5 LC Concepts in Molecular Biology 10.10.2022
RNA processing
We have preveuosly discussed how the
pre-mRNA is capped and the nuclear cap
binding complex CBC is deposited; we
have splicing, introns are rmoved;
there’s a specific complex deposited in
mammalian cells, called the exon-
junctions complex EJC that is deposited
upstream of these exons-exons
junctions.
Furthermore, at the 3’ end most mRNA
are poly-adenylated having a poly-A tail
that can be between 80 – 250 As long;
this is then also bind by the poly A binding protein PABPN1.
All the three major processing steps leave a proteinations wholemark on the rna, this is why we call this an
mrRNP ribonucleoproteinparticles.
RNA modification/editing
Which RNAs are edited and which parts of RNA can be edited?
There are different types of modifications that exist, such
as m7G, m6A where adenosine is methylated, and
pseudouridine.
Modifications in RNA (or any transcript) are introduced
from a writer that installs the modification X (for example pseudouridine), following a reader recognizes
the modification in interest and there’s and effect.
The challenge for a writer and a reader is finding the motive (the specified nucleotide) that is supposed to
be modified.
PSEUDO-URIDINE
It’s known as the fifth RNA nucleotide, most abundant rna modification and present in tRNA, rRNA, and
snRNA, discovered in 2014 to be present even in mRNA.
, 5 LC Concepts in Molecular Biology 10.10.2022
Pseudouridylation in mRNA is also shown to be dynamically regulated upon stress conditions, but tyet no
reader for pseudouridine is known. It’s not clear what it does in the mRNA. It’s suggested that is used to
regulate some function in the cell, stabilizing the mRNA.
There’s a famous paper that says: incorporation of pseudouridine into mRNA yields superior non-
immunogenic vector with increased translational capacity and biological stability. (Karikó & Weissman)
two mRNA vaccines that contain mRNA coding for Spike protein are modified with N1-methyl-
pseudouridine
Pfizer-BioNTech/Comirnaty and Moderna
Therapeutics/Spikevax.
They tested a number of RNA modifications m5C, m6A, s2U, m5U, psi greco (pseudouridinilation), and
asked if they modified the rna with this modifications could still produce proteins.
They measured the cell free translation with 35S-Met methionine, if the rna is not modified we get
translation, if cytocine is modificated there is translation, with m2A and m2U there is no product, with
m5U works well and in psi it is even much more intense.
This is not so much suprising, because uridine baase is rotaded and N
and C are flipped and ribose is conncecting with the C. H bonds remain
unchanged though, allowing the ribosome during translation to simply
recognize it like a uridine molecule.
In an extract they also see that like a 9 fold increase compared to the
non modified mrna this suggested that it increases translation
capabilities.
Even in luciferase, so also in context of vary mrna trascript it shows pretty the same results.
KEY EXPERIMENT
If you put this unmodified rnas into the cells, they trigger an innate immune response; something that we
don’t want (it has to be controlled and however not so strong). With different amounts of mrna injected
they noticed that psi modified mrna
triggers much less response of the
immune system. This sis because
mammalian rna, unlike bacteria, is
abundantly modified and this might
suppress immune response; this is one
way in which we might discriminate self
and non-self.
m6A N6-methyladenosine
is a methylation at the hexocyclic amino
group; but again how you recognize a single
adenosine? There are some consensus sites:
these adenosines are residing in a very
specific motive:
- RRACH motive the A is the
Adenosine that is modified and
downstream to it there is a cytosine
and these two residues are absolutely
conserved.
RNA processing
We have preveuosly discussed how the
pre-mRNA is capped and the nuclear cap
binding complex CBC is deposited; we
have splicing, introns are rmoved;
there’s a specific complex deposited in
mammalian cells, called the exon-
junctions complex EJC that is deposited
upstream of these exons-exons
junctions.
Furthermore, at the 3’ end most mRNA
are poly-adenylated having a poly-A tail
that can be between 80 – 250 As long;
this is then also bind by the poly A binding protein PABPN1.
All the three major processing steps leave a proteinations wholemark on the rna, this is why we call this an
mrRNP ribonucleoproteinparticles.
RNA modification/editing
Which RNAs are edited and which parts of RNA can be edited?
There are different types of modifications that exist, such
as m7G, m6A where adenosine is methylated, and
pseudouridine.
Modifications in RNA (or any transcript) are introduced
from a writer that installs the modification X (for example pseudouridine), following a reader recognizes
the modification in interest and there’s and effect.
The challenge for a writer and a reader is finding the motive (the specified nucleotide) that is supposed to
be modified.
PSEUDO-URIDINE
It’s known as the fifth RNA nucleotide, most abundant rna modification and present in tRNA, rRNA, and
snRNA, discovered in 2014 to be present even in mRNA.
, 5 LC Concepts in Molecular Biology 10.10.2022
Pseudouridylation in mRNA is also shown to be dynamically regulated upon stress conditions, but tyet no
reader for pseudouridine is known. It’s not clear what it does in the mRNA. It’s suggested that is used to
regulate some function in the cell, stabilizing the mRNA.
There’s a famous paper that says: incorporation of pseudouridine into mRNA yields superior non-
immunogenic vector with increased translational capacity and biological stability. (Karikó & Weissman)
two mRNA vaccines that contain mRNA coding for Spike protein are modified with N1-methyl-
pseudouridine
Pfizer-BioNTech/Comirnaty and Moderna
Therapeutics/Spikevax.
They tested a number of RNA modifications m5C, m6A, s2U, m5U, psi greco (pseudouridinilation), and
asked if they modified the rna with this modifications could still produce proteins.
They measured the cell free translation with 35S-Met methionine, if the rna is not modified we get
translation, if cytocine is modificated there is translation, with m2A and m2U there is no product, with
m5U works well and in psi it is even much more intense.
This is not so much suprising, because uridine baase is rotaded and N
and C are flipped and ribose is conncecting with the C. H bonds remain
unchanged though, allowing the ribosome during translation to simply
recognize it like a uridine molecule.
In an extract they also see that like a 9 fold increase compared to the
non modified mrna this suggested that it increases translation
capabilities.
Even in luciferase, so also in context of vary mrna trascript it shows pretty the same results.
KEY EXPERIMENT
If you put this unmodified rnas into the cells, they trigger an innate immune response; something that we
don’t want (it has to be controlled and however not so strong). With different amounts of mrna injected
they noticed that psi modified mrna
triggers much less response of the
immune system. This sis because
mammalian rna, unlike bacteria, is
abundantly modified and this might
suppress immune response; this is one
way in which we might discriminate self
and non-self.
m6A N6-methyladenosine
is a methylation at the hexocyclic amino
group; but again how you recognize a single
adenosine? There are some consensus sites:
these adenosines are residing in a very
specific motive:
- RRACH motive the A is the
Adenosine that is modified and
downstream to it there is a cytosine
and these two residues are absolutely
conserved.
