Transcript Post-transcriptional gene control

Post-transcriptional gene control
Subjects, covered in the lecture
• Processing of eukaryotic pre-mRNA
-capping
-polyadenylation
-splicing
-editing
• Nuclear transport
Processing of eukaryotic pre-mRNA: the
classical texbook picture
Alternative picture: co-transcriptional
pre-mRNA processing
• This picture is more
realistic than the
previous one,
particularly for long
pre-mRNAs
Heterogenous ribonucleoprotein patricles
(hnRNP) proteins
• In nucleus nascent RNA transcripts are associated
with abundant set of proteins
• hnRNPs prevent formation of secondary structures
within pre-mRNAs
• hnRNP proteins are multidomain with one or more
RNA binding domains and at least one domain for
interaction with other proteins
• some hnRNPs contribute to pre-mRNA
recognition by RNA processing enzymes
• The two most common RNA binding domains are
RNA recognition motifs (RRMs) and RGG box
(five Arg-Gly-Gly repeats interspersed with
aromatic residues)
3D structures of RNA recognition
motif (RRM ) domains
Capping
p-p-p-N-p-N-p-N-p….
Capping enzyme
(mCE)
p-p-N-p-N-p-N-p…
GMP
mCE (another subunit)
G-p-p-p-N-p-N-p-N-p…
S-adenosyl
methionine
methyltransferases
CH3
G-p-p-p-N-p-N-p-N-p…
CH3 CH3
The capping enzyme
• A bifunctional enzyme with both 5’-triphosphotase
and guanyltransferase activities
• In yeast the capping enzyme is a heterodimer
• In metazoans the capping enzyme is monomeric
with two catalytic domains
• The capping enzyme specific only for RNAs,
transcribed by RNA Pol II (why?)
Capping mechanism in mammals
Growing
RNA
DNA
Capping enzyme is allosterically controlled by
CTD domains of RNA Pol II and another
stimulatory factor hSpt5
Polyadenylation
•
•
•
•
Poly(A) signal recognition
Cleavage at Poly(A) site
Slow polyadenylation
Rapid polyadenylation
• G/U: G/U or U rich
region
• CPSF: cleavage and
polyadenylation
specificity factor
• CStF: cleavage
stimulatory factor
• CFI: cleavage factor I
• CFII: cleavage factor II
PAP: Poly(A) polymerase
PAP
CPSF
PABPII- poly(A) binding protein II
PABP II functions:
1. rapid polyadenylation
2. polyadenylation termination
Link between polyadenylation and transcription
FCP1 Phosphatase
removes phospates
from CTDs
Pol II gets
recycled
mRNA
Pol II
aataaa
c
t p
d p
PolyA – binding
factors
cap
degradation
p
p
cap
polyA
mRNA gets cleaved
and polyadenylated
cap
splicing,
nuclear
transport
Splicing
The size distribution of exons and introns in
human, Drosophila and C. elegans genomes
Consensus sequences around the
splice site
YYYY
Molecular
mechanism
of splicing
Small nuclear RNAs U1-U6
participate in splicing
• snRNAs U1, U2, U4, U5 and U6 form complexes with 6-10 proteins
each, forming small nuclear ribonucleoprotein particles (snRNPs)
• Sm- binding sites for snRNP proteins
The secondary structure of
snRNAs
Additional factors of exon recognition
ESE - exon splicing enhancer sequences
SR – ESE binding proteins
U2AF65/35 – subunits of U2AF factor, binding to pyrimidine-rich
regions and 3’ splice site
The essential steps in splicing
Binding of U1 and
U2 snRNPs
Binding of U4,
U5 and U6
snRNPs
Rearrangement of
base-pair
interactions
between snRNAs,
release of U1 and
U4 snRNPs
The catalytic core,
formed by U2 and
U6 snRNPs
catalyzes the first
transesterification
reaction
Further
rearrangements
between U2, U6
and U5 lead to
second
transesterification
reaction
The spliced lariat is linearized by debranching
enzyme and further degraded in exosomes
Not all intrones are completely degraded. Some end
up as functional RNAs, different from mRNA
Co-transciptional splicing
mRNA
Pol II
snRNPs
SRs
c
t
d p
p
SCAFs: SR- like
CTD – associated
factors
Intron
cap
Self-splicing introns
• Under certain nonphysiological conditions
in vitro, some introns can get spliced
without aid of any proteins or other RNAs
• Group I self-splicing introns occur in rRNA
genes of protozoans
• Group II self-splicing introns occur in
chloroplasts and mitochondria of plants and
fungi
Group I introns utilize guanosine cofactor, which is not part of RNA
chain
Comparison of secondary structures of group II selfsplicing introns and snRNAs
Spliceosome
• Spliceosome contains snRNAs, snRNPs and many
other proteins, totally about 300 subunits.
