4 LC Concepts in Molecular Biology 07.10.2022
RNA processing
There are many different RNA molecules inside a cell: double stranded rna, circular rna, nuclear rna, rRra, micro rna,
messenger rna… etc.
Rna is heavily processed, it means that POL II transcribes rna but then it goes under other processes such es the
splicing, the 5’ capping, the poly-adenylation, and editing such as base insertions, deletion, modification.
What is the challenge for the cell?
why are there so many modifications?
why do most RNA processing reactions happen co-transcriptionally?
RNA Sorting
All rnas are produced in the nucleus but they function at different locations in the cell. How are rna sorted?
Pre-mRNA processing in eukaryotes
We initially start with a pre-mrna with a 5’ untranslated
region, several exons separated by introns, and the 3’ and 3’
untranslated region. There is 5’ capping, then splicing with
introns removed and exons ligated, polyadenylation.
All of this is different from bacteria, where there is non of
this.
mRNA CAPPING
Initially the rna polymerase start transcribing producing a
very few nucleotides at the beginning and the
synthetized enzyme guanyl-transferase is recruited by the
CTD complex present in the rna POLII (a center that can
be phosphorylated in different ways).
The Guanyl-trsnasferase installs the 5’ Cap, which is
immediately bound by the CBC cap binding complex,
which is at the same time also recruited by a protein
associated to the rna polymerase.
The first nucleotide base 1 transcribed by the rna
polymerase has a tri phosphate (alfa, beta and gamma),
which are bonded by a unhydride bond; following
there is a methylated guanine base at nitrogen
number seven, being positively charged.
Initially the rna is transcribed with the 3phosphate
structure and then one of the phosphates is
removed by a phosphatase obtaining a di2phosphate
at the 5’ end. Then GMP is added reinstalling the
3phosphate structure, at the end the methylated
group is installed.
, 4 LC Concepts in Molecular Biology 07.10.2022
What is the function of the cap at the 5’ end?
5’ cap is important against degradation, esonucleases cannot cut because
there is the cap, it is important for enhancing splicing, polyadenylation,
translatipon and prevent rna decay.
The cap is shortly after bound by the cap binding complex CBC.
CBC
Nuclear cap binding complex is comnposed of NCBP1
(CBC80) and NCBP2(CBP20) and it binds the cap strongly.
The cap is bound and above and below there are two
aromatic plane aminoacids of tyrosine. The m7G base is
sandwiched between two aromatic residues, two
tyrosines (Y) forming a PI (Greek) – PI (Greek)
interactions.
Specificity for the guanine base is established through
hydrogen bonds with Asp (D) and Arg (R) residues from
NCBP2.
The metylated G is regonized a hundred fold better than GpppG; this is because of the positive charged
distributed between the two nitrogen atoms, introduced with the methylation. This creates a cation – Pi
interaction.
This is an example of ìwhat happens in histones and lys that can be methylated such as K27, where there are
writers, readers and erasers.
There are several capping that tare recognized by other cap binding proteins such as cellular cap binding
proteins (CBP20, eIF4E, LARP1) or even virall cap binding proteins (VP39, FluA, Flu B).
How do eukaryotic mrna differ from other rnas?
self-non self recognition
Viral rna don’t have a cap, they have a 3phospate
structure at the beginning that we don’t have. This can be
usefull to our cells to discriminate rnas.
IFIT proteins specifically bind to rna that have 5’ PPP-Rnas,
from viruses and bacteria.
In general, all nucleotides are negatively charged because
of the phosphoribose backbones, and in the 5’ PPP-Rnas is
much more negative at the beginning; so the rna structure
that IFIT5 protein need to recognize is negative charged.
- The 5’ PPP-Rna binds in a positively charged area,
where there are two Arg ( R 253, 186) residues
that directly interact with the triphosphate
structure compensating the charge interacting
electrostatically; but you also see this hydrogen
bonds with that compensates for the recognition.
There are also the first two nucleotides of the rna the N1 and N2 nucleobases 1 and 2, nucleobase 2 has pi-pi
interactions with Phenyl-alanine F337; but in general there seems to be no specific interactions with nucleobase 1
and two and no hydrogen bonds: this is because this is a type of immune system recognition and its better to
recognize a general pattern rather then the sequence of nucleotides that can variate.
