mRNA stability and localization.RNA is critical at many stages of gene expression. How frequently it will be translated, how long it is likely to survive, and where in the cell it will be translated. RNA cis-elements & associated proteins

1. mRNA Stability and
Localization
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Presented by: Sepideh saroughi

2. 1. Introduction
•RNA is critical at many stages of gene expression
•Intermediate in protein synthesis
•How frequently it will be translated, how long it is likely to survive,
and where in the cell it will be translated
•RNA cis-elements & associated proteins
2

3. Features of prokaryotic and eukaryotic mRNAs:
a) Bacterial mRNA
b) Eukaryotic mRNAs
c) Histone mRNAs
3

4. 2. Messenger RNAs Unstable Molecules
1) mRNA instability Action of ribonucleases
2) Ribonucleases differ in their substrate preference and mode
of attack
3) Half-lives
4) Differential mRNA stability
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5. •Unstable molecules
•Ribonucleases: DNA replication, DNA repair, processing of new transcripts &
the degradation of mRNA.
1) Endoribonucleases
2) Exoribonucleases
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6. •Most mRNAs decay stochastically
•Stability half-life
•mRNA decay = mRNA degradation
•Decay rate predominates
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7. 3. Eukaryotic mRNAs mRNPs
1) mRNA associates with population of proteins
2) mRNP proteins have roles in the cytoplasm
3) RNA-binding proteins uncharacterized
4) mRNAs overlapping : regulon
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8. •Eukaryotic mRNAs RNPs
•Nuclear maturation: RNA sequence & complement of proteins + TREX EXIT
•Pioneering round : defective template destroyed
•The “nuclear history” of an mRNA is critical in determining its fate
in the cytoplasm.
•RBPs
•RNA regulon: The set of mRNAs
that share a particular type of RBP
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9. 4. Prokaryotic mRNA Degradation Multiple
Enzymes
1) Initiated by removal of a pyrophosphate from the 5′ terminus
2) Monophosphorylated mRNAs two-step cycle
3) 3′ polyadenylation : facilitate the degradation
4) Main degradation enzymes : degradosome
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10. •In prokaryotes: mRNA degradation occurs during the process of
coupled transcription/translation.
•Prokaryotic ribosomes begin translation even before transcription
is completed
•polyribosome or polysome
•Endonuclease + 3′ to 5′ exonuclease = mRNA degradation
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11. •The major mRNA degradation pathway in E. coli is a multistage
process:
1) pyrophosphate from the 5′ terminus
single phosphate
Monophosphorylate RNaseE
cut near the 5′ end of the mRNA
monocistronic mRNA
2) PNPase(3′ to 5′ exonuclease) upstream fragment
• This two-step ribonuclease cycle is repeated along the
length of the mRNA
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12. • PNPase are unable to progress through double-stranded regions = stem-loop structure at
the 3′ end mRNAs protects
• Degradosome RNase E (N-terminal: endonuclease / C-terminal: scaffold)
PNPase (degradation: endonuclease + exonuclease )
• Inactivation of RNase E mRNA degradation
• Mutations PNPase no effect on overall mRNA stability
• The half-lives of specific mRNAs can be altered by different cellular
physiological states such as starvation or other forms of stress.
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13. 5. Degraded Two
Deadenylation-Dependent Pathways
1) Modifications at both ends of mRNA protect against
degradation
2) mRNA decay pathways Initiated by deadenylation
3) Deadenylation: decapping & exonuclease digestion(5′ or 3′)
4) The decapping enzyme competes with the translation initiation
complex (5′ cap binding)
5) Exosome mRNA digestion(3′)
6) Degradation PBs
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14. •Eukaryotic mRNAs are protected from exonucleases by their
modified ends
•The histone mRNAs in mammals stem-loop structure
•Degradation of the vast majority of mRNAs is deadenylation
dependent
•In both yeast and mammalian cells, the poly(A) tail is initially
shortened by the PAN2/3 complex, followed by a more rapid
digestion of the remaining 60 to 80 A tail by a second complex,
CCR4-NOT, which contains the processive exonuclease CCR4
and at least eight other subunits. 14

