Splicingthe removal of intervening sequences

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Splicing-the removal of intervening sequences

• The majority of eukaryotic genes are split, that
  is, they contain intervening sequences that must
  be removed before the RNA can serve its function.
• Splicing was first discovered in 1977 by Phil
  Sharp at MIT and by Rich Roberts at Cold Spring
  Harbor Labs (for which they won the Nobel Prize
  in 1996)

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Discovery of Splicing

• Both groups were studying adenovirus
  transcription. By heteroduplex analysis and
  DNA/RNA hybridization and S1 nuclease analysis,
  they discovered that the RNA didnt cover as much
  sequence as the DNA

DNA
RNA
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Discovery of splicing

• They also found that the RNA hybridized to
  physically separate regions of the DNA

DNA
RNA
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Three types of splicing

• Group I self splicing introns. The RNA is
  catalytic.
• Group II introns the RNA is catalytic but
  proteins enhance the rate and efficiency of the
  reaction. Lariat formation occurs.
• Pre-mRNA splicing Lariat formation occurs
  requires a large complex termed the spliceosome
  that consists of small nuclear RNAs termed snRNAs
  as well as 50 to 100 proteins that bind to the
  snRNAs and to the pre-mRNA.

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Similarities between the three types of splicing

• Each process has 2 steps
• A) 5 splice site cleavage and ligation
• B) 3 splice site cleavage and ligation
• However, the two reactions result from
  phosphodiester bond exchanges. That is, two
  transesterification reactions occur.
• There is NO endonuclease and NO ligase involved
  in any of the three types of splicing reactions.

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Group I Introns- Self Splicing RNAs

• Group I introns were first discovered in
  Tetrahymena, a ciliated protozoan
• Tom Cech characterized the mechanism of splicing
  in Group I introns for which he won the Nobel
  Prize
• Cech found that there was a 413 nucleotide intron
  in the rRNA of Tetrahymena that was excised from
  the mature rRNA.

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Group I introns

• Similar to the way in which the proteins required
  for polyadenylation were characterized by
  biochemical fractionation of nuclear extracts
  that could carry out the cleavage and
  polyadenylation reactions in vitro, Cech
  developed an in vitro spicing assay in
  Tetrahymena cellular extracts.
• Isolated the pre-rRNA and added the extract,
  which would then be fractionated to determine the
  protein requirements.

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Group I introns

• Surprisingly, the control reaction, that is, no
  extract, also spliced.

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Group I Introns

• Cech considered the possibility that some
  Tetrahymena protein(s) survived the
  phenol-chloroform extraction of the pre-rRNA.
• Therefore, the rDNA was cloned into a plasmid in
  E. Coli. The rDNA sequence was placed under a T7
  bacteriophage promoter and the RNA was
  transcribed in vitro in a test tube.

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Group I Intron Splicing

• Therefore, there was no possibility of
  Tetrahymena proteins being present because the
  rDNA plasmid was propagated in bacteria and the
  pre-rRNA was transcribed in a test tube with
  purified T7 polymerase and NTPs and No other
  proteins were present.
• Yet, the pre-rRNA still excised the 413 nt
  intron.
• Thus, the conclusion was that the RNA was self
  splicing. It did not require any proteins to
  perform the reaction.

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Group I splicing mechanism

• The reaction occurs by two transesterifaction
  reactions.
• First, there is a nucleophilic attack by a
  guanisine. It can be GMP, GDP or GTP -it is not
  an energy source. Only the 3 OH of G is
  required.
• G attacks the 5 splice site and a new
  phosphodiester bond forms with G and the intron.
  The intron is released. It is no longer
  covalently bound to exon 1. There has been a
  phosphodiester bond exchange.

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Group I Self Splicing

• The second reaction occurs when the 3 OH of the
  upstream exon (exon 1) attacks the 5 PO4 of the
  downstream exon.
• This results in the ligation of the upstream exon
  (exon 1) to the downstream exon (exon 2). This is
  the second transesterification reaction.

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Self Splicing Introns

• Self splicing depends on the structural integrity
  of the rRNA precursor.
• Much of the intron is needed for splicing.
• The pre-rRNA has a folded structure with many
  stem loops.
• The folded structure contains weak G-U base pairs
  in addition to strong A-U and G-C base pairs.

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Structure of Self Splicing Introns

• The 5 splice site is aligned with the catalytic
  residues by base pairing between a pyrimidine
  rich region- CUCUCU of the upstream exon and a
  purine rich guide sequence GGGAGGG within the
  intron.

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Self Splicing Introns- G Pocket

• The intron brings together the G and 5 splice
  site so the 3 OH of G can attack the 5 PO4 at
  the 5 splice site.

