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2011 MECHANISMS OF CYTOPLASMIC mRNA TURNOVER IN EUKARYOTES. Kim Keeling, Ph.D. kkeeling@uab.edu Room 456, Bevill Bldg 975-6585. Lecture Overview. 1. General mRNA turnover pathways - Play key role in controlling basal gene expression levels 2. Aberrant RNA turnover pathways

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2011 mechanisms of cytoplasmic mrna turnover in eukaryotes

2011 MECHANISMS OF CYTOPLASMIC mRNA TURNOVER IN EUKARYOTES

Kim Keeling, Ph.D.

kkeeling@uab.edu

Room 456, Bevill Bldg

975-6585

slide2

Lecture Overview

1. General mRNA turnover pathways

- Play key role in controlling basal gene expression levels

2. Aberrant RNA turnover pathways

- Recognize and degrade aberrant mRNAs or mRNAs that are

translated inefficiently

- Increases the quality control of mRNA biogenesis and gene

expression

3. Specialized mRNA turnover pathways

- mRNA turnover acts as a site for regulation in response to

specific signals (hormones, cell cycle, viral infection,

differentiation, nutrient availability, stress)

- AU-rich element mediated (ARE)

4. Specialized sites of cytoplasmic mRNA turnover

- Processing bodies (P bodies)

- Exosome granules for ARE-mediated mRNA turnover

slide3

Determining mRNA Stability Experimentally

Northern blot of ACT1 mRNA

1. Shut off transcription: thiolutin,

actinomycin D, promoters that can

be repressed (such as copper

or tetracycline regulated promoters).

2. Isolate mRNA at several time points

after transcriptional shut-off.

3. Determine the percent of mRNA

remaining at each time point after

transcriptional shut-off.

4. Graph the level of RNA remaining

for each time point on a semi-log

graph. The slope = the rate of

mRNA decay. The time at which

half of the mRNA is degraded is the

half-life of the mRNA.

inhibit

expression

WT

tpa1∆

time (m)

0

10

20

40

0

10

20

40

100

90

80

70

60

50

40

t1/2 =

tpa1

24 min

% ACT1

remaining

30

20

t1/2 =

wild type

12 min

10

0

10

20

30

40

Time (m)

Keeling et al. (2006) Mol Cell Biol 26:5237

slide4

Nucleus

Cytoplasm

mRNAs Are Modified During Nuclear

Processing to Promote Cytoplasmic Stability

1. Addition of 5’ cap and the

CAP binding protein complex

2. Addition of the 3’ poly(A) tail

and multiple poly(A) binding

proteins

3. Circularization of mRNA by

bridging the 5’ CAP and the 3’

poly(A) tail via eIF4G

Goldstrohm & Wickens (2008) Nat Rev Mol Cell Biol 9:337

slide6

The Major Eukaryotic mRNA Turnover Pathways

5’ 3’ pathway

3’ 5’ pathway

CAP hydrolysis

Biochem. Soc. Trans. (2006) 34:35

slide7

The Major Eukaryotic mRNA Turnover Pathways

5’ 3’ pathway

3’ 5’ pathway

CAP hydrolysis

Biochem. Soc. Trans. (2006) 34:35

slide8

Mammalian Deadenylation Occurs in Two Phases

PAN2 complex

Slow

CCR4 complex

Fast

Muhlemann (2005) Nat Struct Mol Biol 12:1024

slide9

Deadenylase Machinery

1) PAN complex:

- First phase

- Slow, distributive

- Stimulated by PAB

- Also trims poly(A) tails

in nucleus

2) CCR4 complex:

- Second phase

- Fast, processive

- Inhibited by PAB

- Independent function

as a transcription factor

3) PARN complex:

- Used for specialized

rapid deadenylation

(ARE-mediated decay)

in mammalian cells

Parker & Song (2004) Nat Struct Mol Biol 11:121

slide10

T

T

T

T

T

T

T

T

T

T

Control RNA

Transcriptional

shut off

Determining Deadenylation Rates Experimentally

RNase H Northern blot

RNase H

+ oligo dT  poly(A)-

PTETOFF

AAAAAAAAAA

Probe

DNA oligo

Northern blot

slow

synchronous

fast

asynchronous

PAN complex

CCR4 complex

Yamashita et al. (2005) Nat Struct Mol Biol 12:1054

slide11

PAN and CCR4 Sequentially Deadenylate mRNAs

Enhanced

2nd phase

Enhanced

1st phase

No difference

Delayed

1st phase

Delayed

2nd phase

No difference

Yamashita et al. (2005) Nat Struct Mol Biol 12:1054

slide12

Deadenylation Is Coupled to Translation Termination

Hosoda et al (2003) JBC 278:38287

  • - eRF3 is a GTPase that mediates translation termination.
  • - The N-terminal domain of eRF3 interacts with the Pab1p C-terminal domain.
  • - This interaction inhibits the ability of Pab1p molecules to oligomerize
  • efficiently on the poly(A) tail. This increases the exposure of the poly(A)
  • tail to deadenylases and turnover.
slide13

eRF3 Binds to Pab1p and Inhibits Its Oligomerization

Hoshino et al. (1999)

Biochem (Mosc) 64:1367

  • - Gel shift assay: radio-labeled RNA is incubated with increasing
  • amounts of Pab1p protein.
  • - A shift in the mobility of the RNA indicates Pab1p protein binding.
  • - Addition of the eRF3 N-terminal domain to the binding assay
  • inhibits the ability of Pab1p to oligomerize on the RNA.
slide14

Hosoda et al (2003)

JBC 278:38287

The eRF3-Mediated Inhibition of Pab1p Results

in Deadenylaton and mRNA Turnover

- Expression of a mutant eRF3 where the N-terminal domain is deleted so

that it can no longer interact with Pab1p results in longer poly(A) tail length.