15. • Two different mRNA degradation pathways:
1) digestion of the poly(A) tail decapping 5′ monophosphorylated exonuclease Xrn1
2) deadenylation poly(A) exonuclease digestion of the body of the mRNA exosome
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16. 6. Other Degradation Pathways Target Specific
mRNAs
1) Four additional degradation pathways
2) Deadenylation-independent decapping poly(A) tail
3) degradation of histone mRNAs 3′ poly(U) tail
4) Degradation of some mRNAs sequence- or structure-
specific endonucleolytic
5) mRNAs are targeted for degradation or translational
repression by microRNAs 16

17. Four other pathways for mRNA degradation:
1) Deadenylation-independent decapping Xrn1
2) Histone mRNAs in mammalian Lsm1–7 , exosome
3) Endonucleotic cleavage digestion 3‘ or 5' decapping
4) microRNA(miRNA) endonucleotic cleavage, deadenylation,
translational repression
•It has been estimated that 50% of all mRNAs could be regulated by
miRNAs.
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18. 18
Summary of key elements of mRNA
decay pathways in eukaryotic cells

19. • The rate of deadenylation and/or other steps in degradation by these pathways
can be controlled by cis-acting elements in each mRNA and trans-acting
factors present in the cell.
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20. 7. mRNA Half-Lives Controlled by Sequences or
Structures Within the mRNA
1) cis-elements degradation
2) Destabilizing elements: mRNA decay , Stabilizing elements: mRNA decay
3) AU-rich elements(AREs) destabilizing elements in mammals
4) DE-binding proteins + decay machinery degradation
5) SEs stable mRNAs
6) mRNA degradation rates variety of signals
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21. What accounts for the large range of half-lives of different mRNAs
in the same cell?
1) cis-elements 3′ UTR
2) Destabilizing elements (DEs)
3) AU-rich element (ARE) pentamer sequence AUUUA
4) Stabilizing elements (SEs) 3′ UTR
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22. 22
• An example of regulated mRNA stabilization occurs for the mammalian transferrin mRNA
• IRE + pro 3′ UTR mRNA is stabilized
• IRE-binding protein mRNA is stabilized
• Microarray studies have shown that
almost 50% of changes in mRNA levels
stimulated by cellular signals are due to
mRNA stabilization or destabilization
events, not to Transcriptional changes.

23. 8. Newly Synthesized RNAs Nuclear Surveillance
System
1) Aberrant nuclear RNAs are identified and destroyed by a surveillance
system
2) Nuclear exosome: processing of normal substrate RNAs &
destruction of aberrant RNAs
3) TRAMP complex facilitated 3′ exonuclease activity
4) Exosome degradation unspliced or aberrantly spliced pre-mRNAs
5) CUTs destroyed in the nucleus 23

24. •All newly synthesized RNAs are subject to multiple processing
steps after they are transcribed
•Surveillance involves two kinds of activities:
1) Identify and tag the aberrant substrate RNA
2) Destroy it
•The destroyer is the nuclear exosome 3′ to 5′ exonuclease
1) RNA processing of some noncoding RNA transcripts: snRNA, snoRNA, and Rrna
2) degradation of aberrant transcripts
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25. For example:
1) Nrd1–Nab3
2) TRAMP
What are the substrates for TRAMP–exosome degradation?
What kinds of abnormalities condemn pre-mRNAs to nuclear destruction?
1) Unspliced or aberrantly spliced pre-mRNAs
2) Improperly terminated, lacking a poly(A) tail.
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26. •Whereas polyadenylation is protective in true mRNAs, it may
actually be destabilizing for cryptic unstable transcripts(CUTs).
•More than three-quarters of RNA Pol II transcripts may be composed
of noncoding RNAs and be subject to rapid degradation by the
exosome!
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27. 9. Control of mRNA Translation : Cytoplasmic
Surveillance Systems
1) Nonsense-mediated decay (NMD): mRNAs with premature stop
codons
2) NMD: UPF and SMG proteins
3) Recognition of a termination codon: unusual 3′ UTR structure & EJCs
4) Nonstop decay (NSD): termination codon
5) No-go decay (NGD): mRNAs with stalled ribosomes in their coding
regions 27