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Structural Integrity of the Intron

• Another part of the intron holds the downstream
  exon in position for the attack by the 3 OH of
  the upstream exon.

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Structure of the Intron is critical

• Cechs group mutated each of the 413 nucleotides
  of the rRNA intron to determine the essential
  regions of the secondary structure.
• It was found that point mutations that disrupt
  the structure abolish splicing.

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Group II splicing

• Group II introns occur mostly in mitochrondrial
  pre-mRNAs in yeast and fungi.
• Self-splicing but the attacking moiety is the 2
  OH of a specific adenylate (A) of the intron.

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Group II Introns

• This attack by an A within the intron results in
  the formation of a Lariat.

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Group II Splicing

• Another difference between group I and group II
  intron splicing is that group I introns are
  auto-catalytic and require NO proteins.
• Group II introns are self splicing and can splice
  in the absence of proteins, BUT splicing rate and
  efficiency is greatly increased in the presence
  of group II specific proteins.
• This suggests that the proteins aid in holding
  the RNA in the correct configuration so that the
  two transesterifications reactions can occur.

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Pre-mRNA Splicing

• The requirement for proteins is even greater in
  pre-mRNA splicing in metazoans.
• Here, the requirement of an elaborate secondary
  structure for the introns would put severe
  evolutionary constraints on the pre-mRNAs, which
  are typically much larger than the group I intron
  pre-rRNAs.

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Pre-mRNA splicing

• Mammalian introns may be as little as 40
  nucleotides (minimum for effective recognition as
  an intron) to as large as several hundred
  thousand nucleotides.
• Therefore, it would not be possible for the
  introns to maintain the rigid secondary structure
  necessary to bring the attacking nucleotides in
  proximity.

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Cis Signals for pre-mRNA Splicing

• 5 Splice site- 9 nt consensusGU begins the
  intron
• 3 Splice site- 3 nt consensusAG ends the intron
• Branch Point- A branch site attacking base- 18
  to 38 nt upstream of the 3 splice site
• Polypyrimidine tract- pyrimidine-rich stretch
  between the branch site and the 3 Splice site

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Spliceosome Assembly
Branch site
UNCURAC
5' splice site
3' splice site
A

• CAG GUAAGU
• A G

(U/C)nN AG G
Intron
Exon 2
Exon 1
Polypyrimidine Tract
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Spliceosome Assembly
Branch site
UNCURAC
5' splice site
3' splice site
A

• CAG GUAAGU
• A G

(U/C)nN AG G
Intron
Exon 2
Exon 1
Polypyrimidine Tract
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Spliceosome Assembly
Branch site
UNCURAC
5' splice site
3' splice site
A

• CAG GUAAGU
• A G

(U/C)nN AG G
Intron
Exon 2
Exon 1
Polypyrimidine Tract
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Spliceosome Assembly
Branch site
UNCURAC
5' splice site
3' splice site
A

• CAG GUAAGU
• A G

(U/C)nN AG G
Intron
Exon 2
Exon 1
Polypyrimidine Tract
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Spliceosome Assembly
Branch site
UNCURAC
5' splice site
3' splice site
A

• CAG GUAAGU
• A G

(U/C)nN AG G
Intron
Exon 2
Exon 1
Polypyrimidine Tract
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In vitro Splicing Reactions
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Conformational rearrangements within the
spliceosome are required to bring the attacking
nucleotides together

• Rearrangements in the snRNA pairing occur
• Bridging proteins (SR) bring protein components
  of the splicesome together.

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Complex formation along the splicing pathway
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Splicing Complex Formation
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Comprehensive proteomic analysis of the human
spliceosome Z. Zhou, L.J. Licklider, S.Gygi R.
Reed NATURE 419 12 SEPTEMBER 2002

• To identify the complete set of proteins that
  constitutes intact functional spliceosomes
  maltose-binding protein affinity chromatography
  was used to isolate spliceosomes in highly
  purified and functional form.
• 2) Using nanoscale microcapillary liquid
  chromatography tandem mass spectrometry 145
  distinct spliceosomal proteins were identified,
  making the spliceosome the most complex cellular
  machine so far characterized.
• 3) The purified spliceosomes comprise all
  previously known splicing factors and 58 newly
  identified components.
• 4) The spliceosome contains at least 30 proteins
  with known or putative roles in gene expression
  steps other than splicing.
• 5) This complexity may be required not only for
  splicing multi-intronic metazoan pre-messenger
  RNAs, but also for mediating the extensive
  coupling between splicing and other steps in gene
  expression.

