Degradation Decay, Turnover of Eukaryotic mRNAs

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Degradation (Decay, Turnover) of Eukaryotic mRNAs
A. Basic processes of mRNA decay

• Deadenylation
• Exonuclease, 3-5 (exosome)
• 5-decapping
• Exonuclease, 5-3 (Xrn1p)

B. mRNA decay in quality control

• Nonsense mediated decay (NMD)
• Nonstop decay
• No-go decay

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Pathways of Nonsense-mediated Decay (NMD)
Nonsense-mediated decay
Decay of aberrant mRNAs with a premature
termination codon (PTC)
How can a cell determine whether a stop codon
is premature? In mammals, it uses splicing history
Exon junction complex (EJC) deposited upon
splicing
If stop codon is far upstream from last EJC
complex, the stop codon is labeled as premature
and NMD is triggered
NMD is dependent on translation, indicating the
the ribosome identifies the stop codon
First, or pioneering round of translation is
thought to be different from subsequent rounds
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Pathways of Nonsense-mediated Decay (NMD)
Yeast use a different mechanism to detect
premature termination codon
Detection relies on large distance between stop
codon and poly(A) sequence (or Pab1p), but
mechanism is unclear
Artificially tethering Pab1p just downstream
from a stop codon abolishes NMD, indicating that
Pab1 is centrally involved
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mRNA Decay Pathway in NMD
Principally decapping and 5-3 exo in both
yeast and mammals
Here, decapping does not require deadenylation
Dcp1/2 recruited by Upf1p
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Nonstop Decay When mRNAs Lack a Termination Codon
Analogous to process carried out by prokaryotic
tmRNA
Nonstop RNA can occur -When polyadenylation
occurs prematurely - When transcription aborts
- Upon incomplete 3-5 decay
Functions of nonstop decay 1. Recycle
ribosomes 2. Degrade RNA
Ski7p binds empty A site and recruits exosome
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No-go mRNA Decay When Translation Stalls
Established experimentally by long hairpin
(Parker and colleagues, 2006)
Requires protein Dom34, related to release
factor
Binding of Dom34 and Hbs1 presumably recruit
endonuclease (or proposed that ribosome itself is
the endonuclease)
Decay then proceeds 3-5 by exosome (for the
5 fragment) and 5-3 by Xrn1p (for the 3
fragment)
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Key Points
1. RNA decay is critical for gene regulation, RNA
quality control, and viral defense. Here we
discussed the first two roles.
2. The basic process of RNA decay is
deadenylation followed either by 1) continued
exonuclease digestion 3 to 5 by the exosome, or
2) decapping. If decapped, the mRNA is typically
degraded 5 to 3 by Xrn1p.
3. Complex systems are present to ensure the
degradation of damaged mRNAs that are unable to
be translated accurately into protein. These
pathways include nonsense-mediated decay (NMB),
nonstop decay, and no-go decay.
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Protein Degradation In the Eukaryotic Cytoplasm
Degradation is an important component of the
cellular regulatory machinery. Destruction of a
protein assures that it is completely and
irreversibly inactivated.
Cellular proteolysis machinery must
-Processively degrade protein in a sequence
non-specific manner -Be very tightly regulated
so as to avoid degrading non-targeted proteins
Solution Proteases are sequestered inside a
chambered machine termed the proteasome
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Protein Degradation In the Eukaryotic Cytoplasm
1. Structures and properties of the proteasome
2. Tagging proteins for destruction by attachment
of ubiquitin
3. What determines which proteins are
ubiquitinated The N-end rule
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Chambered Proteases In Prokaryotes and Eukaryotes
Pickart and Cohen, Nat. Rev. Mol. Cell Biol.
(2004) 5, 177-187
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Electron-microscopy Images of Yeast Proteasome
? and ? rings each have seven subunits
? subunits control passage of polypeptide
substrates into ? region and may allow repeated
cycles of action
Three of seven ? subunits have protease active
sites, and there are two ? rings per proteasome
complex.
The entire proteasome therefore has six active
sites of three different types
Lid and base additions at end contain ATPases
Pickart and Cohen, Nat. Rev. Mol. Cell Biol.
(2004) 5, 177-187