28. •Some kinds of mRNA defects can be assessed only during translation
Substrates for cytoplasmic surveillance systems:
1) Nonsense-mediated decay (NMD)
2) Nonstop decay (NSD)
3) No-go decay (NGD)
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29. How are PTCs distinguished from
the normal termination codon
further downstream?
Effect of tethering a PABP
near a premature
termination codon
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30. The list of normal NMD substrates:
1) mRNAs with especially long 3′ UTRs
2) mRNAs encoding selenoproteins
3) unknown number of alternatively spliced mRNAs
• NMD may turn out to be an important rapid decay pathway for a variety of
short-lived mRNAs
• Nonstop decay (NSD) targets mRNAs that lack an in-frame termination codon
• Targeting nonstop substrates SKI proteins
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31. •No-go decay (NGD) targets mRNAs with ribosomes stalled in the
coding region codon
•Targeting of the mRNA involves recruitment of two proteins,
Dom34 and Hbs1, which are homologous to eRF1 and Erf3,
respectively.
•mRNA degradation is initiated by an endonucleolytic cut, and the 5′
and 3′ fragments are digested by the exosome and Xrn1.
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32. 10. Translationally Silenced mRNAs : RNA Granules
1) RNA granules: translationally silenced mRNA + proteins
2) Germ cell granules & neuronal granules : translational repression
and transport
3) Processing bodies (PBs): mRNA decay components
4) Stress granules (SGs) accumulate in response to stress: Inhibition
of translation
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33. • The occurrence in germ cells and neurons of macroscopic, cytoplasmic
particles containing mRNA has been known for many years.
• One similarity among all of the known RNA granules is that they harbor
untranslated mRNAs and about 50 to 100 different proteins, depending on
granule type.
•RNA granules = mRNPs + protein
•Germ cell granules (maternal mRNA granules) oocytes
Repression deadenylation
Activation polyadenylation
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34. • Neuronal granules: similar to maternal mRNA granules
• Processing bodies (PBs): contains proteins involved in mRNA decay
• mRNAs silenced via RNAi and miRNA pathways are present in PBs
• PABPs are not found in PBs, suggesting that deadenylation precedes mRNA
localization into these structures.
• PABPs: dynamic, increasing and decreasing in size and number, and even
disappearing, under different cellular and experimental conditions that affect
translation and decay
• Not all resident mRNAs are doomed for destruction, though; some can be
released for translation 34

35. • Stress granule (SG): only accumulate in response to stress-induced
inhibition of translation initiation
• SGs lack components of the RNA decay machinery, which PBs have, but
include many translational initiation components that PBs lack.
• Both types of particle can coexist in one cell
• the size and numbers of both increase under stress conditions
• mRNAs may be exchanged between the two types of particles
• Granule mRNAs are normally in a dynamic equilibrium with the
population of mRNAs being translated
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36. 11. Eukaryotic mRNAs Localized to Specific
Regions of a Cell
1)Localization of mRNAs serves diverse functions in single cells and
developing embryos
2)Three mechanisms for the localization of mRNA
3)Localization: cis-elements target mRNA / trans-factors mediate
4)The predominant active transport mechanism involves the directed
movement of mRNPs along cytoskeletal tracks
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37. • Most mRNAs are probably translated in random locations
• mRNA localization: 3 key functions
1) specific mRNAs in the oocytes
2) asymmetric cell divisions
3) mechanism for the compartmentalization
of the cell intospecialized regions
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38. • mRNA localization is particularly important for highly polarized cells such
as neurons
• Most mRNAs are translated in the neuron cell body, many mRNAs are
localized to its dendritic and axonal extensions: β-actin mRNA
• Localization of mRNA at neuronal postsynaptic sites seems to be essential
for modifications accompanying learning
• Glial cells myelin sheath
• Plants localize mRNAs to the cortical region of cells and to regions of
polar cell growth
• mRNA localization involves transport from one cell to another. 38

39. • Maternal mRNPs in Drosophila are synthesized and assembled in
surrounding nurse cells and are transferred to the developing oocyte
through cytoplasmic canals
• Plants can export RNAs through plasmodesmata and transport them for
long distances via the phloem vascular system
• 3 mechanisms for the localization of mRNA:
1) The mRNA is uniformly distributed but degraded at all sites except the site of
translation
2) The mRNA is freely diffusible but becomes trapped at the site of translation
3) The mRNA is actively transported to a site where it is translated 39

