Splicing RNA: MechanismsSplicing of Group I and II intronsSelf-splicing in pre-rRNA in Tetrahymena : T. Cech et al. 1981Self-splicing by a phosphoester transfer mechanismA catalytic activity in Group I intronGroup I intron catalyzes cleavage and nucleotide additionThe intron folds into a particular 3-D structureActive sites in Group I intron self-splicingDomains of the Group I intron ribozymeRNAs that function as enzymesHammerhead ribozymesDesign hammerhead ribozymes to cleave target RNAsMechanism of hammerhead ribozymePhosphotransfers for Group I vs. Group II & pre-mRNASplicing of pre-mRNAInitiation of phosphoester transfers in pre-mRNASplicing of pre-mRNA, step 1Splicing of pre-mRNA, step 2Investigation of splicing intermediatesRNase H + oligonucleotides complementary to different regions give very different productsAnalysis reveals a lariate structure in inter-mediateInvolvement of snRNAs and snRNPssnRNPsSm proteins may form ring around snRNAsPredicted structure of assembled Sm proteinsAssembly of spliceosomeSpliceosome assembly and catalysisCatalysis by U6/U2 on branch oligonucleotide in vitroRNA editingAddition of nucleotides by editingWhat is a gene?Mammalian example of editingSplicing RNA: MechanismsSplicing of Group I and II introns•Introns in fungal mitochondria, plastids, Tetrahymena pre-rRNA•Group I–Self-splicing–Initiate splicing with a G nucleotide–Uses a phosphoester transfer mechanism –Does not require ATP hydrolysis.•Group II–self-splicing–Initiate splicing with an internal A–Uses a phosphoester transfer mechanism–Does not require ATP hydrolysisSelf-splicing in pre-rRNA in Tetrahymena : T. Cech et al. 1981Exon 1 Exon 2Intron 1 Exon 1 Exon 2 Intron 1+pre-rRNASpliced exonIntron circleIntron linearpre-rRNANuclear extractGTP+ + + +- + - +- + + -•Products of splicing were resolved by gel electrophoresis:Additional proteinsare NOT needed forsplicing of this pre-rRNA!Do need a G nucleotide (GMP, GDP, GTP or Guanosine).Self-splicing by a phosphoester transfer mechanismExon 1Exon 2Intron 1Exon 1 Exon 2Intron 1+PPPPGOHUUGAPUPUPPGOHGAPN15 N16G AN15POHCircular intron+A catalytic activity in Group I intron•Self-splicing uses the intron in a stoichiometric fashion.•But the excised intron can catalyze cleavage and addition of C’s to CCCCCGroup I intron catalyzes cleavage and nucleotide addition5'pCCCCCC-OH+2 pCCCCC-OHpCCCC-OH + pCCCCCC-OHGGGAGG5'3'GOH5'pCCCCC-OHGGGAGG5'3'GOHC-5'pCCCCC-OHGGGAGG5'3'GOHGGGAGG5'GOHC-3'5'pCCCC-OHThe intron folds into a particular 3-D structure•Has active site for phosphoester transfer•Has G-nucleotide binding siteActive sites in Group I intron self-splicingGGGAGGGGGAGG5'GGGGAGG5'3'GOHGGGAGGG+G414G-OH3'CUCUCU5'G-binding siteSubstrate binding siteIGSex1ex24143'1st transferCUCUCU5'ex1GOHex2ex1ex2+4142nd transferUUUACCUG3rd transfer4145'GUUUACCUDomains of the Group I intron ribozymeRNAs that function as enzymes•RNase P•Group I introns•Group II introns•rRNA: peptide bond formation•Hammerhead ribozymes: cleavage•snRNAs involved in splicingHammerhead ribozymes•A 58 nt structure is used in self-cleavage•The sequence CUGA adjacent to stem-loops is sufficient for cleavage CUGAGACCGGGGCCAAAACUCGAGUCACCACUGGUGUBond that is cleaved.5'3'CUGA is required for catalysisDesign hammerhead ribozymes to cleave target RNAsCUGAGACCGGGGCCAAAACUCGUAAGAGUCACCACUGGUGUBond that is cleaved.5'3'substrate strandenzyme strandPotential therapy for genetic disease.Mechanism of hammerhead ribozyme•The folded RNA forms an active site for binding a metal hydroxide•Abstracts a proton from the 2’ OH of the nucleotide at the cleavage site. •This is now a nucleophile for attack on the 3’ phosphate and cleavage of the phosphodiester bond.Phosphotransfers for Group I vs. Group II & pre-mRNA2’GHO3’Exon 1Exon 2OHGOHExon 1+2GAHO2’Exon 1Exon 2OHAExon 1+22’ A++Group IGroup II and pre-mRNASplicing of pre-mRNA•The introns begin and end with almost invariant sequences: 5’ GU…AG 3’•Use ATP to assemble a large spliceosome•Mechanism is similar to that of the Group II fungal introns:–Initiate splicing with an internal A–Uses a phosphoester transfer mechanism for splicingInitiation of phosphoester transfers in pre-mRNA•Uses 2’ OH of an A internal to the intron•Forms a branch point by attacking the 5’ phosphate on the first nucleotide of the intron•Forms a lariat structure in the intron•Exons are joined and intron is excised as a lariat•A debranching enzyme cleaves the lariat at the branch to generate a linear intron•Linear intron is degradedSplicing of pre-mRNA, step 1Splicing of pre-mRNA, step 2Investigation of splicing intermediates5' 3'RNARNase H5'oligodeoxyribonucleotide+|||||In vitro splicing reaction: nuclear extracts + ATP+ labeled pre-mRNAResolve reaction intermediates and products on gels.Some intermediates move slower than pre-mRNA.Suggest they are not linear.Use RNase H to investigate structure of intermediate.RNase H cuts RNA in duplex with RNA or DNA.RNase H + oligonucleotides complementary to different regions give very different products5'3'precursor RNAexon 1intron exon 21 2 3 4splicing reactionexons joined in a linear molecule excised intron, non-linear moleculeMap of positions of oligodeoxyribonucleotides that annealed to different regions of the excised intron. This is not the structure of the excised intron.+5'5'5'3'3'3'3'3'+5'RNase HAnalysis reveals a lariate structure in inter-mediate5'3'precursor RNAexon 1intronexon 21234splicing reactionexcised intron, non-linear moleculeMap of positions of oligodeoxyribonucleotides that annealed to different regions of the excised intron. +5'5'3'3'3'3'3'+5'After annealing with the oligo, the heteroduplexes were treated with RNase HAnswer:exons joined in a linear molecule1234GUAGAoligo 1oligo 2oligo 3oligo 4Involvement of snRNAs and snRNPs•snRNAs = small nuclear RNAs•snRNPs = small nuclear ribonucleoprotein particles•Antibodies from patients with the autoimmune disease systemic lupus erythematosus (SLE) can react with proteins in snRNPs–Sm proteins•Addition of these antibodies to an in vitro pre-mRNA splicing reaction blocked splicing.•Thus the snRNPs were implicated in splicingsnRNPs•U1, U2, U4/U6, and U5 snRNPs–Have snRNA in each: U1, U2, U4/U6, U5–Conserved from yeast to human–Assemble into spliceosome–Catalyze splicing•Sm proteins bind “Sm RNA motif” in snRNAs–7 Sm proteins: B/B’, D1, D2, D3, E, F,