RNA

SPLICING
MECHANISM
Linda K.
 INTRODUCTION

 RNA polymerase cannot distinguish coding regions of the gene from non-
coding regions and transcribe everything
 Eukaryotic genes are interrupted by non-coding regions called introns/
intervening sequences
 The process of removing the non-coding RNA from the original transcript (pre-
mRNA) and joining the coding sequences (exons) together is called splicing.
 SPLICE SITE RECOGNITION

 For accurate splicing, the splicing machinery must know exactly at which site
to cut the mRNA  must recognize the border between introns and exons
 Consensus sequence: Conserved nucleotide sequence, which is common in
mRNA  GU at 5‘ end of the intron and AG at 3‘ end of the intron is present in
most pre-mRNAs
 The first splicing protein (ribonucleoprotein) has a complementary RNA strand,
which recognizes and basepairs with the consensus sequence.
 Mutation in the consensus sequence results in genetic disorders like beta-
thalassemia.
 In addition, there is a polypyrimidine tract near the 3‘ splice site.
SPLICE SITE RECOGNITION
 SPLICING MACHINERY

 Splicing of pre-mRNA is done by a macromolecular machine called the spliceosome.

 Spliceosome consists of different proteins and small nuclear ribonucleoproteins
(snRNP) called ‘‘snurps‘‘.
 The spliceosome is only assembled during the event of splicing, as the individual
proteins attach to the pre-mRNA.
 Removal of an intron requires several snRNP particles: U1, U2, U4, U5 and U6 (U4 &
U6 are bound to each other by complementary basepairing  U4/U6 duplex)
 BBP : Branch point Binding Protein
 Both the RNA and the protein components of the spliceosome catalyze the chemical
reactions required for splicing.
 STEPS OF PRE-mRNA SPLICING

 ASSEMBLY PATHWAY:
 STEP 1: snRNP U1 attaches to the 5‘ end of the intron-exon boundary. The nucleotide
sequence of U1 is complementary to the 5‘ splice site and basepairs with it.
 U2AF (U2 Auxiliary factor) has 2 subunits:
Larger subunit U2AF65 recognizes and binds to the polypyrimidine tract.
Smaller subunit U2AF35 binds to the 3‘ splice site.
U2AF65 helps BBP to bind to the branch site at the middle of the intron, where an
adenosine nucleotide (A) is present.
 This arrangement of proteins and RNA is called early complex (E).
STEPS OF PRE-mRNA SPLICING
 STEPS OF PRE-mRNA SPLICING

 STEP 2: U2 snRNP binds to the branch site with the help of U2AF, displacing BBP.

 This arrangement is called the A complex.

 The adenosine residue at the branch site is extruded out from the RNA helix. This A
residue later reacts with the 5‘ splice site.
STEPS OF PRE-mRNA SPLICING
 STEPS OF PRE-mRNA SPLICING

 STEP 3: U4 & U6 along with U5 join the complex, where the U4/U6 duplex is bound
together by complementary basepairing and U5 is more loosely associated.
 U4, U6 and U5 are together called the tri-snRNP particle.
This arrangement is called the B complex.
STEPS OF PRE-mRNA SPLICING
 STEPS OF PRE-mRNA SPLICING

 STEP 4: U1 leaves the complex and U6 replaces it at the 5‘ splice site.

STEPS OF PRE-mRNA SPLICING
 STEPS OF PRE-mRNA SPLICING

 CATALYSIS PATHWAY

 STEP 5: U4 detaches from the complex, so that U6 and U2 can interact and produce
the active site.  C complex
STEPS OF PRE-mRNA SPLICING
 STEPS OF PRE-mRNA SPLICING

 STEP 6: 5‘ splice site and the branch site are brought together.
 STEPS OF PRE-mRNA SPLICING

 STEP 7: 2‘ OH group of adenosine present at the branch site attacks the

phosphodiester bond between the first exon and the G at the beginning of the intron.
 A free 5‘ exon and a branched intron-3‘ exon intermediate is formed.
 The branched intron is called a ‘‘lariat‘‘.
STEPS OF PRE-mRNA SPLICING
 STEPS OF PRE-mRNA SPLICING

 STEP 8: 3‘OH group at the end of the first exon attacks the phosphodiester bond
between the lariat and the second exon.
 STEPS OF PRE-mRNA SPLICING

 STEP 9: The intron is released in the lariat form.

 The 2 exons are bound together with the help of snRNP U5.
 Spliceosome is disassembled and snRNPs are also released and recycled for the next
splicing.
 The lariat RNA is degraded.

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 ALTERNATIVE SPLICING

 Many genes in eukaryotes encode RNAs that can be spliced in alternative ways, which
results in different mRNAs and thus different protein products (isoforms).
Alternative splicing results in the production of different proteins from a single gene.
 40% of Drosophila genes and 90% of human genes undergo alternative splicing.
 This type of splicing ensures that different cell types produce different proteins in
response to different conditions.
Muscle Protein Troponin T
FIVE WAYS TO SPLICE RNA
 MECHANISMS ENSURING MUTUALLY EXCLUSIVE
SPLICING

There are several mechanisms to ensure that when one exon is chosen, the other is not:
 Steric hindrance: If the splice sites within the intron are too close together, splicing
factors (snRNPs) do not have the space for binding. So exons in that region are excluded.
 MECHANISMS ENSURING MUTUALLY EXCLUSIVE
SPLICING

 Combinations of Major and Minor Splice sites: Minor spliceosomes recognize consensus
sequences that are only present in some rarely occurring introns. They identify and bind
to introns with AU at the 5‘ splice site and AC at the 3‘ splice site (Major spliceosomes
recognize GU at 5‘splice site and AG at the 3‘ splice site of the intron).
 When an intron contains a combination of major and minor splice sites, neither one of the
spliceosomes can remove the intron and mutual exclusion is achieved.
 MECHANISMS ENSURING MUTUALLY EXCLUSIVE
SPLICING

 Non-sense Mediated Decay: When 2 exons are both included, it produces an mRNA that
contains a premature stop codon. These are degraded by NMD.
 CONTROL OF ALTERNATIVE SPLICING

 What stimulates recognition of splicing signals in a certain circumstance and the inhibition
of the signals in another context?
Exons contain sequences called exonic splicing enhancers (ESEs), which stimulate splicing
and exonic splicing silencers (ESSs), that inhibit splicing (intronic splicing enhancers and
silencers also exist).
First mechanism: A1 hnRNP binds to an ESS within an exon. Bound A1 molecules spreads on
the entire exon and hides the splicing signals from the splicing machinery, preventing the
binding of snRNPs.
Second mechanism: A1 binds to an intronic silencing element near the branchpoint in the
intron. This prevents U2 snRNP from binding.
Third mechanism: A1 binds to the intronic silencing elements in the introns, that are
flanking the exon. These 2 A1 molecules interact and produce a RNA loop, which isolates the
exon and hides it from the splicing machinery.
CONTROL OF ALTERNATIVE SPLICING
 EXAMPLE OF ALTERNATIVE SPLICING

 In Drosophila the products of 3 genes in the sex determination pathway are

subjected to alternative splicing.
 Female specific splicing of the tra-transcript gives an active product that causes
female specific splicing of the dsx pre-mRNA, producing a female fly.
 Male specific splicing of the tra- transcript gives an inactive product that allows
male specific splicing of the dsx pre-mRNA, producing a male fly.
VIDEO