SPLICING

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SPLICING
Lecture 4
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Rate 40nt/sec
Poly A, 5 cap

• Eukaryotic genes are mosaics of Int (non coding)
  and Exons (coding)
• Exons typically small (150 bp average)
• Introns can be small or huge and MANY
• DHFR Gene 31 kb, 6 exons, 2 kb mRNA (coding DNA
  lt10)

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RNA Splicing

• Primary transcript pre-mRNA
• Must be processed
• Splicing converts pre-mRNA to mRNA
• Alternative splicing can increase gene diversity
• Estimated 60 of genes are alt. spliced!
• One gene could encode 1000s of splice variants!
• Accuracy is CRITICAL, mistakes not tolerated

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Mechanisms

• Consensus sequences in the transcript are key to
  precise splicing outcomes

Consensus site _at_ splice junctions HIGHLY
conserved especially GU and AG
Branch point mid intronnear poly Pyr tract
Donor site
Acceptor Site
NOTE THAT THE CONSENSUS ELEMENTS ARE IN INTRONS
AND NOT EXONS (CONSTRAINED BY CODING SEQUENCE)
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Intron excision involves formation of a lariat
structure

• Splicing is a continuum
• 2 successive transesterifications
• Phosphodiester linkages break/reseal in a coupled
  reaction
• Rxn can be visualized as a 2-step process
• 1st is 2OH at conserved A residue
• 2nd is formation of lariat and splice product

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Nucleophilic attack _at_ P
Result Freed 5 end of intron joins A to make
the branch site in lariat
1st rxn
3 way junction2 OH Link at A
Nucleophilic attack _at_ P in splice site junction
2nd rxn
2 Products are made as a result
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Key points

• No net increase in phosphodiester bonds
• 2 bonds are broke and 2 are made
• No energy input required in transesterification
  reactions
• However, ATP is consumed
• Required for maintenance/assembly of splicing
  machinery in vivo

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If no net energy input, what makes splicing
reaction irreversible?

• Entropically driven by
• Breaking a single RNA transcript in two creates
  disorder (favorable)
• Rearrangement of ion clouds in process
• Exicised intron rapidly degraded
• Thus, cannot go back or reverse the splicing
  reaction

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Trans-splicing

• Exons from different transcripts are fused
• Rare in animals but does occur
• More common in C. elegans, trypanosomes

No lariat a Y structure is formed instead
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Splicesomes

• Large complexes or molecular machines carry out
  splicing in vivo

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Splicing machines RNPs

• gt150 proteins
• 5 RNAs
• Small nuclear RNAs (snRNAs) U1,2,4,5,6
• Ca. 100 and 300 nt long complexed with protein
  (snRNP or snurps)
• RNPs and misc. ptns come and go in process
• Process mediated primarily by RNA catalysis with
  protein support
• Akin to a ribosome

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snRNP Roles

• Recognize 5 splice site and branch site
• Bring these sites into proximity
• Catalyze the splicing reaction

Discuss in detail
RNA-RNA RNA-protein Protein-Protein
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• Different snRNPs recognize same (or overlapping)
  sites in transcript
• Here U1 and U6 shown to bind to splice site
  (donor)

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• snRNP U2 binds branch site

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• RNA pairing between snRNP U2 amd U6 is shown
• Brings 5 splice site and branch site into
  proximity

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Branch point binding protein

• Here BBP (not part of splicesome) recognizes A
  region and is displaced by U2 during the reaction
  sequence

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Other protein roles

• U2AF binds poly-pyr tract helps BBP bind to
  branch
• RNA-annealing factors
• Help load snRNPs onto transcript
• DEAD Box helicases
• Use ATPase to dissociate RNA duplexes
• Facilitate alternative RNA-RNA interactions

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• Mechanistic overview
• U1 snRNP binds 5 splice site
• U2AF binds Pyr tract and 3 splice site (U2AF has
  2 subunits)
• U2AF interacts with BBP to help stabilize this
  interaction
• U2 snRNA binds A branch site and displaces BBP
  A complex
• A residue extrudes and made available to bond w.
  5 splice site
• A complex reorganized to bring together all 3
  splice sites
• U4 and U6 snRNAs along with U5 join to form the
  tri-snRNP complex
• Entry of tri-snurp complex defines formation of
  B complex
• 7. U1 exits and is replaced by U6 ( C complex)
  or active site.