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Model of Eukaryotic Proteasome
Zwickl and Baumeister, Ann. Rev. Biochem. (1999)
68, 1015-1068
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Subunit Composition of the Yeast Proteasome
ATPases
Pickart and Cohen, Nat. Rev. Mol. Cell Biol.
(2004) 5, 177-187
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Substrate Proteins Are Unfolded As They Enter the
Proteasome
Very large changes in substrate protein
stability give only small changes in degradation
rate
ATPases in 19S portion actively unfold proteins
to allow them to enter the proteasome
Mechanism is apparently to push or pull the
polypeptide chain into the cavity. Entry rate is
sensitive to structural features at initial
engagement site but not global stability
Peptides with intra-chain crosslinks can be
degraded, indicating that more than one
polypeptide chain can enter at the same time, or
that an internal loop can be the initial
engagement site
Once inside, the protein is extensively
degraded. The active sites hydrolyze peptide
bonds with high efficiency and little
specificity. Fragments are presumably trapped by
the lids until they are cleaved many times and
are very short.
Pickart and Cohen, Nat. Rev. Mol. Cell Biol.
(2004) 5, 177-187
15
Protein Degradation In the Eukaryotic Cytoplasm
1. Structures and properties of the proteasome
2. Tagging proteins for destruction by attachment
of ubiquitin
3. What determines which proteins are
ubiquitinated The N-end rule
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Pathway For Ubiquitination and Degradation By the
Proteasome
Zwickl and Baumeister, Ann. Rev. Biochem. (1999)
68, 1015-1068
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Activation of Ubiquitination and Attachment to a
Target Protein
Ubiquitin is first activated by reaction with
ATP, catalyzed by E1 enzyme
Ubiquitin is then attached to E1 with a
thio-ester linkage, with AMP as leaving group
In the next step, ubiquitin is transferred to
E2, again with thio-ester linkage
Last, ubiquitin is transferred to Lys of target
protein in a process directed by E3
Pickart, Cell (2004) 116, 181-190
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ATP Is Used in Two Steps of the Pathway For
Destruction of Protein
ATP is necessary both for ubiquitination
process and for unfolding of substrate proteins
Pickart, Cell (2004) 116, 181-190
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While the Protein Substrate Is Destroyed,
Ubiquitin Is Spared
Ubiquitin is released, but only after substrate
is engaged and translocation begins
Rpn11 mediates ubiquitin release by breaking
its covalent connection with the substrate
protein. In 26S proteasome, the reaction is
strictly dependent on ATP. ATP-dependent
translocation is apparently necessary to
translocate the polypeptide into position for
removal of the ubiquitin
Substrates that contain a non-cleavable
ubiquitin variant G76V are ultimately
proteolyzed, including the ubiquitin, but
cleavage is much slower
Pickart and Cohen, Nat. Rev. Mol. Cell Biol.
(2004) 5, 177-187
20
Protein Degradation In the Eukaryotic Cytoplasm
1. Structures and properties of the proteasome
2. Tagging proteins for destruction by attachment
of ubiquitin
3. What determines which proteins are
ubiquitinated The N-end rule
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What Targets a Protein For Destruction? The N-end
Rule
Initially discovered by A. Varshavsky in 1986
Varshavsky, PNAS (1996) 93, 12142-9
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Amino Acids Are Classified As Stabilizing or
Destabilizing
secondary
tertiary
Varshavsky, PNAS (1996) 93, 12142-9
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What Targets a Protein For Destruction? The N-end
Rule
Secondary and tertiary destabilizing residues are
enzymatically converted into primary ones
Varshavsky, PNAS (1996) 93, 12142-9
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Key Points
1. Eukaryotic proteins are degraded in the
cytoplasm by a large, complex machine called the
proteasome. Proteins are protected from random
encounters with the proteasome by being denied
entry into the soluble inside chamber, where the
protease active sites are sequestered.
2. The process of degrading proteins depends on
ATP, both for tagging the proteins with ubiquitin
and for unfolding the proteins as they enter the
proteasome.
3. The N-terminal amino acid of proteins is an
important determinant of their susceptibility to
degradation by the proteasome.
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