BIOL 200 Lecture Notes - Rna Editing, Base Pair, Polyadenylation

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6 Apr 2012
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Naveen Sooknanan McGill Fall 2011
1
RNA Processing:
As we stated before, RNA polymerase II is the only one of its kinda to have a carboxyterminal
domain (CTD)
The CTD consists of the aa pattern YSPTSPS repeated
26 times in yeast and around 52 times in humans
o Is the CTD is removed in yeast, the organism dies, and the
same is assumed to happen for humans
The CTD is attached to Pol II by a linker peptide which is 28nm in
length
The CTD is a crucial part of Pol II because it is the source of two very
important phosphorylation events which essentially carry out RNA
processing of class II gene transcripts. These phosphorylation events are
carried out by the PNK activity of TFIIH
The first phosphorylation occurs on the 5th aa, a serine, on every repeat of the CTD. This
causes a capping enzyme to bind to the CTD and install a cap onto the 5’ end of the pre-
mRNA as it is being transcribed
o This occurs after pol II has synthesized around 25 nucleotides
o This cap protects the new pre-mRNA from being digested by exonucleases
o After this phosphorylation occurs, pol II becomes much more active and enters an
elongation phase
The second phosphorylation occurs on the 2nd aa of the sequence, also a serine, at every
repeat on the CTD
o This occurs during elongation and recruits the splicing machinery necessary for
splicing out noncoding regions of the pre-mRNA
Pol I and Pol III don’t have CTDs, so their gene products do not undergo capping
o They do, however, undergo post transcriptional modification which will be
discussed later
Capping involves the addition of a 7’ methylguanylate cap to the 5’ end of the pre-mRNA as
soon as it exits the transcription complex
This ensures that the mRNA is not damaged by exonucleases and
happens so quickly because to the close proximity between the 5’ end
of the mRNA and the capping enzyme on the CTD
o This 7’ MG cap comes from a GTP substrate can creates an
odd 5’ 5’ phosphodiester linkage which exonucleases can’t
recognize
Animals and higher plants have a second security barrier in which the
second base is methylated on the 2’ hydroxyl group
Vertebrates have a further security feature which methylates the 2’
hydroxyl group of the third base
Along with evasion of exonuclease digestion, the capping also allows for the recognition
of various nuclear export proteins as well as translation factors in the cytoplasm
Naveen Sooknanan McGill Fall 2011
2
The transition from pre-mRNA to mRNA is carried out by a mechanism called splicing. Unlike
bacterial genes, most eukaryotic genes have introns which must be spliced out before the mRNA
is translated.
While introns were initially considered to be junk DNA, it is found that they can actually
code for important regulatory functions as well as aid in alternative splicing events
Splicing was discovered by hybridization events in which a finals mRNA was annealed to its
corresponding gene
To the scientists surprise, the mRNA was actually much smaller than the gene
For example, in the hexon gene, DNA segments which coded for introns looped out of
the double stranded structure because they were spliced out of the final mRNA
Because of this size discrepancy, it was proved that genes pre-mRNA must undergo some
splicing event to reach a mature state
One important characteristic in splicing is the high level of conservation of intron borders. This
helps splicing machinery know where to carry out its splicing events to form mature mRNA
This is helpful for prediction of mRNA sequences
from a gene without conducting the actual
sequencing
An AG is highly conserved at 3’ end the 5’ exon, followed by a GU at the 5’ end of the
intron
o This is known as the 5’ splice site
Somewhere near the end of the intron is a 100% conserved A which is called the branch
point
The last 2 bases at the 3’ end of the intron are always AG, usually followed by a G in the
3’ exon
o This corresponds to the 3’ splice site
This conversation can be seen as the “GU-AG rule”
The splicing event of an intron is carried out by two sequential trans-esterification reactions,
creating a lariat intron and two seamlessly joined exons
The first reaction involves a nucleophilic attack by the 2’
hydroxyl group of the branch point towards the 5’ phosphate of
the first intron nucleotide. This is followed by the 3’ phosphate of
the 3’ exon attacking the branch point forming a lariat intron
structure (loop)
o This produces a strange 2’ – 5’ linkage
The second reaction involved the 3’ hydroxyl group of the 5’
exon attacking the 3’ phosphate of the 3’ exon which lets go of
the lariat structure
o This creates a fully excised lariat intron and two
seamlessly joined exons
These reaction in theory use no energy because the energy gained
from breaking old bonds is equal to the energy required for the
formation of new ones
Naveen Sooknanan McGill Fall 2011
3
Intron splicing can be seen in vitro by radiolabelling the pre-mRNA and
taking various samples at various times and looking at differences in size and
number of different pieces
This is done by running the mRNA sample through an acrylamide gel
As the pre-mRNA is being spliced, excised lariat introns, which are
smaller, will start to appear farther down in the gel than the mRNA
This also allows us to determine the size of various splicing
intermediates as well as the final mRNA product
The trans-esterification reactions done above are catalyzed by various factors making up the
splicing machinery. Among these are 5 small nuclear
ribonucleoprotein particles (snRNPs): U1, U2, U4, U5 and U6, as
well as 6-10 other proteins
Ribonucleic proteins are complexes containing both RNA
and protein subunits
U1 snRNP is able to base pair with the 5’ splice site while U2 is able to base pair with a
conserved region around the branch point
o The branch point itself does NOT base pair with U2 snRNP and forms a bulge
which is important in the recruitment of other factors
The protein parts of these snRNPs help stabilize the RNA component and keep them in
place
One interesting phenomenon with U1 binding is that a mutation in
the 5’ splice site sequence of the pre-mRNA will result in failure to
initiate the splicing reactions
By introducing a compensatory mutation into the U1 snRNP binding sequence so that it
matches the mutant pre-mRNA, the u1 snRNP cab bind as it
normally would and splicing ability is restores
o This can be done in vitro and, more surprisingly, in
vivo
Some genes, such as rRNA genes of protozoans, are able to undergo splicing reactions without
the help of any proteins. These are known as group 1 genes. They represent a tiny minority of the
overall picture, however.
These genes represent our ancestral genes and this evidence shows that the secondary
structure of out ancestral genes may have been able to undergo splicing all by themselves
o This proves that RNA itself can have enzymatic properties, and not just proteins
The majority of genes, however, need the assistance of a ribonucleoprotein called a spliceosomes
The spliceosomes acts as a catalyst for the two trans esterification
reactions and actually contains U1 and U2 snRNPs listed above
This has actually been proved photographically by electron
microscopy, where a large lump, the spliceosome, is seen excising an
intron from a piece of mRNA
The formation of the spliceosome involves the sequential binding of various
snRNPs to the pre-mRNA