Samenvatting - Advanced molecular biology (NWI-BBO17C)

Post-transcriptional gene regulation
1. Transcription
2. RNA processing
3. mRNA translation
4. Replication
Transcripts are produced by three RNA polymerases
- Pol I >> rRNA (transcription of ribosomal RNA)
- Pol II >> mRNA, miRNA, snRNA (transported to the cytoplasm (snRNA goes back into the
nucleus after)
- Pol III >> tRNA, scRNA, snoRNA, 5S rRNA

1. What is approximately the percentage of mRNA in relation to total RNA in rapidly growing
mammalian cells?
The minority of the RNA, this is because there is a lot of ribosomes in the cell with a long
lifespan. The protein is not needed after an amount of time, the protein and mRNA is
degraded, so mRNA does not have a long lifetime.
2. What is the fraction of pre-mRNA synthesis of the total RNA synthesized in replicating
mammalian cells?
About 80%. The activity of Pol II is highest compared to the other two. Because of the low
activity of mRNA, this 80% is only responsible for a small part of the total mRNA

RNA editing = changing the nucleotide sequence of the RNA
mRNA can be silenced by miRNA, this means that it will be present but will not be translated because
of the miRNA.

Part of the newly synthesized RNA can already be translated while it is still being synthesized.

Three major forms of pre-mRNA processing
- Capping >> functional aspect so that it wouldn’t get degraded, consists of an addition of
nucleotides attached to the sequence in an irregular way. Transcription, 5’ capping
- Polyadenylation >>
- Splicing >>
>> these only happen in eukaryotic cells

Capping
- Attached to the first nucleotide
- Three phosphates linking the nucleotide
- Linked to the 5’ end, so 5’ to 5’ linkage
- A number of methyl groups added that aren’t usually present at RNAs
o Methyl groups to the 2’ of the first and second base of the RNA
- Combination of four enzymatic steps
1. Linkage to the gamma phosphate is broken
2. Gamma phosphate of the GTP is linked to the beta phosphate of the mRNA
3. Methyl donor molecule is needed to provide the methyl groups >> molecule that
is a combination of an amino acid and nucleotide
What is the function of the cap at the 5’ end of mRNAs?
- Protection
- Transport, the cap will be bound by cap binding proteins, and these cap binding proteins can
contribute to the transport proteins
- Translation process, the cap binding proteins are initiation factors for translation

, hnRNP proteins (heterogeneous nuclear ribonucleoprotein)
- Virtually all RNAs are coated with RNA-binding proteins
Hardly any RNA that isn’t bound by proteins
The DNA Is fluorescent white
The extent by which the RNA’s are bound with
protein (the red parts)

Generated by the immunofluorescence assay




RNA-binding motifs
RRM (RNA recognition motif) >> located on the protein
- RNA binding proteins often contain multiple RNA-binding domains: either of the same class
or of distinct
classes of
RNA-binding
motifs

Contain 2 RRM
domains, in both
cases the RNA that
gets bound is
included.

Blue is positive and
red is negative

Mainly the blue is in contact with the RNA
There are different ways in which the RRM can interact with RNA
- Other conserved RNA-binding motifs: RGG box; KH motif (K-homology motif)
- Charge of the arginine will contribute to the binding
RNA-binding assays
Electrophoretic mobility shift assay (EMSA)
RNA immunoprecipitation (RIP/CIP)
Can be done directly (native) or after cross linking
Cleavage and polyadenylation of pre-mRNAs
After RNA synthesis , there can still be cleavage of the RNA
- Poly (A) signal: very conserved but the main sequence. Downstream is a sequence that is rich
in G/U.
- CPSF: cleavage and polyadenylation specificity factor
- CStF: cleavage stimulatory factor
- CFI, CFII: cleavage factors
- Next step: binding of PAP = poly (A) polymerase