mRNA processing Lecture Note Presentation.ppt
- 1. Molecular Biology Fourth Edition Chapter 14 Messenger RNA Processing I: Splicing Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
- 2. 14-2 14.1 Genes in Pieces • Consider the sequence of the human -globin gene as a sentence: This is bhgty the human -globin qwtzptlrbn gene. • Two italicized regions make no sense – Contain sequences unrelated to the globin coding sequences surrounding them – Called intervening sequences, IVSs – Usually called introns • Parts of the gene making sense – Coding regions – Exons • Some lower eukaryotic genes have no introns
- 3. 14-3 Evidence for Split Genes • Most higher eukaryotic genes coding for mRNA and tRNA are interrupted by unrelated regions called introns • Other parts of the gene, surrounding the introns, are called exons • Exons contain the sequences that finally appear in the mature RNA product – Genes for mRNAs have been found with anywhere from 0 to 362 exons – tRNA genes have either 0 or 1 exon
- 4. 14-4 RNA Splicing • Introns are present in genes but not in mature RNA • How does the information not find its way into mature RNA products of the genes? – Introns are never transcribed • Polymerase somehow jumps from one exon to another – Introns are transcribed • Primary transcript result, an overlarge gene product is cut down by removing introns • This is correct process • Process of cutting introns out of immature RNAs and stitching together the exons to form final product is RNA splicing
- 5. 14-5 Splicing Outline • Introns are transcribed along with exons in the primary transcript • Introns are removed as the exons are spliced together
- 6. 14-6 Stages of RNA Splicing • Messenger RNA synthesis in eukaryotes occurs in stages • First stage: – Synthesis of primary transcript product – This is an mRNA precursor containing introns copied from the gene if present – Precursor is part of a pool of large nuclear RNAs – hnRNAs • Second stage: – mRNA maturation – Removal of introns in a process called splicing
- 7. 14-7 Splicing Signals • Splicing signals in nuclear mRNA precursors are remarkably uniform – First 2 bases of introns are GU – Last 2 are AG • 5’- and 3’-splice sites have consensus sequences extending beyond GU and AG motifs • Whole consensus sequences are important to proper splicing • Abnormal splicing can occur when the consensus sequences are mutated
- 8. 14-8 14.2 Mechanism of Splicing of Nuclear mRNA Precursors • Intermediate in nuclear mRNA precursor splicing is branched – looks like a lariat • 2-step model – 2’-OH group of adenosine nucleotide in middle of intron attacks phosphodiester bond between 1st exon and G beginning of intron • Forms loop of the lariat • Separates first exon from intron – 3’-OH left at end of 1st exon attacks phosphodiester bond linking intron to 2nd exon • Forms the exon-exon phosphodiester bond • Releases intron in lariat form at same time
- 9. 14-9 Simplified Mechanism of Splicing • Excised intron has a 3’- OH group • Phosphorus atom between 2 exons in spliced product comes from 3’-splice site • Intermediate and spliced intron contain a branched nucleotide • Branch involves 5’-end of intron binding to a site within the intron
- 10. 14-10 Signal at the Branch • Along with consensus sequences at 5’- and 3’-ends of nuclear introns, branchpoint consensus sequences also occur • Yeast sequence invariant: UACUAAC • Higher eukaryote consensus sequence is more variable • Branched nucleotide is final A in the sequence
- 11. 14-11 Spliceosomes • Splicing takes place on a particle called a spliceosome • Yeast spliceosomes and mammalian spliceosomes have sedimentation coefficients of 40S and 60S • Spliceosomes contain the pre-mRNA – Along with snRNPs and protein splicing factors – These recognize key splicing signals and orchestrate the splicing process
- 12. 14-12 snRNPs • Small nuclear RNAs coupled to proteins are abbreviated as snRNPs, small nuclear ribonuclear proteins • The snRNAs (small nuclear RNAs) can be resolved on a gel: – U1, U2, U4, U5, U6 – All 5 snRNAs join the spliceosome to play crucial roles in splicing
- 13. 14-13 U1 snRNP • U1 snRNA sequence is complementary to both 5’- and 3’-splice site consensus sequences – U1 snRNA base-pairs with these splice sites – Brings the sites together for splicing is too simple an explanation • Splicing involves a branch within the intron
- 14. 14-14 Wild-Type and Mutant U1 snRNA • Genetic experiments completed • Base pairing between U1 snRNA and 5’-splice site of mRNA precursor – Is necessary – Not sufficient for binding
- 15. 14-15 U6 snRNP • U6 snRNP associates with the 5’-end of the intron by base pairing through the U6 RNA • Occurs first prior to formation of lariat intermediate • Character may change after first step in splicing • Association between U6 and splicing substrate is essential for the splicing process • U6 also associates with U2 during splicing
- 16. 14-16 U2 snRNP • U2 snRNA base-pairs with the conserved sequence at the splicing branchpoint • This base pairing is essential for splicing • U2 also forms base pairs with U6 – This region is called helix I – Helps orient snRNPs for splicing • 5’-end of U2 interacts with 3’-end of U6 – This interaction forms a region called helix II – This region is important in splicing in mammalian cells, not in yeast cells
- 17. 14-17 Yeast U2 Base Pairing with Yeast Branchpoint Sequence Source: 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.
