Multiple Steps in Gene Expression
after Transcription
1.
2.
3.
4.
5.
6.
7.
Transcription
5’ Capping
3' maturation: cleavage & polyadenylation
Splicing
Transport of RNA to Cytoplasm
Stabilization/Destabilization of mRNA
Translation
Relative cellular RNA abundance
• Ribosomal RNAs (rRNAs)
• Transfer RNAs (tRNAs)
• Messenger RNAs (mRNAs)
The rest (~3%):
• Signal recognition particle (SRP) RNA
• Small nuclear RNAs (snRNAs)
• Small nucleolar RNAs (snoRNAs)
• Micro RNAs (miRNAs)
~ 90%
~ 5%
~ 2%
Likely order of events in producing a
mature mRNA from a pre-mRNA.
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 nt of the RNA.
3. Occurs before the pre-mRNA is 30 nt long
Fig. 15.3
or “RNA triphosphatase”
Capping:
order of
events and
enzymes
AdoMet = S-adenosylmethionine,
the methyl donor
Product is Cap 1
Fig. 15.4
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
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
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!
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)
An Unexpected Mechanism for
Polyadenylation of pre-mRNA
1.
2.
3.
4.
Transcription extends
beyond mRNA end
Transcript is cut at 3’ end of
what will become the
mRNA (in green)
PolyA Polymerase adds
~250 As to 3’ end
“Extra” RNA (in red)
degraded
Fig. 15.12
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
Run-on
transcription
assay with
isolated
nuclei
Fig. 5.33
Only gene Y is transcribed.
Fig. 15.14
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).
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
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
Fig. 15.21, 2 ed.
U-rich*
* Absent from the globin gene
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.
Polyadenylation: The Proteins
Proteins required in mammals for cleavage
and polyadenylation of a new transcript.
James Manley
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
Model for the pre-cleavage complex
Fig. 15.19
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
2 Phases to polyadenylation
Hela-cell nuclear extract was incubated with radioactively-labeled RNA
substrates, which were then separated by gel electrophoresis.
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).
Conclusion: the AAUAAA not needed for phase 2, only phase 1.
Fig. 15.21
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
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)
Fig. 15.27,3rd ed.
CstF stimulation factor
CPSF specificity factor
CFI and CFII
PAP II
PAB II
Fig. 15.25