A complex
B complex
U4 exits and U2 takes over to complete
order not well known
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How did splicing evolve?

• Its complicated lots of players
• Probably evolved from self splicing mechanisms
  with catalytic RNA
• Summary of 3 classes of RNA Splicing

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Nuclear pre-mRNA

• Abundance
• Very common used in most eukarya
• Mechanism
• Transesterifications branch A site
• Catalytic mechanism
• Major spliceosome

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Group II Introns

• Abundance
• Rare some eukaryotic genes from organelles
• Prokaryotic mechanism
• Mechanism
• Transesterifications branch A site
• Catalytic mechanism
• RNA encoded by intron ( Ribozyme mediated)

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Group I Introns

• Abundance
• Rare nuclear rRNA in some eukaryotes
• Organelles genes
• A few prokaryotic genes
• Mechanism
• Transesterifications branch G site
• Catalytic mechanism
• RNA encoded by intron ( Ribozyme mediated)
• NOTE Not a true enzyme catalytic event!
  mediate only one round of events

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Group I Introns Releases Linears

• Different pathway to splicing
• Uses free G (not branch _at_ A)
• G residue bound to RNA and its 3OH presented to
  splice site.
• Gp I introns have an internal guide sequence that
  pairs with 5 splice site
• Directs nucleophilic site of G attack

G binding pocket forms on RNA
free 3 end of exon attacks 3 splice site
RIBOZYMES
linear byproduct
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Gp I Introns can act as ribozymes

• Provide free G in excess (there is a terminal G
  at 3 end of intron)
• Any RNA with homology to Internal guide seq.
  (IGS) will be degraded
• By modifying IGS, we can target specific mRNAs
  for degradation
• Thereby modulate gene expression in cells.

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Gp I introns Most of the RNA essential for
self-splicing reactions

• Usually 400-1000 nt long
• Most or all essential
• Because folding of RNA is especially critical
• In vivo ptn factors important in stabilizing
  proper configuration of RNA backbone
• In vitro VERY high salt concentrations can
  compensate (self-splicing rxns can occur in vitro)

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What is high ionic concentration and why is it
required

• Molar amounts of divalent cations. This is huge
  and NOT physiological!

VERY high repulsive charges at surface of folded
RNA! Must be shielded to make proximity possible.
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Similar chemistry in self-splicing type II
introns and spliceosome meidated pre-mRNA splicing

• Evolutionary consideration. Perhaps gp II
  introns (self-splicing) were ancestral
• Evolved into ptn mediated splicing (but still
  retained catalytic functions)
• Proteins as snRNPs made it efficient
• Note folding similarities in gp II and pre-mRNA
  introns

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• Early on proteins in spliceosome supplanted the
  self-splicing character of gp II introns.

Dotted region 4 other domains not
shown Similarity is quite striking!
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Accuracy of splicing

• Spliceosome factors (snRNPs) aid in fidelity
• But other aspects increase fidelity
• Average gene 6-8 exons
• Some have MANY more (few hundred)
• Average exon 150 bp
• Average intron 2-3 kb (up to 800 kb!)

exons must be accurately selected in a huge
excess of intronic sites
Accuracy matters!
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Splice site errors two types
Resembles a splice site but really is not real
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Splice site fidelity improved in two ways

• Txnal loading as pre-mRNA is made, processing
  proteins (snRNPs, spliceosome) recruited from CTD
  of Rpol to transcript.
• Sequential exposure of each 5 and 3 splice
  junction increases fidelity
• Minimizes exon skipping and error site selection

FIRST
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Splice site fidelity improved in two ways

• Splice sites NEAR exons recognized preferentially
• SR (serine-arg rich) proteins bind to exon
  splicing enhancers within exons
• SR ptns also interact with spliceosome features
• Splicing machinery recruits and works more
  efficiently in this way