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AdML-M3 pre-mRNA contains three hairpins that
bind to the MS2MBP fusion protein used for
affinity purification (MS2 is a bacteriophage
coat protein MBP is maltose binding protein).
AdML pre-RNA, which lacks these hairpins, was
used as a negative control. After adding the
MS2MBP fusion protein to the two pre-mRNAs,
spliceosomes were assembled in vitro, isolated by
gel filtration, affinity-selected by binding to
amylose resin, and eluted with maltose under salt
conditions optimal for splicing. The products of
the first and second catalytic steps of splicing
were detected in the gel filtration fraction for
both AdML and AdML-M3 pre-mRNAs (lanes 2 and 5).
In contrast, after binding to and elution from
the amylose affinity resin, only the splicing
products of AdML-M3 spliceosomes were detected
(lanes 3 and 6). Thus, the spliceosomes were
highly purified.
c) Proteins from an RNase-A-treated aliquot of
the gel filtration fraction or final elution were
separated on 412 SDSPAGE and stained with
silver.
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Three-dimensional structure of C complex
spliceosomes by electron microscopy M. S. Jurica,
D. Sousa, M. J. Moore N. Grigorieff NATURE
STRUCTURAL MOLECULAR BIOLOGY (MARCH 2004) The
spliceosome is a multimegadalton RNA-protein
machine that removes noncoding sequences from
nascent pre-mRNAs. Spliceosomes that were
arrested before the second chemical step of
splicing (C complex) in which U2, U5 and U6
snRNAs are stably associated, were isolated.
Using electron microscopy, images of C complex
spliceosomes under cryogenic conditions were
obtained and a three-dimensional structure of a
core complex to a resolution of 30 Å was
determined. The structure reveals a particle of
dimensions 27 22 24 nm with a relatively open
arrangement of three primary domains.
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EM of individual spliceosomes assembled on Ftz-M3
pre-mRNA.
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Functional coupling of RNAP II transcription to
spliceosomeassembly

• R. Das, K. Dufu, B. Romney, M. Feldt, M. Elenko
  and R. Reed
• Genes Dev. 2006 20 1100-1109

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Functional coupling of RNAP II transcription to
spliceosomeassembly

• An efficient in vitro system was established to
  determine how RNA polymerase II (RNAP II)
  transcription is functionally coupled to pre-mRNA
  splicing.
• The data show that nascent pre-mRNA synthesized
  by RNAP II is immediately and quantitatively
  directed into the spliceosome assembly pathway.
• In contrast, nascent pre-mRNA synthesized by T7
  RNA polymerase is quantitatively assembled into
  the nonspecific H complex, which consists of
  heterogeneous nuclear ribonucleoprotein (hnRNP)
  proteins and is inhibitory for spliceosome
  assembly.
• Consequently, RNAP II transcription results in a
  dramatic increase in both the kinetics of
  splicing and overall yield of spliced mRNA
  relative to that observed for T7 transcription.
• RNAP II mediates the functional coupling of
  transcription to splicing by directing the
  nascent pre-mRNA into spliceosome assembly.

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RNAP II transcription and pre-mRNA splicing in
vitro
32P-UTP and the CMV DNA template were incubated
under transcription/splicing conditions for 15
min. Actinomycin D was added and incubation was
continued for the time specified.
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Transcription by RNAP II is functionally coupled
tospliceosome assembly
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Yields of spliced mRNA are dramatically enhanced
in the RNAP II transcription/splicing system.
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Linking Splicing to RNAP II Transcription
Stabilizes Pre-mRNAs and Influences Splicing
Patterns

• M.J. Hicks, C.-R. Yang, M.V. Kotlajich, and K.J.
  Hertel
• PLoS Biol 4(6) e147, 2006.

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• Using an in vitro transcription/splicing assay,
  it was demonstrated that an association of RNA
  polymerase II transcription and pre-mRNA splicing
  is required for efficient gene expression.
• RNAP II synthesized RNAs containing functional
  splice sites were protected from nuclear
  degradation, presumably because the local
  concentration of the splicing machinery was
  sufficiently high to ensure its association over
  interactions with nucleases.
• Other RNA polymerases (T7 polymerase) did not
  provide similar protection from nucleases, the
  link between transcription and RNA processing is
  RNAP II-specific.
• The connection between transcription by RNAP II
  and pre-mRNA splicing guarantees an extended
  half-life and proper processing of nascent
  pre-mRNAs

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Linking Splicing to RNAP II Transcription
Increases the Efficiency of Pre-mRNA Splicing
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The Link between RNAP II Transcription and
Splicing Increases the Stability of Nascent
Transcripts
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Pre-mRNA Splicing

• A simple reaction of two phosphodiester bond
  exchanges requires hundreds of proteins and 5
  snRNPS
• Splicing is coupled to transcription and occurs
  co-transcriptionally