NucleicAcids20 RNA Splicing.pdf

Recap of lecture • CRISPR regions are structured, recognizable DNA sequences in non-eukaryotes • CRISPR DNA sequences are key part of adaptive immunity in non-eukaryotes • CRISPR accessory Cas proteins required for integration of foreign DNA • Foreign DNA is captured and turned against itself...

Recap of lecture • CRISPR regions are structured, recognizable DNA sequences in non-eukaryotes • CRISPR DNA sequences are key part of adaptive immunity in non-eukaryotes • CRISPR accessory Cas proteins required for integration of foreign DNA • Foreign DNA is captured and turned against itself by integrating it into genome • Animals defend against detrimental RNA using CRISPR-like piRNAs and Piwis • CRISPR RNA modified, used by Cas proteins to target DNA • CRISPR/Cas9 now used to create knock-out or knock-in mutations • Mutated Cas9 nucleases increase possibilities of genetic engineering • Permanent human genetic alteration now a reality • New CRISPR Prime Editing greatly improves prospects for genetic surgery • What are exons and intron exactly? • How do you get rid of introns? • What proteins are required to get rid of them? • How do these proteins cooperate to do this? • Are these proteins even strictly required? • How are mistakes avoided in deleting introns? • How are deviations from the norm dealt with? BIOC 3041 Nucleic Acids Biochemistry Lecture 20 – RNA Splicing Structure of Eukaryotic Genes • Prokaryotic genes encode RNAs that are translated to protein from 5′ to 3′ • Eukaryotic genes encode RNAs that first need modification (but not always!) • Exons are (usually!) Expressed splicing removes intervening introns • Some eukaryotic genes lack introns… • Intron content per gene increases with complexity of the eukaryote • Intron size variable, but usually larger than exons • mRNAs can be spliced in multiple ways to yield different proteins from same gene (by process of ‘alternative’ splicing, results in isoforms) Eukaryotic gene transcripts usually need to have long introns spliced out, leaving protein-encoding exons Sequences in the RNA determine splicing sites • Transcripts must be precisely spliced - or frameshift mutations occur! • Two splice sites exist for each intron, a 5’ (donor) splice site and a 3’ (acceptor) splice site at each end of intron (note consensus sequences!) 5’ (donor) splice site 3’ (acceptor) splice site • The third part needed is the branch point sites (A) followed by a stretch of pyrimidines 11 (N=any base, Y=any pyrimidine, R=any purine) • Most conserved sequence is GU for 5’ splice site and AG for 3’ splice site • Note that most of conserved splice site sequence is in intron itself so no restrictions apply to an exon’s (protein-coding) sequence 5’ donor, 3’ acceptor and internal branch sites are key to proper intron splicing Intron removed in a “lasso” (lariat) form as flanking exons joined • Nucleophilic attack by 2′ OH group of branch site Adenine on the 5′ phosphoryl group at beginning of the intron • The 3′ OH group at 3′ end of upstream exon performs a nucleophilic attack on the phosphoryl group at Guanine’s 5′ end of downstream exon • The reaction products are an intron with a lariat structure, plus a correctly spliced RNA Sequential transesterification events allows intron self-splicing, self-ligation Intron removed in a “lasso” (lariat) form as flanking exons joined • The structure of the 3-way junction formed during the splicing reaction • Need to rotate it to see linkages made 3′ 2′ • 2 bonds broken and 2 made…. …. no ATP needed (at least here…) • The spliced out intron is rapidly destroyed to prevent reverse reaction Lariat structure is intermediary in splicing of intron that requires no ATP RNA splicing carried out by large complex- ‘spliceosome’ • mRNA splicing reaction needs a huge molecular machine – “spliceosome” • Made of 150 proteins + 5 RNAs (similar size as ribosome, is a ribonucleoprotein) • requires ATP for function, RNA performs some of functions…like ribosome • The 5 small nuclear RNAs (snRNAs) called U1, U2, U4, U5, U6 • snRNAs bound to proteins = small nuclear RiboNucleoprotein Proteins snRNPs have several functions when bound to spliceosome off and on; 1) recognize 5′ splice site and branch point site 2) bring 5′ splice site and branch point site together 3) catalyse the RNA cleavage and joining reactions • Requires RNA•RNA, RNA•protein, and protein•protein interactions www.youtube.com/watch?v=aVgwr0QpYNE Spliceosome is huge ribonucleoprotein with transient snRNPs doing splicing Some RNA-RNA hybrids formed during the splicing reaction • RNA-RNA hybrids form between the U# snRNAs and pre-mRNA by base pairing • U1 binds 5′ splice site early, preparing it for U6 later • U2 snRNA binds branch site • U2 (+branch site) binds U6 (+5′ splice site) to bring the lariat together • Can have non-snRNP protein participate too, U2AF (U2 Auxilliary Factor) helps BBP (Branch point Binding Protein) recognize polypyrimidine stretch • BBP then displaced by U2 snRNA that binds • Helicases using ATP unwind RNA-RNA complexes, drive molecular rearrangements RNA-RNA hybrids and proteins help drive splicing reactions Assembly, rearrangements, catalysis in the spliceosome • The 5′ splice junction is recognized by the U1 snRNP • 3′ splice junction is recognized by a U2AF subunit, the‘35’ part • U2AF’s other subunit (“65” part) recognizes the pyrimidines (Y11), U2AF also interacts with BBP • U2 binds to the branch site and displaces BBP (bulge helps) Early