SECOND
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Legitimate sites validated as real by being close
to exons

• SR recruits U2AF and U1 as shown
• SR factors bind to ESE as shown
• SR essential for splicing of pre-mRNA

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Alternative splicing

• Single genes can produce multiple products
• A few to 100s or even 1000s!
• Example with troponin gene
• 5 introns
• 2 alt. splice mRNAs ea. With 4 exons

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Alternative splicing

• Can be regulated or constitutive
• Some spl. Sites used only sometimes
• Exons can be extended or skipped or fused
• Introns can even be included in final mRNA
• Another example is SV40 T-Ag which makes two
  versions small t and large T.
• This is a constitutive process

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SV40 T-antigen gene and constitutive splicing
5SST is for T5sst is for t3SST is for both
T and t antigens made in infections

• t ag longer mRNA but includes a stop codon
• A host SR protein complex (SF2/ASF) tends to
  dictate final ratios of Tt in infected cells

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5 ways to alternatively splice an RNA
Most Silencers bound by hnRNP type proteins

• Above are regulated by Activators/Repressors
• Bind to exonic or intronic splicing enhancers
  (ESE, ISE)
• Or exonic, intronic splicing repressors (ESR,
  ISR)
• Enhancers can recruit splcing machinery (U2AF)
• Silencers simply bind to a site (ESR or ISR) and
  sterically block access of splicing factors (SR
  proteins for example)

Blocked
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Alternative splicing and gene regulation

• Most alt spl. Gives rise to isoforms
• Alt. spl. Can be use to turn off a gene
• In this case only 1 gene product is actually
  produced
• A STOP codon may be included in mRNA
• Such partial products are usally malfolded and
  degraded

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Origin of Introns?

• Introns may have existed in lower prokaryotes
  first and then lost later (intron early model)
• Introns may simply have arisen later in higher
  eukaryotes (intron late model)
• Possibly by transposition mechanism

Not clear which
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Exon shuffling

• Evolutionary advantages of introns
• Increase genetic potential (alt. splcing)
• Exons can be reshuffled to make NEW genes
• Evidence for a reshuffling mechanism
• Exons are small and correspond to ptn domains
• Evidence that some genes evolved by exon
  duplication (more likely too happen with introns
  and modular exons)
• Exons often found in otherwise unrelated genes
• Exons recycled into other genes

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Exon shuffling

• Final point Exons are short, introns huge
• Recombination or gene disruption more likely in
  introns and not exons.
• Splicing mech universality of 5/3 splice
  joints allows exons to interchangeleads to new
  gene products without discarding orignal!

Cells do the expt!
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Modular Exon Design
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Gene made up of parts (exons) of otherwise
unrelated genes
THIS IS VERY COOL
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RNA Editing

• Another way to alter mRNA sequence
• Two mechanisms
• Deamination
• Guide RNA-directed U insertion/deletion

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Deamination

• Only in certain tissues and regulated
• CAA codon converted to UAA stop
• Gives a truncated mRNA and protein

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(No Transcript)
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Guide RNA (gRNAs) Edits

• Mitochondria and trypanosomes
• Poly U added or deleted in mRNA
• Significant change in coding!

coxII Gene in trypanosome
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Guide RNA (gRNAs) Edits

• gRNAs separate genes encode
• 40-80 nt long
• Involves guide, anchor, cut and ligation
• End result U repeats can be added anywhere and
  alter coding potential

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mRNA Transport

• Nuclear to cytoplasm tranport
• mRNA is capped, poly A, intronless must egress
  nuclear for translation
• Regulated process (small RNA is nuclear)
• Processed mRNA has an army of associated
  proteins Identified as ready to exit nucleus
• Introns protein factors define retention in
  nucleus

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Export

• mRNA goes through nuclear pore complex
• Small (50 kDa or less) go through easily
• mRNAfactors too big and must be actively
  transported
• Requires energy GTPase called Ran

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Has right collection of proteins distinguishes
it form other RNA species
Example Factors at exon-exon borders in direct
contact
recycle