Molecular biology - Chapter 14: Messenger rna processing I: Splicing

Molecular BiologyFourth EditionChapter 14Messenger RNA Processing I:SplicingLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.114.1 Genes in PiecesConsider the sequence of the human b-globin gene as a sentence:This is bhgty the human b-globin qwtzptlrbn gene.Two italicized regions make no senseContain sequences unrelated to the globin coding sequences surrounding themCalled intervening sequences, IVSsUsually called intronsParts of the gene making senseCoding regionsExonsSome lower eukaryotic genes have no introns2Evidence for Split GenesMost higher eukaryotic genes coding for mRNA and tRNA are interrupted by unrelated regions called intronsOther parts of the gene, surrounding the introns, are called exonsExons contain the sequences that finally appear in the mature RNA productGenes for mRNAs have been found with anywhere from 0 to 362 exonstRNA genes have either 0 or 1 exon3RNA SplicingIntrons are present in genes but not in mature RNAHow does the information not find its way into mature RNA products of the genes?Introns are never transcribedPolymerase somehow jumps from one exon to anotherIntrons are transcribedPrimary transcript result, an overlarge gene product is cut down by removing intronsThis is correct processProcess of cutting introns out of immature RNAs and stitching together the exons to form final product is RNA splicing4Splicing OutlineIntrons are transcribed along with exons in the primary transcriptIntrons are removed as the exons are spliced together5Stages of RNA SplicingMessenger RNA synthesis in eukaryotes occurs in stagesFirst stage:Synthesis of primary transcript productThis is an mRNA precursor containing introns copied from the gene if presentPrecursor is part of a pool of large nuclear RNAs – hnRNAs Second stage:mRNA maturationRemoval of introns in a process called splicing6Splicing SignalsSplicing signals in nuclear mRNA precursors are remarkably uniformFirst 2 bases of introns are GULast 2 are AG5’- and 3’-splice sites have consensus sequences extending beyond GU and AG motifsWhole consensus sequences are important to proper splicingAbnormal splicing can occur when the consensus sequences are mutated714.2 Mechanism of Splicing of Nuclear mRNA PrecursorsIntermediate in nuclear mRNA precursor splicing is branched – looks like a lariat2-step model2’-OH group of adenosine nucleotide in middle of intron attacks phosphodiester bond between 1st exon and G beginning of intronForms loop of the lariatSeparates first exon from intron3’-OH left at end of 1st exon attacks phosphodiester bond linking intron to 2nd exonForms the exon-exon phosphodiester bondReleases intron in lariat form at same time8Simplified Mechanism of SplicingExcised intron has a 3’-OH groupPhosphorus atom between 2 exons in spliced product comes from 3’-splice siteIntermediate and spliced intron contain a branched nucleotide Branch involves 5’-end of intron binding to a site within the intron9Signal at the BranchAlong with consensus sequences at 5’- and 3’-ends of nuclear introns, branchpoint consensus sequences also occurYeast sequence invariant: UACUAACHigher eukaryote consensus sequence is more variableBranched nucleotide is final A in the sequence10SpliceosomesSplicing takes place on a particle called a spliceosomeYeast spliceosomes and mammalian spliceosomes have sedimentation coefficients of 40S and 60SSpliceosomes contain the pre-mRNA Along with snRNPs and protein splicing factorsThese recognize key splicing signals and orchestrate the splicing process11snRNPsSmall nuclear RNAs coupled to proteins are abbreviated as snRNPs, small nuclear ribonuclear proteinsThe snRNAs (small nuclear RNAs) can be resolved on a gel:U1, U2, U4, U5, U6All 5 snRNAs join the spliceosome to play crucial roles in splicing12U1 snRNPU1 snRNA sequence is complementary to both 5’- and 3’-splice site consensus sequencesU1 snRNA base-pairs with these splice sitesBrings the sites together for splicing is too simple an explanationSplicing involves a branch within the intron13Wild-Type and Mutant U1 snRNAGenetic experiments completedBase pairing between U1 snRNA and 5’-splice site