Eukaryotic%20mRNA%20Transcripts%20are%20Processed

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Eukaryotic mRNA Transcripts are Processed
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A Simple Eukaryotic Gene
Transcription
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Processing of eukaryotic mRNA

• RNA processing achieves three things
• 1) Removal of Introns
• 2) Addition of a 5 cap
• 3) Addition of a 3 tail
• The mRNA then moves out of the nucleus and is
  translated in the cytoplasm.

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Translational control
Structure of eukaryotic and prokaryotic mRNAs
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Model of eukaryotic ribosome
rRNAs are believed to play a catalytic role in
protein synthesis. After removal of 95 of the
ribosomal proteins, the 60S subunit can catalyze
formation of peptide bonds. Ribosomal proteins
are now believed to help fold the rRNAs properly
and to position the tRNAs.
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• Small large ribosomal subunits.
• A Binding site for
• the mRNA is present on small subunit.
• Two binding sites
• (P A) bind tRNAs on large subunit.
• P site holds the tRNA carrying the growing
  polypeptide chain.
• A site holds the tRNA with the next AA to be
  added.
• Ribosomes hold the mRNA and tRNAs together and
  connect the amino acids at the A site to the
  growing polypeptide.

Ribosome Structure
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• Structure of tRNA
• Aligns each amino acid with the corresponding
  codon
• 70-80 nt long
• 3 end has the 5- CCA sequence to which aa
  are linked
• The opposite end contains the anticodon loop
• Contains modified bases

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Many RNA Viruses have capped genomic RNAs similar
to eukaryotic host mRNAs
Most eukaryotic mRNAs are capped at the 5
end during nuclear processing. The terminal 5
phosphate is first removed by a 5
triphosphatase. Guanyltransferase transfers GMP
from GTP to the 5 end of the mRNA to add the
GpppN cap structure. The 5 terminal inverted
G residue is then modified by methylation.
Many RNA viruses replicate in the cytoplasm and
must use a viral dependent capping mechanism
supplied by the RNA-Dependent-RNA Polymerase.
The Cap structure, m7GpppN, is most common in
viral and mammalian mRNAs.
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• Three distinct stages of translation
•  Initiation
•  Rate limiting step
•  Requires hydrolysis of ATP and GTP
•  Results in formation of a complex containing
  the mRNA, the ribosome and the initiator Met-tRNA
•  A. 5 end (Cap) dependent initiation
• The initiation complex binds to the 5 cap
  structure and scans in a 5 to 3 direction
  until initiating AUG is encountered
• B. Internal ribosome entry
•   Initiation complex binds upstream of initiation
  codon

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5 end (cap) dependent initiation

• The first step is the recognition of the 5 cap
  by eIF4F, which consists of three proteins,
  eIF4E, eIF4G and eIF4A.
• Cap binding protein, eIF4E, binds to cap
• The N-terminus of eIF4G binds eIF4E and the
  C-terminus binds eIF4A
• The 40S subunit binds to eIF4G via eIF3

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Cap-Dependent Initiation of Protein Synthesis in
Eukaryotes
An initiation complex forms at the cap with the
40S ribosomal subunit and other translation
initiation factors.
The 40S complex then scans down the 5
untranslated region to the first AUG codon.
A GTP hydrolysis step by eIF5 triggers GDP
binding of eIF2 and release of initiation
proteins.
The 60S subunit joins the complex and the 80S
ribosome initiates translate the ORF.
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Elongation
eEF1a
GTP
Ribosome selects aminoacylated tRNA eEF1a and
GTP are bound to aminoacylated tRNA Ribosome
catalyzes formation of a peptide
bond Translocation is dependent on eEF2 and GTP
hydrolysis Many ribosomes may translate mRNAs
simultaneously on the same strand.
P A
eEF2
GTP
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• Termination
• Translation is terminated
• at one of three stop codons (UAA, UAG UGA).
• Termination codon at the A site is recognized
  by the release factor instead of a tRNA
• The release factor binds the termination codon
• The peptide chain is then released followed by
  dissociation of the tRNA and the ribosome

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Closed loop model

• The 5 end dependent initiation is stimulated
  by the poly(A) binding protein Pabp1p, which
  interacts with eIF4G
• This interaction circularizes the mRNA and
  facilitates formation of the initiation complex
• Mechanism to ensure that only intact mRNA is
  translated

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5end (cap) independent initiation
Poliovirus

• mRNAs of picornaviruses lack 5 cap
• Ribosomes bind internally rather than at the
  mRNA 5 end
• 5 end of poliovirus RNA promotes internal
  binding of 40S subunit at internal ribosome entry
  site (IRES)
• In poliovirus infected cells eIF4G is cleaved,
  inactivating translation of cellular mRNAs
• The initiation in the IRES does not depend on the
  presence of a cap structure, but requires
  C-terminal fragment of eIF4G to recruit the 40S
  subunit through interaction with eIF3.

