Post-transcriptional gene control

1
Post-transcriptional gene control
2
Subjects, covered in the lecture

• Processing of eukaryotic pre-mRNA
• -capping
• -polyadenylation
• -splicing
• -editing
• Nuclear transport

3
(No Transcript)
4
Processing of eukaryotic pre-mRNA the classical
texbook picture
5
Alternative picture co-transcriptional pre-mRNA
processing

• This picture is more realistic than the previous
  one, particularly for long pre-mRNAs

6
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)

7
3D structures of RNA recognition motif (RRM )
domains
8
Capping
p-p-p-N-p-N-p-N-p.
p-p-N-p-N-p-N-p
G-p-p-p-N-p-N-p-N-p
9
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?)

10
Capping mechanism in mammals
Growing RNA
Capping enzyme is allosterically controlled by
CTD domains of RNA Pol II and another stimulatory
factor hSpt5
DNA
11
Polyadenylation

• Poly(A) signal recognition
• Cleavage at Poly(A) site
• Slow polyadenylation
• Rapid polyadenylation

12

• 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

13
PAP Poly(A) polymerase
14
(No Transcript)
15
PAP
CPSF
16
PABPII- poly(A) binding protein II
17

• PABP II functions
• rapid polyadenylation
• polyadenylation termination

18
Link between polyadenylation and transcription
mRNA
Pol II
ctd
p
p
PolyA binding factors
cap
19
Splicing
20
The size distribution of exons and introns in
human, Drosophila and C. elegans genomes
21
Consensus sequences around the splice site
YYYY
22
Molecular mechanism of splicing
23
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

24
The secondary structure of snRNAs
25
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
26
The essential steps in splicing
Binding of U1 and U2 snRNPs
Binding of U4, U5 and U6 snRNPs
27
Rearrangement of base-pair interactions between
snRNAs, release of U1 and U4 snRNPs
28
The catalytic core, formed by U2 and U6 snRNPs
catalyzes the first transesterification reaction
29
Further rearrangements between U2, U6 and U5 lead
to second transesterification reaction
30
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
31
Co-transciptional splicing
mRNA
Pol II
ctd
p
SRs
p
snRNPs
SCAFs SR- like CTD associated factors
Intron
cap
32
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

33
Group I introns utilize guanosine cofactor, which
is not part of RNA chain
34
Comparison of secondary structures of group II
self-splicing introns and snRNAs
35
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?

36

• 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

37
One gene several proteins

• Cleavage at alternative poly(A) sites
• Alternative promoters
• Alternative splicing of different exons
• RNA editing

38
Alternative splicing, promoters poly-A cleavage
39
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

40
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

41
Guide RNAs (gRNAs) are required for editing
42
Organization of pre-rRNA genes in eukaryotes
43
Electron micrograph of tandem pre-rRNA genes
44
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
• removing introns from pre-rRNA
• methylation of 2 OH groups at specific sites
• converting of uridine to pseudouridine

45
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

46
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

47
Assembly of ribosomes
48
Processing of pre-tRNAs
RNase P cleavage site
49
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

50
Macromolecular transport across the nuclear
envelope
51
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

52
Two different kinds of nuclear location sequences
Proteins which are transported into nucleus
contain nuclear location sequences
basic
hydrophobic
importin a
importin b
importin b
nuclear import
53
Artifical fusion of a nuclear localization signal
to a cytoplasmatic protein causes its import to
nucleus
54
Mechanism for nuclear import
55
Mechanism for nuclear export
56
Mechanism for mRNA transport to cytoplasm
57
Example of regulation at nuclear transport level
HIV mRNAs
58
After mRNA reaches the cytoplasm...

• mRNA exporter, mRNP proteins, nuclear cap-binding
  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

59
(No Transcript)