Docstoc

RNA Splicing

Document Sample
RNA Splicing Powered By Docstoc
					         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
  cleavage 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
  according 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
  RNA
• Reasons 1. loop out the exon
•          2. cooperative molecule to cover
•             the whole exon
• Both render the exon “invisible “to splice
  machinery!
  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
  the need of the increasing the rate of replication
  and cell division
• 2.intron late moder
• Introns are inserted later in eukaryotes by
  transposon-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
  region of target RNA site for editing
• 2. second, determines exactly where the
  Us 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

				
DOCUMENT INFO
Shared By:
Categories:
Tags:
Stats:
views:16
posted:1/27/2012
language:English
pages:44