40. • Active transport is the predominant mechanism for localization:
• All three molecular motor types are exploited: dyneins & kinesins & myosins
4 components:
1) cis-elements on the target mRNA
2) trans-factors that directly or indirectly attach the mRNA to the correct motor protein
3) trans-factors that repress translation
4) an anchoring system at the desired location.
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41. • Only a few cis-elements, sometimes called zipcodes, have been characterized
• A large number of trans-factors have been associated with localized mRNA
transport and translational repression
• Staufen protein: This multitalented factor has multiple domains that can
couple complexes to both actin- and microtubule-dependent transport pathways
• Cotranscriptional binding of the zipcode element by the protein ZBP1 is
required for localization
• mRNA is committed to localization before it is even processed and exported
from the nucleus
• β-actin mRNA localization is dependent on intact actin fibers in fibroblasts and
intact microtubules in neurons
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42. Newly exported ASH1 mRNA is attached
to the myosin motor Myo4 via a complex
with the She2 and She3 proteins. The motor
transports the mRNA along actin filaments
to the developing bud
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43. 5' and 3' modifications controlling RNA
degradation
• RNA degradation is a key process in the regulation of gene expression. In all organisms,
RNA degradation participates in controlling coding and noncoding RNA levels in response
to developmental and environmental cues.
• The constant control of RNA quality prevents potential deleterious effects caused by the
accumulation of aberrant non-coding transcripts or by the translation of defective
messenger RNAs (mRNAs).
• any functional RNA (including a successful pathogenic RNA) must be protected from the
scavenging RNA degradation machinery. Yet, this protection must be temporary, and it
will be overcome at one point because the ultimate fate of any cellular RNA is to be
eliminated. This special issue focuses on modifications deposited at the 5' or the 3'
extremities of RNA, and how these modifications control RNA stability or degradation.
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44. • Although the decay of eukaryotic RNAs can be initiated by endoribonucleolytic cleavages,
it is mainly carried out by exoribonucleases
• The m7G cap initially is recognized by the nuclear cap binding complex, which facilitates
mRNA export from the nucleus and is later replaced by the essential translation initiation
factor, eIF4E.
• At the other end of the mRNA, the poly(A) tail is bound by poly(A) binding proteins
(PABPs), which facilitate mRNA export from the nucleus and enhance protein synthesis
through interactions with translation initiation factors
• Deadenylated mRNAs can be uridylated or stored in a dormant state to be later re-
adenylated to activate protein synthesis TUTases & non-canonical poly(A)
polymerases
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45. • Although in bacteria, bulk RNA decay is initiated by endonucleolytic
events, the ends of RNA molecules also play an important role in
controlling RNA fate. Secondary structures may impede the progression
of 3' to 5' exoribonucleases and polyadenylation accelerates the
degradation of RNA decay intermediates facilitating the action of 3' to 5'
exoribonucleases. Moreover, the triphosphate moiety present at the 5'
end of the primary transcripts restricts endonucleolytic cleavage by the
RNaseE and its conversion to monophosphate accelerates the decay rates
of many, but not all, transcripts.
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46. • Charenton: 5‘ end cap structure Nudix(family hydrolase Dcp2)
• Warkocki: uridylation by TUT7/TUT4 in the cytoplasm induces decay of
various RNA species and protects cells against viruses and LINE-1
retrotransposons
• Zigackova: The key factors involved in the uridylation and degradation of
RNAs, i.e. TUTases and 30 –50 exoribonucleases recognizing uridylated
RNAs.
• Meaux: How uridylation induces decay of replication-dependent histone
mRNAs in mammals? the serine/threonine protein kinase Smg1 & the RNA
helicase Upf1
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47. The role of nonsense-mediated mRNA decay (NMD)
in regulating gene expression
• NMD results from improper translation termination at stop codons occurring in places of
the mRNP that lack necessary termination stimulating signals and/or contain termination
inhibitors.
• The most well understood is the major cytoplasmic poly(A) binding protein (PABPC1),
which acts as a potent NMD suppressor when bound in the vicinity of the stop codon.
• Slow or stalled translation termination allows the core NMD factor UPF1 – an
ATPdependent RNA helicase – and the SMG1 complex to interact via the release factors
eRF1 and eRF3 with the stalled ribosome. Interaction of UPF2 and possibly UPF3B with
the SMG1- UPF1 complex activates the SMG1 kinase, resulting in the phosphorylation of
several serineglutamine (SQ) and threonine-glutamine TQ motifs in the N and C-terminal
part of UPF1 that flank its helicase domain. 47