of mRNA precursor Is necessary Not sufficient for binding14U6 snRNPU6 snRNP associates with the 5’-end of the intron by base pairing through the U6 RNAOccurs first prior to formation of lariat intermediateCharacter may change after first step in splicingAssociation between U6 and splicing substrate is essential for the splicing processU6 also associates with U2 during splicing15U2 snRNPU2 snRNA base-pairs with the conserved sequence at the splicing branchpointThis base pairing is essential for splicingU2 also forms base pairs with U6This region is called helix IHelps orient snRNPs for splicing5’-end of U2 interacts with 3’-end of U6This interaction forms a region called helix IIThis region is important in splicing in mammalian cells, not in yeast cells16Yeast U2 Base Pairing with Yeast Branchpoint SequenceSource: Adapted from Parker, R., P. G. Sliciano, and C. Guthrie, Recognition of the TACTAAC box during mRNA splicing in yeast involves base pairing to the U2-like snRNA. Cell 49:230, 1987.17U5 snRNPU5 snRNA associates with the last nucleotide in one exon and the first nucleotide of the next exonThis should result in the two exons lining up for splicing18U4 snRNPU4 base-pairs with U6Its role seems to be to bind U6 When U6 is needed in a splicing reaction U4 is removedU6 gene is split by an mRNA-type intron in at least two yeast species19snRNP Involvement in mRNA SplicingSpliceosomal complex contains:Substrate U2U5U6The complex ready for the 2nd step in splicing can be drawn as a group II intron at same stage of splicingSpliceosomal snRNPs substitute for elements at center of catalytic activity of group II introns at same stage of splicing20Spliceosome Catalytic ActivityCatalytic center of spliceosome appears to include Mg2+ and a base-paired complex of 3 RNAs:U2 snRNAU6 snRNABranchpoint region of the intronProtein-free fragments of these RNAs can catalyze a reaction related to the first step in splicing21Spliceosome Assembly and FunctionSpliceosome is composed of many components – proteins and RNAThese components assemble stepwiseThe spliceosome cycle:AssemblyFunction Disassembly By controlling assembly of the spliceosome, a cell can regulate quality and quantity of splicing and so regulate gene expression22Spliceosome CycleAssembly begins with binding of U1 to splicing substrate forming a commitment complex, a unit committed to to splicing out the intron U2 joins the complex next, followed by the othersU2 binding requires ATPU6 dissociates from U4 and displaces U1 at the 5’-splice siteThis step is ATP-dependentActivates the spliceosomeAllows U1 and U4 to be released23snRNP StructureAll snRNP’s have the same set of 7 Sm proteinsCommon targets of antibodies in patients with systemic autoimmune diseasesSm protein binds to a common Sm site on the snRNAs: AAUUUGUGGU1 snRNP has 3 specific proteins70K has an Mr of 52 kDA has an Mr of 31 kDC has an Mr of 17.5 kDSm proteins form a doughnut-shaped structure with a hole through the middle, like a flattened funnel24Sm Site and RNAFive snRNPs participate in splicing All contain a common set of 7 Sm proteins and several other proteins that are specific to snRNPStructure of U1 snRNP reveals that the Sm proteins form a doughnut-shaped structure to which the other proteins are attached25A Minor SpliceosomeA minor class of introns with variant but highly conserved 5’-splice sites and branchpoints can be spliced with the help of a variant class of snRNAsCells can contain minor snRNAs:U11 performs like U1U12 acts like U2U4atac and U6atac perform like U4 and U6 respectively26Commitment, Splice Site Selection and Alternative SplicingsnRNPs do not have enough specificity and affinity to bind exclusively and tightly at exon-intron boundariesAdditional splicing factors are needed to help snRNPs bindSome splicing factors are needed to bridge across introns and exons and so define these RNA elements273’-Splice Site SelectionSplicing factor Slu7 is required for correct 3’-splicing site selectionWithout Slu7, splicing to correct 3’-splice site AG is suppressed and splicing to aberrant AG’s within 30 nt of the branchpoint is activatedU2AF is also required for 3’-splice site recognition65-kD