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Five different types of IRES sequences are found
on viral RNAs  Type I-entero and rhinoviruses
(poliovirus) Initiation codon is located past
the 3 end of the IRES   40S binds to IRES scans
to AUG Type II-cardio and apthoviruses
(EMCV) Initiation codon is at the 3 end of the
IRES 40S binds at or near AUG no scanning
occurs Type III- hepatitis A virus initiation
codon is located past the 3 end of the
IRES requires all of initiation proteins,
including eIF4E Type IV- hepatitis C virus The
3 end of the hepatitis C virus IRES extends
beyond the AUG codon Type V-cricket paralysis
virus IRES ends at the initiation codon,
although it is not an AUG codon, no initiation
factors are required initiation codon is placed
at the A site instead of the P site
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Four types of IRES
Hepatitis C
polivirus
Encephelomyocarditis virus (EMCV)
Cricket paralysis virus
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Hepatitis C virus IRES

• 40S ribosomal subunit binds directly to
  Hepatitis C virus IRES in the absence of most
  initiation factors
• A dramatic change in the conformation of the 40S
  subunit occurs when it binds Hepatitis C virus
  IRES setting the AUG at the P site
• eIF3 also binds to the Hepatitis C virus IRES

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Bicistronic mRNA assay for IRES elements
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Viral translation strategies
Polyprotein synthesis   Picornaviruses- Entire
() sense RNA genome is translated into a single
large polyprotein. Processing is carried out by
two virus encoded proteases 2A pro and 3C
pro.   Flaviviruses- Viral precursor proteins are
processed by cellular proteases. The () sense
RNA genome is translated into a polyprotein
precursor processed by viral serine protease and
by host signal peptidase.   Potyvirus group of
plant viruses- Potato virus Y and tobacco etch
virus contain a () sense genome RNA of around
10,000 bases which has a single open reading
frame. This polyprotein is processed by viral
encoded proteases.
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Polyprotein processing in enteroviruses and
flaviviruses
Poliovirus
Flavivirus
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Leaky scanning   Although majority of
eukaryotic mRNAs are monocistronic, some viral
mRNAs encode overlapping reading frames. The
first start site is in a poor context, some
ribosomes can bypass it and initiate at the
second AUG, which has a better context. This
will result in translation of two different
proteins.
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Reinitiation   Rare in eukaryotes, but very
common in prokaryotic cellular and viral mRNAs.
Some eukaryotic mRNAs contain upstream AUG codons
that terminate before the downstream reading
frame. The upstream open reading frames may be
translated, with reinitiation occurring at the
downstream open reading frame.  
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Reinitiation of translation in influenza B virus
In influenza B virus mRNA, M2 initiation codon is
part of the termination codon for M1 protein. M2
synthesis is not efficient and dependent on M1
synthesis
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Suppression of termination   Suppression of
termination occurs during translation of may
viral mRNAs as a means of generating a second
protein with extended carboxy terminus. In
retroviruses, gag and pol genes are encoded by a
single mRNA and separated by an amber termination
codon UAG. Translational suppression of the
amber codon allows synthesis of the gag pol
precursor.   A similar strategy is used by
tobacco mosaic virus to translate its replicase
proteins.   Translation suppression is mediated
by suppressor tRNAs that can recognize
termination codons and insert a specific amino
acid. The nucleotide sequence 3 of the
termination codon also plays an important role in
the efficiency of translational suppression.
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Suppression of termination codons in alphaviruses
and retroviruses
Pseudonot
alphavirus
retrovirus
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Ribosomal frameshifting   Ribosomal frameshifting
is a process in which ribosomes move to a
different reading frame and continue translation
in that reading frame.   It was discovered in
cells infected with Rous sarcoma virus and has
since been described for many other viruses
including other retroviruses, () strand RNA
viruses and herpes simplex virus.   Requires a
slippery sequence X-XXY-YYZ (in Rous sarcoma
virus A-AAU-UUA) and an RNA secondary structure
called a pseudoknot five to eight nucleotides
downstream.   Two tRNAs in the zero reading frame
slip back by one nucleotide to the 1 phase and
each tRNA base pairs with the mRNA in the first
two nucleotides of each codon.   As a result of
the frameshift a Gag-pol fusion is produced at
about 5 of the level of Gag protein.  
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Ribosomal frameshifting
Rous sarcoma virus mRNA encodes Gag and Pol
proteins that overlap in a 1 reading frame
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REGULATION OF TRANSLATION DURING VIRAL
INFECTION   Interferons are produced in response
to viral infection as part of the rapid innate
immune response Interferons bind to cell
surface receptors and activate transcription of
antiviral genes Two interferon induced genes
encode RNase L and protein kinase RNA-activated
(Pkr) RNase L degrades RNA Pkr phosphorylates
eIF2a, inhibiting translation initiation  Pkr is
a serine threonine kinase composed of an
N-terminal regulatory domain and a C-terminal
catalytic domain   Pkr is activated by the
binding of dsRNA to two dsRNA binding motifs at
the N-terminus of the protein.  
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Model of activation of Pkr
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  Viral regulation of Pkr Viruses use at least
five different mechanisms to block Pkr activation
or to stop activated Pkr from inhibiting
translation   inhibition of dsRNA binding-
adenovirus VA RNA binds Pkr blocks its
activation by dsRNA   vaccinia virus E3L
protein sequesters ds RNA   inhibition of Pkr
dimerization influenza virus P58 hepatitis C
virus NS5A   inhibitors of kinase function-
vaccinia virus K3L protein has homology to
N- terminus of eIF2-a    
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Regulation of eIF4F activity by different viruses