hnRNA Processing II Pre mRNA Splicing Lecture 5 Part B SPLICING Need for PreBmRNA Splicing introns consItute 90 of vertebrate genes exons must be joined accurately in order to produce translatable mRNA introns exons must join accurately to make translatable mRNA hnRNA Processing Splicing INTRONS Discovery discovered via electron process of dicovery microscopy EM DNA mRNA hybrid examined by EM gene segments DISPLACED looped out DBloops DBloop segment of gene w o its corresponding sequence in the mRNA 1 Isolate mature mRNA 2 BaseCpair mRNA to DNA of gene 3 Prepare DNA for visualizaIon 4 Observe via EM DNA mRNA hybrid examined w EM DCloops displaced looped out no sequence in mRNA Genome Introns Discovery DBLOOP Importance of Discovery vertebrate genes SPLIT preBmRNA loses info i e introns during maturaIon proteinCcoding genes NOT coClinear w mRNA vertebrate genes split preCmRNA loses info i e introns mRNA DNA not coClinear w mRNA Genome Introns DCLoop Why Do We Have Split Genes exon shu ing w introns b tw exons chance of recombining exons increases b c exons are further apart from one another evoluIonary Ime scale alternaUve splicing splicing paiern of introns varies allowing one preCmRNA to give rise to mulIple proteins happening as we speak i e in real Ime features of a proteinCcoding gene regulatory DNA sequences introns exons exon shu ing alternaIve splicing features of proteinCcoding gene Genome Introns Exons Split Genes EXON SHUFFLING evoluIonary Ime scale occurs DNA level genome contains a repertoire of exons exons from di erent genes may be combined to form a new gene exon shu ing could produce proteins w evoluIonary DNA level exons from di erent genes combined to form new gene exon shu ing generate proteins w di erent funcIons LDL receptor di erent funcIons EX LDL receptor has 18 exons 9 related to EGF preCcursor 5 related to C9 complement 3 unique having introns b tw exons increases likelihood of exon shu ing DNA level combine exons to form new gene makes proteins w di erent funcIons LDL receptor Genome Exon Shu ing real Ime scale 1 preCmRNA gives rise to mulIple ALTERNATIVE SPLICING proteins real Ime 1 preCmRNA mulIple proteins exons from 1 preCmRNA joined in di erent combos cell type decides combo of exons used Ctropomyosin gene used exons from 1 preCmRNA are joined in di erent combos cell type speci es combo of exons EX Ctropomyosin gene 1 tropomyosin gene gives rise to several di erent tropomyosin proteins 1 preCmRNA mulIple proteins exons joined in various combos combo of exons decided by cell type Genome AlternaIve Splicing SPLICEOSOME Where Splicing Occurs spliceosome 50SC60S complex of RNA protein contains snRNAs bases proteins snRNAs U1 U2 U4 U5 U6 complexed to proteins in snRNPs spliceosome 50SB60S size of a ribosome subunit a complex of RNA protein contains snRNAs Small Nuclear RNAs 100C300 bases PROTEINS 100 di erent proteins snRNAs U1 U2 U4 U5 U6 U4 U5 U6 triple snRNP snRNAs complexed to proteins in snRNPs Small Nuclear Ribonucleoproteins 50SC60S RNA protein snRNAs U1 U2 U4 U5 U6 bases proteins snRNPs hnRNA Processing Splicing Spliceosome 2 boundaries splice sites SS 5 SS 3 SS INTRONS In Vertebrate PreBmRNA 4 features 2 boundaries splice sites SS 5 SS 3 SS 4 features GUBAG GU 5 SS AG 3 SS branch point upstream of 3 SS polyBpryrimidine region downstream of branch point 2 1 GUBAG rule GU 5 SS AG 3 SS branch point CUPurAPyr i e CUA GAC U 20C50 bases upstream of 3 SS polyBpyrimidine region pPyr 15 bases or longer downstream of branch point 3 splice sites 5 SS 3 SS 4 features GUCAG branch point polyCpyrimidine region hnRNA Processing Splicing Introns PREBmRNA SPLICING Overview DO NOT need ATP b c G is low transphosphorylaIon break 1 PO4 bond but also make 1 PO4 bond so it s net neutral exonC1 is free w 3 OH group 5 3 bonds 2 3 5 OH aiacks 3 SS lariat gets degraded graphic of overview of preCmRNA splicing hnRNA Processing Splicing SPLICING Factor Recruitment U1 U2 U5 U4 U6 assemble sequenIally on preCmRNA U2AF U2 Accessory Factor binds pPyr tract U2AF BBP Branch Point Binding Protein cooperaIvely bind preCmRNA both U2AF BBP needed for U2 snRNP to associate w branch point U1 snRNA binds 5 SS summary U1 5 SS U2 branch point U1 U2 U5 U4 U6 assemble on preCmRNA U2AF BBP for U2 U1 binds 5 SS hnRNA Processing Splicing Factor Recruitment U1 U2 ON PREBmRNA snRNAs Base Pair to PreBmRNAs U1 pairs w 5 SS U2 binds branch point invariant 2 branch point A nucleoIde bulges out U1 binds 5 SS U2 binds branch point hnRNA Processing Splicing U1 U2 on PreCmRNA TRIPLE snRNP U456 Recruitment U6 girl comes to dance w U4 boy U5 other guy friend tags along recruitment of triple snRNP U456 hnRNA Processing Splicing Triple snRNP U456 SPLICEOSOMES Dynamic Macromolecular Structures protein preBmRNA preCmRNA snRNA interacIons RNABRNA hybrids form unwound new ones form RNA helicases ATP important protein preCmRNA preCmRNA snRNA RNACRNA hybrids RNA helicases ATP hnRNA Processing Splicing Flexibility of InteracIons U6 U2 RecogniUon of PreBmRNAs Dynamic U6 displaces U1 from 5 SS U2 replaces BBP branch point 5 SS 2 snRNAs branch point 1 protein 1 snRNA U1 U2 U5 U4 U6 assemble on preCmRNA U2AF BBP for U2 U1 binds 5 SS hnRNA Processing Splicing U6 U2 U6 U2 U6 Pairs w Either U4 or U2 But NOT Both same nonaCmer used to form 2 RNACRNA hybrids RNA helicase important U2 pairs w both U6 branch point in spliceosome U6 pairs w U2 releases U4 U4 is kicked out of triple snRNP b c U6 is w U2 now U6 comes to dance w U4 but dumps U4 for U2 dance U2 pairs w U6 branch point spliceosome U6 pairs w U2 releases U4 hnRNA Processing Splicing U6 U2 U6 U2 At AcUve Site U6 pairs w U2 displaces U4 U6 posiIoned near branch point U6 binds 5 SS displaces U1 e ect 5 SS brought close U5 takes care of this exon doesn t let it get away to branch point 3 RNA molecules baseIpairing enzyme acCvity U6 pairs w U2 displaces U4 U6 binds 5 SS displaces U1 hnRNA Processing Splicing U6 U2 3 OH on 2 aiacks 5 SS to make branch point MechanisUc Stages of PreBmRNA Splicing Top Image intron is circularized exonC1 is released Boeom Image exons are joined lariat is excised 5 OH aiacks 3 SS joining the exons releasing the lariat intron circularized exonC1 released exons joined lariat excised hnRNA Processing Splicing 2 Stages of cis Splicing TRANSESTERIFICATIONS Splicing Involves Transesteri caUons 2 OH of A branch point aiacks phosphodiester of G 5 SS intron circularized branch exonC1 released has free point 3 OH 3 OH of exonC1 reacts w 3 SS exons