U2AF subunit binds to polypyrimidine tract upstream of 3’-splice site and 35-kD subunit binds to the 3’-splice site AG28CommitmentCommitment to splice at a given site is determined by an RNA-binding proteinThis protein binds to splicing substrate and recruits other spliceosomal componentsThe first component to follow is U1SR proteins SC35 and SF2/ASF commit splicing on human b-globin pre-mRNA and HIV tat pre-mRNAPart of the commitment involves attraction of U1 in some cases29Bridging Proteins and CommitmentYeast commitment complex has a branchpoint bridging protein (BBP) binds to:U1 snRNP protein at the 5’-end of the intronMud2p near the 3’-end of the intronRNA near the 3’-end of the intronBridges the intron and could play a role defining intron prior to splicingMammalian BBP is SF1, may serve same bridging function 30Yeast Two-Hybrid Assay31Intron-Bridging Protein-Protein InteractionsBranchpoint bridging protein binds to U1 snRNP proteinComparison of yeast to mammalian complexes is seen at right32Role of the RNA Polymerase II CTDC-terminal domain of the Rpb1 subunit of RNA polymerase II stimulates splicing of substrates that use exon definitionThis does not apply to those that use intron definition to prepare for splicingCTD binds to splicing factors and could assemble the factors at the end of exons to set them off for splicing33Alternative SplicingTranscripts of many eukaryotic genes are subject to alternative splicingThis splicing can have profound effects on the protein products of a geneCan make a difference between:Secreted or membrane-bound proteinActivity and inactivityProducts of 3 genes in sex determination pathway of the fruit fly are subject to alternative splicing34Sex-Specific SplicingFemale-specific splicing of tra transcript gives:An active product that causes female-specific splicing of dsx pre-mRNAThis produces a female fruit flyMale-specific splicing of tra transcript gives:An inactive product that allows male-specific splicing of dsx pre-mRNAThis produces a male fruit fly35Tra and Tra-2Tra and its partner Tra-2 act in conjunction with one or more other SR proteins to commit splicing at the female-specific splice site on the dsx pre-mRNACommitment is probably the basis of most, if not all, alternative splicing schemes36Alternative Splicing PatternsAlternative splicing of the same pre-mRNA gives rise to very different productsAlternative splicing patterns occur in over half of human genesMany genes have more than 2 splicing patterns, some have thousands37Types of Alternative SplicingBegin transcripts at alternative promotersSome exons can simply be ignored resulting in deletion of the exonAlternative 5’-splice sites can lead to inclusion or deletion of part of an exonAlternative 3’-splice sites can lead to inclusion or deletion of part of an exonA retained intron can be retained in the mRNA if it is not recognized as an intronPolyadenylation causes cleavage of pre-mRNA and loss of downstream exons38Silencing of SplicingWhat stimulates recognition of signals under only some circumstances?Exons can contain sequences – Exonic splicing enhancers (ESEs) stimulate splicingExonic splicing silencers (ESSs) inhibit splicing39Reporter Construct Detects ESS Activity4014.3 Self-Splicing RNAsSome RNAs could splice themselves without aid from a spliceosome or any other proteinTetrahymena 26S rRNA gene has an intron, splices itself in vitroGroup I introns are a group of self-splicing RNAsAnother group, Group II introns also have some self-splicing members41Group I IntronsGroup I introns can be removed in vitro with no help from proteinReaction begins with attack by a guanine nucleotide on the 5’-splice siteAdds G to the 5’-end of the intronReleases the first exonSecond step, first exon attacks the 3’-splice siteLigates 2 exons togetherReleases the linear intronIntron cyclizes twice, losing nucleotides each time, then linearizes a last time42Linear Introns43Group II IntronsRNAs containing group II introns self-splice by a pathway using an A-branched lariat intermediate, like spliceosome lariatsSecondary structures of the splicing complexes involving spliceosomal systems and group II introns are very similar44