complex ‘A’ complex Bulge induced Spliceosome assembly, rearrangements, catalysis • A rearrangement occurs in the complex U4 & U6 along with U5 (tri-RNP particle) & joins the Complex • U2AF lost • U4 and U6 snRNAs are paired • U6 replaces U1 at the 5’ splice site and U1 leaves the Complex Assembly, rearrangements, catalysis in the spliceosome • U4 is released from the complex • U6 now interacts with U2 to form the active site • 5′ splice site and the ‘A’ branch point brought close together to get “C(ut) complex” • The 5′ splice site and the 3′ splice sites are brought together by U5 • The second transesterification occurs www.youtube.com/watch?v=YgmoHtLGb5c good but 5min so watch it at home www.youtube.com/watch?v=U_5yJYRvh8A Spliceosome-mediated processing or pre-mRNA involves U# RNA and proteins sequentially assembling, dissasembling on pre-mRNA to make active sites Self-splicing introns show RNA can catalyze RNA splicing • pre-mRNAs that don’t require spliceosome called “self-splicing” introns • Splicing classes differ in abundance, branch point sites used, the source of genes they splice (nuclear, organellar, & prokaryotic) • Catalytic mechanism is always transesterification Ribozymes! Spliceosome mostly used for processing pre-mRNA, ribozyme can replace it Group I introns release a linear intron rather than a lariat Group I introns release a linear intron rather than a lariat Self-splicing introns very well conserved to maintain function, & are usually shorter -smaller than Group II -uses free G nucleotide or nucleoside Both use A-site branch site Use G-site branch site Splicesomes may have evolved from Group II self-splicing introns these have same structure as spliceosomes’s snRNP (RNA part) Splicing type tells you if spliceosome needed, branch site used, final intron type How does the spliceosome find the splice sites reliably? • Splicing should work in linear fashion so pre-mRNA properly spliced • U# snRNPs act as a guard – make sure the pre-mRNA is able to bind properly • Multiple introns (363 is record for man) and different splicing possibilities (38,000 in 1 fly gene!) plus huge introns (800kb max) means this is difficult • If exons are misassembled, can miss exons (key domains!) • Can also get incomplete exons if ‘pseudo’-splice site used instead of real one Proper splicing can be very difficult with dire effects but snRNPs help out SR proteins recruit spliceosome components to the 5′ end and 3′ splice sites Two ways to ensure accuracy; 1) Transfer of splicing factors from RNA polymerase CTD to the 5′ splice site • Poised & ready to react with next 3′ site Reduces exon skipping • Does not mean that exons are spliced in order (that they are spliced matters) 2) Ensure that only splice sites next to exons are selected • SR proteins (Serine-aRginine rich) bind to Exonic Splicing Enhancers (ESE) • Recruit components of splicing machinery (U2AF proteins to 3′ splice site, U1 snRNP to 5′ splice site) Special proteins that bind ‘homing’ DNA sequences direct splicing to proper sites Exons from different RNA molecules can be fused together by trans-splicing • May want exon skipping • Alternative splicing is where an exon may be joined to another exon that isn’t necessarily the nextmost one (but to yet another one downstream) • Can in rare cases have an exon from one mRNA (#1) joined to an exon of a completely different mRNA (#2) II I • Same chemistry as before, but no larait formed • “Y”-shaped RNA spliced out instead I • Trans-splicing occurs in almost all mRNAs of trypanosomes to add in leader sequence (an exception to the rarity rule…), also C. elegans worm II ‘Trans-splicing’ exons of two separate mRNA results in a Y-intron, not lariat A small group of introns are spliced by an alternative spliceosome composed of a different set of snRNPs • Most eukaryotes use spliceosome to process pre-mRNA, but a ‘minor’ form exists too • To confer more specificity, U1 and U2 can be replaced by U11 and U12 that will direct spliceosome to another target mRNA • Target splice sequences are different from that of the ‘major’ spliceosome (AU – AC at 5′ and 3′ sites) • These AT-AC spliceosomes may be an intermediate between group II introns and full-blown major spliceosome Minor spliceosome recognizes distinct 5′, 3′ splice sites but uses same process Recap of lecture • Eukaryotic gene transcripts usually need to have long introns spliced out, leaving protein-encoding exons • 5′ donor, 3′ acceptor and internal branch sites are key to proper intron splicing • Sequential transesterification events allows intron self-splicing, self-ligation • Lariat structure is intermediary in splicing of intron that requires no ATP • Spliceosome is huge ribonucleoprotein with transient snRNPs doing splicing • RNA-RNA hybrids and proteins help drive splicing reactions • Spliceosome-mediated processing or pre-mRNA involves U# RNA and proteins sequentially assembling, dissasembling on pre-mRNA to make active sites • Spliceosome mostly used for processing pre-mRNA, ribozyme can replace it • Splicing type tells you if spliceosome needed, branch site used, final intron type • Proper splicing can be very difficult with dire effects but snRNPs help out • Special proteins that bind ‘homing’ DNA sequences direct splicing to proper sites • Minor spliceosome recognizes distinct 5’, 3’ splice sites but uses same process www.youtube.com/watch?v=3rlziMRQZoU watch this video and the reading… Reading – MBPGF p 398-408, MBOG7 pg 467-483, MBOG6 pg 415-432