• This makes it the most complicted macromolecular
machine known to date.
• But why is spliceosome so extremely complicated if
it only catalyzes such a straightforward reaction as
an intron deletion? Even more, it seems that some
introns are capable to excise themselves without aid
of any protein, so why have all those 300 subunits?
• No one knows for sure, but there might be at least
4 reasons:
• 1. Defective mRNAs cause a lot of problems for
cells, so some subunits might assure correct
splicing and error correction
• 2. Splicing is coupled to nuclear transport, this
requires accessory proteins
• 3. Splicing is coupled to transcription and this
might require more additional accessory proteins
• 4. Many genes can be spliced in several alternative
ways, which also might require additional factors
One gene – several proteins
•
•
•
•
Cleavage at alternative poly(A) sites
Alternative promoters
Alternative splicing of different exons
RNA editing
Alternative splicing, promoters &
poly-A cleavage
RNA editing
• Enzymatic altering of pre-mRNA sequence
• Common in mitochondria of protozoans and plants and
chloroplasts, where more than 50% of bases can be altered
• Much rarer in higher eukaryotes
Editing of human apoB pre-mRNA
The two types of editing
1) Substitution editing
• Chemical altering of individual nucleotides
• Examples: Deamination of C to U or A to I
(inosine, read as G by ribosome)
2) Insertion/deletion editing
•Deletion/insertion of nucleotides (mostly uridines)
•For this process, special guide RNAs (gRNAs) are
required
Guide RNAs (gRNAs) are required for editing
Organization of pre-rRNA genes
in eukaryotes
Electron micrograph of tandem
pre-rRNA genes
Small nucleolar RNAs
•
•
~150 different nucleolus restricted RNA species
snoRNAs are associated with proteins, forming small
nucleolar ribonucleoprotein particles (snoRNPs)
• The main three classes of snoRNPs are envolved in
following processes:
a) removing introns from pre-rRNA
b) methylation of 2’ OH groups at specific sites
c) converting of uridine to pseudouridine
What is this pseudouridine good for?
Uridine ( U )
Pseudouridine ( Y )
• Pseudouridine Y is found in RNAs that have a tertiary
structure that is important for their function, like rRNAs,
tRNAs, snRNAs and snoRNAs
• The main role of Y and other modifications appears to be
the maintenance of three-dimensional structural integrity in
RNAs
Where do snoRNAs come from?
• Some are produced from their own promoters by
RNA pol II or III
• The majority of snoRNAs come from introns of
genes, which encode proteins involved in ribosome
synthesis or translation
• Some snoRNAs come from intrones of genes,
which encode nonfuctional mRNAs
Assembly of
ribosomes
Processing of pre-tRNAs
RNase P
cleavage
site
Splicing of pre-tRNAs is different
from pre-mRNAs and pre-rRNAs
• The splicing of pre-tRNAs is catalyzed by
protein only
• A pre-tRNA intron is excised in one step,
not by two transesterification reactions
• Hydrolysis of GTP and ATP is required to
join the two RNA halves
Macromolecular transport across the
nuclear envelope
The central channel
• Small metabolites, ions and globular
proteins up to ~60 kDa can diffuse freely
through the channel
• Large proteins and ribonucleoprotein
complexes (including mRNAs) are
selectively transported with the assistance
of transporter proteins
Proteins which are transported into nucleus contain
nuclear location sequences
Two different kinds of nuclear location sequences
basic
hydrophobic
importin a
importin b
importin b
nuclear import
Artifical fusion of a nuclear localization signal to a
cytoplasmatic protein causes its import to nucleus
Mechanism for nuclear “import”
Mechanism for nuclear “export”
Mechanism for mRNA transport to cytoplasm
Example of regulation at nuclear transport level:
HIV mRNAs
After mRNA reaches the cytoplasm...
• mRNA exporter, mRNP proteins, nuclear capbinding complex and nuclear poly-A binding
proteins dissociate from mRNA and gets back to
nucleus
• 5’ cap binds to translation factor eIF4E
• Cytoplasmic poly-A binding protein (PABPI)
binds to poly-A tail
• Translation factor eIF4G binds to both eIF4E and
PABPI, thus linking together 5’ and 3’ ends of
mRNA