RNA Splicing

• Prokaryotic genes: the codon is immediately adjacent to another

• Eukaryotic genes: consists of blocks of coding sequences separated by noncoding sequences

• Thus! RNA splicing is necessary to eliminated these noncoding sequences.

outline

• The chemistry of RNA splicing

• The spliceosome machinery

• Splicing pathways

• Alternative splicing

• Exon shuffling

• RNA editing

• mRNA transport

• RNA splicing: the process of removal of introns from the pre-mRNA.

• * the significance of precision of splicing

: since the translation is conducted in a fixed frame, even one base lost or insertion would result in the change of the reading frame, which would further result in error protein…

The chemistry of RNA splicing

• 1. structural foundation

A. 3’ splice site:

B. 5’ splice site

C. branch point site

• 2. Process of reaction 2’ OH of A at branch site

as nucelophile to attack the phosphoryl group of G in the 5’ splice site

phosphodiester bond clea

vage between intron and exon at 5’ splice site

• Freed 5’ end joined to the A within the branch site

• 5’ exon as a new nucleophile to attack the phosphoryl group at 3’ splice site

5’ exon and 3’ exon are spliced ; intron lariat is released

• Consequence: two phosphodiester bonds formation at the cost of another two phosphodiester bonds equivalent bargain!

• However , large amount of ATP required . For what? Splicing machinary

outline

• The chemistry of RNA splicing

• The spliceosome machinery

• Splicing pathways

• Alternative splicing

• Exon shuffling

• RNA editing

• mRNA transport

The spliceosome machinery

Process : U1 attached to 5’ splice site BBP binds to A within branch

site U2AF binds to Py tract U2 binds to branch site aided by

U2AF to replace BBP A residue extruded as bulge

resulting form the base pair between U2 and branch site

• Tri-snRNP particle: U6 U4 U5 recruited to join in the complex

• U2AF released

• U6 binds to 5’ splice site replacing U1

• U4 released, allowing interactions between U6 and U2 to form active site

• First transesterification

• Second transesterification then two exons linked

Self-splicing introns

• Definition : the intron itself folds into specific conformation within the precursor RNA and catalyze the chemistry of its own release .

• without the involvement of other RNA or protein

• 1. Group I Intro 2. Group II Intron

Group I intron release a linear intron rather than a lariat

• Essential difference

• free G nucleotide

• Nucleophile used

• A residue branch site

Group I self-splicing

• Internal guide sequence base-pairs with 5’ splice site determining precisely which site for nucleophile attack by the G nucletide

Compares• Represents tendency of evolution • For self-splicing, intron sequences are restricted accordi

ng to the need of formation of specific configration for self-splicing

• For pre-mRNA spliceosome, the requirment above of intron sequence is released with the help of spliceosome

Three class of RNA Splicing

How does the spliceosome find the splice sites reliably?

• Splice-site recognition is prone to errors• 1. splice site could be skipped• 2.close sequence but not legitimate splice sites

mistakenly recognized . pseudo splice site

Guard against inappropriate splicing

• 1. active site formation only formed on RNA sequences that pass the testing of being recognized by multiple elements during spliceosome assembly

• 2.co-transcriptional loading process: correct 3’ splice site could be recognized before the formation of competing sequence downstreams

• 3. ESE(exonic splicing enhancers): SR proteins bind to ESE and then recruit component of spliceosome(U2AF&U1) around the ESE.

outline

• The chemistry of RNA splicing

• The spliceosome machinery

• Splicing pathways

• Alternative splicing

• Exon shuffling

• RNA editing

• mRNA transport

Alternative splicing

• Single genes could produce multiple products by alternative splicing

• Constitutive: more than one product is always made

• regulated : different forms are generated at different times under different conditions

Constitutive splicing of T antigen genes

Alternative splicing is regulated by activators and repressors

• Activator :

• directing the splice machinery to related exons to activate splicing

• Repressor:

• inability to recruit the splice machinery but blocking splice site to prevent splicing

One example for activator

• SR protein

• One domain (RNA-recognizing motif) for binding to RNA

• Another domain (RS domain ) to interact with splicing machinery

• Recognizing the splice site with RRM and then use RS domain to recruit splice machinery to activate splicing

One example for repressor

• hnRNP (heterogeneous nuclear ribonucleoprotein)

• hnRNPA 1 binds to an exonic silencer

• hnRNPA promote cooperative binding of additional molecule to cover the enhancer site

• SC35(SR) fails to activate splicing

Another mammalian splicing repressor

• hnRNP I • Through binding to Py tract• Consequence: exclude one exon from mature R

NA • Reasons 1. loop out the exon• 2. cooperative molecule to cover • the whole exon• Both render the exon “invisible “to splice machin

ery!

The consequence of alternative splicing

• 1. generation of diverse proteins( isoforms)

form only one gene.

2. regulating the use of intron, which, if retained in the mRNA, the latter would never be transported outside the nucleus thus would never be translated.

outline

• The chemistry of RNA splicing

• The spliceosome machinery

• Splicing pathways

• Alternative splicing

• Exon shuffling

• RNA editing

• mRNA transport

Exon shuffling

• Why would introns only presents in eukaryotes but not in bacterias???

• Two theory• 1.intron early model• introns did exist in bacteria but are excised to t

he need of the increasing the rate of replication and cell division

• 2.intron late moder• Introns are inserted later in eukaryotes by transp

oson-like mechanism.

Evidence for exon shuffling

• 1. borders between exons and introns coincide with the boundaries of different domains within proteins

• 2. proteins composed of repeated unit are thought to arise through the exon duplication

• 3. exons found in one gene turn out to be the recombination of exons in different otherwise genes

outline

• The chemistry of RNA splicing

• The spliceosome machinery

• Splicing pathways

• Alternative splicing

• Exon shuffling

• RNA editing

• mRNA transport

RNA editing

• Another way of altering the sequence of am mRNA

• Two mechanism

• 1. deamination

• 2.guide RNA-directed uridine insertion or deletion

deamination

• C U

• As a consequence

• Codon CAA could be changed into UAA (stop codon)

• As a result

• A full-length protein being truncated

Aslo A insoine

• ADAR (adenosine deaminase acting on RNA)

• Insoine could base pair with C

• Thus, protein translated could altered

Uridine insertion

• How? • Guide RNAs (gRNA)• Three domains• 1. 5’ end, anchor, directs gRNA to the regi

on of target RNA site for editing• 2. second, determines exactly where the U

s will be inserted within the edited sequence

• 3.3’ end ,poly-U stretch

• Anchor region basie pair with target site

• A residues are bulge out

• Endonuclease cuts at mismatch

• UTP added to be inserted in the blank region and linked by ligase

outline

• The chemistry of RNA splicing

• The spliceosome machinery

• Splicing pathways

• Alternative splicing

• Exon shuffling

• RNA editing

• mRNA transport

mRNA transport

• Three prerequisite for transportation out of the nucleus

• 1. 5’-capped • 2.intron-free• 3.3’-polyadenylated

summary

• 1.the chemistry of RNA splicing and the RNA splicing pathway, related spliceosome machinery

• 2. diversities of self-splicing and difference and relationship among them

• 3.the features and significances of alternative splicing , activation and repression of alternative splicing

• 4.the evidences bolstering exon shuffling and its evolutionary means

• 5.the mechanism and pathway of RNA editing