The document summarizes several key steps in gene expression after transcription in eukaryotic cells. These include 5' capping, 3' cleavage and polyadenylation of pre-mRNA, splicing, transport of mRNA from the nucleus to cytoplasm, and translation. It focuses on the mechanisms and protein factors involved in RNA capping and 3' end processing, including the AAUAAA polyadenylation signal, GU/U-rich elements, and the roles of CPSF, CstF, PAP, and PAB proteins. Transcription is shown to extend beyond the polyadenylation site, and the polyA tail is added co-transcriptionally in two phases requiring different protein complexes and the AAUAAA
5. RNA Capping
1. Post-transcriptional (i.e., G not encoded)
2. Involves adding a 7MeGuanosine nt to the
first (RNA) nt in an unusual way, and
often methylation of the first few
nucleotides of the RNA.
3. Occurs before the pre-mRNA is 30 nt long
8. Cap Functions
Cap provides:
1. Protection from some ribonucleases*
2. Enhanced translation*
3. Enhanced transport from nucleus
4. Enhanced splicing of first intron for
some pre-mRNAs
*Also functions of the polyA-tail
9. Post-transcriptional Processes II:
Pre-mRNA Polyadenylation
• Most cytoplasmic mRNAs have a polyA tail (3’
end) of 50-250 Adenylates
– a notable exception is histone mRNAs
• Discovered in 1971 (J. Darnell et al.)
• Added post-transcriptionally by an enzyme,
polyA polymerase(s)
• Turns over (recycles) in cytoplasm
10. Functions of the PolyA-Tail
1. Promotes mRNA stability
- De-adenylation (tail shortening) can
trigger rapid degradation of the RNA
2. Enhances translation
- promotes recruitment by ribosomes
- bound by a polyA-binding protein in
the cytoplasm, PAB1
- synergistic stimulation with Cap!
11. Methods: The firefly-luciferase mRNAs (with the 5’ and 3’
UTRs from a plant gene) were electroporated into
protoplasts. Then, luciferase mRNA, and luciferase activity
levels were measured at select times. (from D. Gallie)
12. An Unexpected Mechanism for
Polyadenylation of pre-mRNA
1. Transcription extends
beyond mRNA end
2. Transcript is cut at 3’ end
of what will become the
mRNA (in green)
3. PolyA Polymerase adds
~250 As to 3’ end
4. “Extra” RNA (in red)
degraded
13. Evidence for Transcription Past
the End of a Cellular mRNA
• Nuclear run-on transcription assay using
Friend erythroleukemic cells treated with
DMSO (stimulates globin gene transcription)
– Newly synthesized RNAs are hybridized to
DNA regions that span the globin gene,
including regions downstream
– The amount of newly synthesized RNA
complementary to each gene region is thus
quantified
15. Transcription beyond the polyA site.
After run-on transcription (in the presence of 32P-UTP) with nuclei from
Friend Erythroleukemic cells, the labeled RNA was hybridized to DNA
fragments A-F that span the globin gene. The relative molarities of
newly synthesized RNA that hybridized to each fragment are given.
s.d. is the standard deviation.
Notice that there is just as much RNA transcribed from just
downstream of the gene (fragment E) as there is from the
gene itself (fragments B-D).
Fig. 15.14
16. Polyadenylation (PolyA) Signal
• AAUAAA (in mammals and plants)
– Located ~20-30 bp from the polyA-tail
– There are other hexamers that will
provide this function, but less efficiently.
• Mutagenesis and in vivo expression studies
revealed 2 other motifs downstream of
AAUAAA that promote 3’ processing.
1. GU-rich stretch
2. U-rich stretch
17. What if there are multiple possible
signals?
Competition experiment:
1. The synthetic polyadenylation site (SPA) below
was inserted downstream of the normal one in a
globin gene (giving a globin gene with 2 signals).
GU-rich U-rich*
* Absent from the globin gene
18. 2. The globin gene with two 3’ processing signals
was introduced into HeLa cells, and the 3’ end of
the mRNA determined by S1 mapping.
Result:
• SPA was mainly used for polyAdenylation
Conclusion
• The stronger set of elements (signals) in the SPA
outcompeted the native 3’-processing signal,
which lacks the U-rich motif.
19. Polyadenylation: The Proteins
Proteins required in mammals for cleavage
and polyadenylation of a new transcript.
Proteins required for efficient cleavage of pre-mRNA:
1. CPSF (cleavage & polyadenylation specificity
factor), binds the AAUAAA
2. CstF (cleavage stimulation factor) binds to the G/U
rich region cooperatively with CPSF
3. CFI and CFII (cleavage factors I and II), RNA-binding
proteins
4. PAP (polyA polymerase)
5. nRNAP II (the CTD of the very large RPB1 subunit)
stimulates cleavage
21. Polyadenylation: Mechanism
• Occurs in 2 phases
–Phase 1: requires AAUAAA and ~8 nt
downstream (3’)
–Phase 2 : Once ~10 As are added,
further adenylation does not require
the AAUAAA
22. 2 Phases to polyadenylation
Fig. 15.21
Substrates:
1. 58-nt RNA from SV40
that ends with AAUAAA
plus 8 nt ( ).
2. Same as (1), but with
a 40-nt polyA-tail (A40).
3. Same as (1), but with
a random 40-nt at the 3’
end (X40).
The series with an X
contain a mutated
AAUAAA (AAGAAA).
Hela-cell nuclear extract was incubated with radioactively-labeled RNA
substrates, which were then separated by gel electrophoresis.
Conclusion: the AAUAAA not needed for phase 2, only phase 1.
23. Proteins Required for
Polyadenylation
Phase I:
1. CPSF
2. PolyA polymerase (PAP II)
Phase II:
1. PolyA polymerase (PAP II)
2. PolyA Binding Protein II (PAB II)
- PAB II binds to short A-tail
- Helps PAP II synthesize long tails
24. Nuclear PolyA Polymerase (long
form = PAP II)
RBD - RNA binding domain
NLS - nuclear localization signal
PM - polymerase module
S/T- serine/threonine rich regions (yellow)