- 18. 14-18 U5 snRNP • U5 snRNA associates with the last nucleotide in one exon and the first nucleotide of the next exon • This should result in the two exons lining up for splicing
- 19. 14-19 U4 snRNP • U4 base-pairs with U6 • Its role seems to be to bind U6 • When U6 is needed in a splicing reaction U4 is removed • U6 gene is split by an mRNA-type intron in at least two yeast species
- 20. 14-20 snRNP Involvement in mRNA Splicing • Spliceosomal complex contains: – Substrate – U2 – U5 – U6 • The complex ready for the 2nd step in splicing can be drawn as a group II intron at same stage of splicing • Spliceosomal snRNPs substitute for elements at center of catalytic activity of group II introns at same stage of splicing
- 21. 14-21 Spliceosome Catalytic Activity • Catalytic center of spliceosome appears to include Mg2+ and a base-paired complex of 3 RNAs: – U2 snRNA – U6 snRNA – Branchpoint region of the intron • Protein-free fragments of these RNAs can catalyze a reaction related to the first step in splicing
- 22. 14-22 Spliceosome Assembly and Function • Spliceosome is composed of many components – proteins and RNA • These components assemble stepwise • The spliceosome cycle: – Assembly – Function – Disassembly • By controlling assembly of the spliceosome, a cell can regulate quality and quantity of splicing and so regulate gene expression
- 23. 14-23 Spliceosome Cycle • Assembly 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 others • U2 binding requires ATP • U6 dissociates from U4 and displaces U1 at the 5’-splice site – This step is ATP-dependent – Activates the spliceosome – Allows U1 and U4 to be released
- 24. 14-24 snRNP Structure • All snRNP’s have the same set of 7 Sm proteins – Common targets of antibodies in patients with systemic autoimmune diseases – Sm protein binds to a common Sm site on the snRNAs: AAUUUGUGG • U1 snRNP has 3 specific proteins – 70K has an Mr of 52 kD – A has an Mr of 31 kD – C has an Mr of 17.5 kD • Sm proteins form a doughnut-shaped structure with a hole through the middle, like a flattened funnel
- 25. 14-25 Sm Site and RNA • Five snRNPs participate in splicing • All contain a common set of 7 Sm proteins and several other proteins that are specific to snRNP • Structure of U1 snRNP reveals that the Sm proteins form a doughnut-shaped structure to which the other proteins are attached
- 26. 14-26 A Minor Spliceosome • A 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 snRNAs • Cells can contain minor snRNAs: – U11 performs like U1 – U12 acts like U2 – U4atac and U6atac perform like U4 and U6 respectively
- 27. 14-27 Commitment, Splice Site Selection and Alternative Splicing • snRNPs do not have enough specificity and affinity to bind exclusively and tightly at exon-intron boundaries • Additional splicing factors are needed to help snRNPs bind • Some splicing factors are needed to bridge across introns and exons and so define these RNA elements
- 28. 14-28 3’-Splice Site Selection • Splicing factor Slu7 is required for correct 3’-splicing site selection • Without Slu7, splicing to correct 3’-splice site AG is suppressed and splicing to aberrant AG’s within 30 nt of the branchpoint is activated • U2AF is also required for 3’-splice site recognition • 65-kD U2AF subunit binds to polypyrimidine tract upstream of 3’-splice site and 35-kD subunit binds to the 3’-splice site AG
- 29. 14-29 Commitment • Commitment to splice at a given site is determined by an RNA-binding protein • This protein binds to splicing substrate and recruits other spliceosomal components • The first component to follow is U1 • SR proteins SC35 and SF2/ASF commit splicing on human -globin pre-mRNA and HIV tat pre-mRNA • Part of the commitment involves attraction of U1 in some cases