48. • The endonuclease SMG6 and the heterodimer SMG5/SMG7 have been found to interact
with phosphorylated UPF1 and to induce the degradation of the targeted mRNA.
• Recent evidence suggests that in human cells NMD is predominantly initiated through a
SMG6-mediated endonucleolytic cleavage near the stop codon and as a backup system
through SMG7-mediated recruitment of the CCR4/NOT complex, which deadenylates the
mRNA, leading to decapping followed by 5’-to-3’ exonucleolytic degradation by XRN1.
• Interestingly, it seems that not all proteins known to be required for NMD in
mammalian cells have homologues in other organisms
• It was discovered early on that the presence of an exon-exon junction located >50
nucleotides downstream of the stop codon was a hallmark of mammalian mRNAs subject
to NMD
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49. • It was later found that these exon-exon junctions function as enhancers of NMD efficacy
and that long 3’ UTRs lacking exon-exon junctions can also trigger NMD
NMD factors are essential for normal embryonic development and viability
• There is evidence that some NMD factors function in additional cellular pathways apart
from NMD. For example, UPF1 is involved in telomere maintenance, cell cycl progression
and degradation of histone mRNAs and SMG1 and SMG6 were also reported to play a role
in telomere maintenance
• There is evidence suggesting that NMD inactivation is responsible for the observed
developmental defects.
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50. Tight regulation of NMD activity is required for normal spermatogenesis
• Several lines of evidence support a role for NMD in germ cell development and male
fertility
• Conditional ablation of UPF2 from embryonic Sertoli cells (SC) – somatic cells that nourish
sperm cells through spermatogenesis – in mice leads to testicular atrophy and male sterility
due to depletion of SC and germ cells (GC)
NMD is involved in the integrated stress response
• By downregulating stress-responsive genes in unstressed cells, NMD activity may prevent
the triggering of a stress response upon innocuous stimuli. However, following exposure to
strong stressors, specific mRNAs can evade NMD and are upregulated as part of the stress
response.
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51. • Autophagy is a surveillance mechanism that rids the cells of cytoplasmic deleterious proteins
and also serves as a source of amino acids during metabolic stress. Many cellular stressors,
like amino acid deprivation and hypoxia, inhibit NMD and activate autophagy, and an
inverse correlation between these two pathways was reported.
• Inhibition of NMD activates autophagy, in a partially ATF4-dependent manner, and increases
intracellular amino acid levels, while NMD hyperactivation blunts stress-induced autophagy
• NMD inhibition by stress-induced eIF2α phosphorylation leads to stabilization of SLC7A11,
increase in intracellular glutathione levels and establishment of an adaptive response to
oxidative stress.
• It was recently reported that NMD inhibition is also part of the pro-apoptotic response to
stress
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52. RNA viruses are targeted by or able to evade the host NMD machinery
• Since viral genomes present targets for NMD, it is unsurprising that viruses have evolved
mechanisms to protect them from degradation by the host cell NMD machinery. For
example, Hepatitis C Virus (HCV) was shown to inhibit NMD in hepatoma cell lines
NMD factors are autoregulated by the NMD pathway
• Huang: suppression of NMD by NMD factor knockdowns resulted in a significant increase
in NMD factor mRNA levels of UPF1, UPF2, UPF3B, SMG1, SMG5, SMG6 and SMG7.
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53. NMD factor mutations in human diseases and the therapeutic potential of
modulating NMD activity
• somatic mutations in the UPF1 gene are frequently found in pancreatic adenosquamous
carcinoma (ASC) and in inflammatory myofibroblatic tumors (IMTs)
• In both cases, the mutations reduce UPF1 protein levels and result in impaired NMD in
the tumor tissue relative to adjacent normal tissue
• NMD exerts important biological functions that reach beyond mRNA surveillance.
Although we are still in the beginning of unravelling the different regulatory circuits in
which NMD is implicated, the existing evidence suggests that NMD affects gene
expression in a diverse range of biological contexts. The unifying principle in all these
cases appears to be the occurrence of stop codons in mRNP environments that are not
optimal for fast enough and/or correct translation termination, leading to the degradation
of the respective mRNA. 53

54. 54

55. 1. Lewin’s-Genes-XII-2018
2. Molecular Biology of the Cell Sixth Edition
3. 5' and 3' modifications controlling RNA degradation: from safeguards to
executioners(2018)
4. Beyond quality control: The role of nonsense-mediated mRNA decay
(NMD) in regulating gene expression(2017)
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