RNA processing
There are many different RNA molecules inside a cell: double stranded rna, circular rna, nuclear rna, rRra, micro rna,
messenger rna… etc.
Rna is heavily processed, it means that POL II transcribes rna but then it goes under other processes such es the
splicing, the 5’ capping, the poly-adenylation, and editing such as base insertions, deletion, modification.
What is the challenge for the cell?
why are there so many modifications?
why do most RNA processing reactions happen co-transcriptionally?
RNA Sorting
All rnas are produced in the nucleus but they function at different locations in the cell. How are rna sorted?
Pre-mRNA processing in eukaryotes
We initially start with a pre-mrna with a 5’ untranslated
region, several exons separated by introns, and the 3’ and 3’
untranslated region. There is 5’ capping, then splicing with
introns removed and exons ligated, polyadenylation.
All of this is different from bacteria, where there is non of
this.
mRNA CAPPING
Initially the rna polymerase start transcribing producing a
very few nucleotides at the beginning and the
synthetized enzyme guanyl-transferase is recruited by the
CTD complex present in the rna POLII (a center that can
be phosphorylated in different ways).
The Guanyl-trsnasferase installs the 5’ Cap, which is
immediately bound by the CBC cap binding complex,
which is at the same time also recruited by a protein
associated to the rna polymerase.
The first nucleotide base 1 transcribed by the rna
polymerase has a tri phosphate (alfa, beta and gamma),
which are bonded by a unhydride bond; following
there is a methylated guanine base at nitrogen
number seven, being positively charged.
Initially the rna is transcribed with the 3phosphate
structure and then one of the phosphates is
removed by a phosphatase obtaining a di2phosphate
at the 5’ end. Then GMP is added reinstalling the
3phosphate structure, at the end the methylated
group is installed.
, 4 LC Concepts in Molecular Biology 07.10.2022
What is the function of the cap at the 5’ end?
5’ cap is important against degradation, esonucleases cannot cut because
there is the cap, it is important for enhancing splicing, polyadenylation,
translatipon and prevent rna decay.
The cap is shortly after bound by the cap binding complex CBC.
CBC
Nuclear cap binding complex is comnposed of NCBP1
(CBC80) and NCBP2(CBP20) and it binds the cap strongly.
The cap is bound and above and below there are two
aromatic plane aminoacids of tyrosine. The m7G base is
sandwiched between two aromatic residues, two
tyrosines (Y) forming a PI (Greek) – PI (Greek)
interactions.
Specificity for the guanine base is established through
hydrogen bonds with Asp (D) and Arg (R) residues from
NCBP2.
The metylated G is regonized a hundred fold better than GpppG; this is because of the positive charged
distributed between the two nitrogen atoms, introduced with the methylation. This creates a cation – Pi
interaction.
This is an example of ìwhat happens in histones and lys that can be methylated such as K27, where there are
writers, readers and erasers.
There are several capping that tare recognized by other cap binding proteins such as cellular cap binding
proteins (CBP20, eIF4E, LARP1) or even virall cap binding proteins (VP39, FluA, Flu B).
How do eukaryotic mrna differ from other rnas?
self-non self recognition
Viral rna don’t have a cap, they have a 3phospate
structure at the beginning that we don’t have. This can be
usefull to our cells to discriminate rnas.
IFIT proteins specifically bind to rna that have 5’ PPP-Rnas,
from viruses and bacteria.
In general, all nucleotides are negatively charged because
of the phosphoribose backbones, and in the 5’ PPP-Rnas is
much more negative at the beginning; so the rna structure
that IFIT5 protein need to recognize is negative charged.
- The 5’ PPP-Rna binds in a positively charged area,
where there are two Arg ( R 253, 186) residues
that directly interact with the triphosphate
structure compensating the charge interacting
electrostatically; but you also see this hydrogen
bonds with that compensates for the recognition.
There are also the first two nucleotides of the rna the N1 and N2 nucleobases 1 and 2, nucleobase 2 has pi-pi
interactions with Phenyl-alanine F337; but in general there seems to be no specific interactions with nucleobase 1
and two and no hydrogen bonds: this is because this is a type of immune system recognition and its better to
recognize a general pattern rather then the sequence of nucleotides that can variate.
