ Right (acceptor) 3’ splice site U A C U A A C (Yeast) Py 80 NPy 80 Py 87 Pu 75 APy 95 (Animal-Subscripts indicate percent frequency) The branch sequence allows identification of the 3’ splice site © 2000 Timothy G. Standish

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

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

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

Common Splicing Mechanism Yee ha! Lariat Formation Intron U A G Exon 2 AG 3’ 3’ on Ex 1 5’ © 2000 Timothy G. Standish

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

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

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

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

The Spliceosomes are structures that form within the nucleus to remove introns from eukaryotic hn. RNA This structure is large, on the order of a ribosome subunit Like the ribosome, spliceosomes are composed of both protein and RNA © 2000 Timothy G. Standish

© 2000 Timothy G. Standish