- 30. 14-30 Bridging Proteins and Commitment • Yeast commitment complex has a branchpoint bridging protein (BBP) binds to: – U1 snRNP protein at the 5’-end of the intron – Mud2p near the 3’-end of the intron – RNA near the 3’-end of the intron • Bridges the intron and could play a role defining intron prior to splicing • Mammalian BBP is SF1, may serve same bridging function
- 32. 14-32 Intron-Bridging Protein-Protein Interactions • Branchpoint bridging protein binds to U1 snRNP protein • Comparison of yeast to mammalian complexes is seen at right
- 33. 14-33 Role of the RNA Polymerase II CTD • C-terminal domain of the Rpb1 subunit of RNA polymerase II stimulates splicing of substrates that use exon definition • This does not apply to those that use intron definition to prepare for splicing • CTD binds to splicing factors and could assemble the factors at the end of exons to set them off for splicing
- 34. 14-34 Alternative Splicing • Transcripts of many eukaryotic genes are subject to alternative splicing – This splicing can have profound effects on the protein products of a gene – Can make a difference between: • Secreted or membrane-bound protein • Activity and inactivity • Products of 3 genes in sex determination pathway of the fruit fly are subject to alternative splicing
- 35. 14-35 Sex-Specific Splicing • Female-specific splicing of tra transcript gives: – An active product that causes female-specific splicing of dsx pre-mRNA – This produces a female fruit fly • Male-specific splicing of tra transcript gives: – An inactive product that allows male-specific splicing of dsx pre-mRNA – This produces a male fruit fly
- 36. 14-36 Tra and Tra-2 • Tra 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-mRNA • Commitment is probably the basis of most, if not all, alternative splicing schemes
- 37. 14-37 Alternative Splicing Patterns • Alternative splicing of the same pre-mRNA gives rise to very different products – Alternative splicing patterns occur in over half of human genes – Many genes have more than 2 splicing patterns, some have thousands
- 38. 14-38 Types of Alternative Splicing • Begin transcripts at alternative promoters • Some exons can simply be ignored resulting in deletion of the exon • Alternative 5’-splice sites can lead to inclusion or deletion of part of an exon • Alternative 3’-splice sites can lead to inclusion or deletion of part of an exon • A retained intron can be retained in the mRNA if it is not recognized as an intron • Polyadenylation causes cleavage of pre-mRNA and loss of downstream exons
- 39. 14-39 Silencing of Splicing • What stimulates recognition of signals under only some circumstances? • Exons can contain sequences – – Exonic splicing enhancers (ESEs) stimulate splicing – Exonic splicing silencers (ESSs) inhibit splicing
- 40. 14-40 Reporter Construct Detects ESS Activity
- 41. 14-41 14.3 Self-Splicing RNAs • Some RNAs could splice themselves without aid from a spliceosome or any other protein • Tetrahymena 26S rRNA gene has an intron, splices itself in vitro – Group I introns are a group of self-splicing RNAs – Another group, Group II introns also have some self-splicing members
- 42. 14-42 Group I Introns • Group I introns can be removed in vitro with no help from protein • Reaction begins with attack by a guanine nucleotide on the 5’-splice site – Adds G to the 5’-end of the intron – Releases the first exon • Second step, first exon attacks the 3’-splice site – Ligates 2 exons together – Releases the linear intron • Intron cyclizes twice, losing nucleotides each time, then linearizes a last time
- 44. 14-44 Group II Introns • RNAs containing group II introns self- splice by a pathway using an A-branched lariat intermediate, like spliceosome lariats • Secondary structures of the splicing complexes involving spliceosomal systems and group II introns are very similar