This mutant also leads to an increased mRNA half-life = decreased turn-

over rate.

- This suggests that normally, eRF3 mediates mRNA turnover by decreasing

Pab1p oligomerization on poly(A) tails which leads to deadenylation and

mRNA decay. This represents one way to mediate general mRNA decay.

slide15

The Major Eukaryotic mRNA Turnover Pathways

5’ 3’ pathway

Pan2, Ccr4

CAP hydrolysis

Biochem. Soc. Trans. (2006) 34:35

slide16

Decapping Enzymes

Dcp1

- Dcp2p = catalytic subunit

- Dcp1p = stimulates Dcp2p

- Hedls = also needed to

stimulate Dcp2p activity in

mammals

- In yeast, Dcp1p and Dcp2p

can directly interact with each

other to stimulate decapping

- Dcp2p is differentially expressed

in mammalian tissues

- Another decapping enzyme was

identified in the cytoplasm of

mammalian cells called Nudt16.

No ortholog found in yeast, C.

elegans or Drosophila.

- Nudt16 and Dcp2 appear to each

work on subsets of cellular

transcripts.

Hedls

Yeast interactions

Human interactions

Dcp2

Simon et al. (2006) TIBS 31:241

Nudt16

Taylor & Peculis (2008) NARS 36:6021

slide17

Control of the Decapping Reaction

  • 1. Poly(A) tail status

- Poly(A) binding protein inhibits decapping by physically interacting with

the 5’ cap complex (via eIF4G) to stabilize the cap and enhance translation.

2. CAP status

- The CAP protein complex must dissociate from the cap prior to decapping.

3. Translation status

- A transition must take place from a translation competent state to a state

where translation does not take place (change in mRNP structure).

4. Assembly of the decapping complex

- Specific factors are required for the localization, assembly, and activation

of the decapping complex.

slide18

Parker & Song (2004) Nat Struct Mol Biol 11:121

Decapping Activators

Pat1p

- Interacts with both poly(A) [+] and poly(A) [-] transcripts

- Seeds decapping complex onto mRNA

- Interacts with Dcp1p, Dcp2p, Lsm1-7p, Dhh1p, Xrn1p

- Along with the Lsm complex, represses translation

Lsm1-7p

- 7 ring complex interacts with poly(A) [-] mRNA

- Facilitates the assembly of the decapping complex

in cytoplasmic and nuclear decapping

- Interacts with Dcp1p, Dcp2p, Dhh1p, Pat1p, Xrn1p

Dhh1p

- ATP dependent helicase

- Binds directly to mRNA substrate

- Interacts with Dcp1p, Dcp2p, Lsm1-7p, Ccr4p, Pop2p, Edc1p, Edc2p

Edc1/Edc2

- Binds directly to mRNA substrate

- Enhances decapping activity

- Interacts with Dcp1 and Dcp2

- Enhances alterations in mRNA stability in response to nutrient changes

slide19

The Major Eukaryotic mRNA Turnover Pathways

5’ 3’ pathway

Pan2, Ccr4

Dcp2, Nudt16

CAP hydrolysis

Biochem. Soc. Trans. (2006) 34:35

slide20

The Major Eukaryotic mRNA Turnover Pathways

5’ 3’ pathway

Pan2, Ccr4

Dcp2, Nudt16

CAP hydrolysis

Xrn2/Rat1

= nuclear

exonuclease

Biochem. Soc. Trans. (2006) 34:35

slide21

The Major Eukaryotic mRNA Turnover Pathways

Pan2, Ccr4

CAP hydrolysis

Biochem. Soc. Trans. (2006) 34:35

slide22

Exosome Components

Core Subunits: all essential

Rrp4p

Rrp40p

Csl4p

Rrp41p/Ski6p

Rrp42p

Rrp43p

Rrp44p/Dis3p

Rrp45p

Rrp46p

Mtr3p

Associated factors:

Mtr4p essential ATP-dependent helicase

Ski2p non-essential ATP-dependent helicase

Ski3p non-essential TPR repeat

Ski8p non-essential WD repeat

Ski7p non-essential GTPase

Nuclear subunits:

Rrp6p non-essential nuclear subunit

Parker & Song (2004) Nat Struct Mol Biol 11:121

slide23

The Exosome Gains Access to mRNAs for

Degradation Via Ski7p and the SKI Complex

- Ski7p bridges an interaction between the mRNA-bound SKI complex

and the exosome.

- In the nucleus, a group of factors called the TRAMP complex is the

bridge between mRNAs and the exosome.

Araki et al. (2001) EMBO J 20:4684

slide24

The Major Eukaryotic mRNA Turnover Pathways

Pan2, Ccr4

Dcp2, Nudt16

CAP hydrolysis

Biochem. Soc. Trans. (2006) 34:35

slide25

Final Step of mRNA Decay: Degrading the 5’ Cap

- The scavenger decapping enzyme hydrolyzes di-

and tri-phosphorylated CAPs to the mono form.

- Substrates are less than 10 nucleotides in length.

Parker & Song (2004) Nat Struct Mol Biol 11:121

slide26

Summary of the General Eukaryotic

mRNA Decay Pathways

Meyer et al. (2004)

Crit Rev Biochem Mol Biol 39:197

slide27

Nuclear mRNA Decay Also Utilizes Components of the 5'  3' and 3' 5' mRNA Turnover Pathways

Moore (2002) Cell 108:431

TRAMP

Xrn2p

Nuclear

Yes

Yes

Xrn2p/Rat1p

TRAMP

slide28

2) Aberrant mRNA Decay Pathways

A. Nonsense-mediated mRNA decay (NMD)

- Degrades mRNAs with premature stop codons

B. Nonstop mRNA decay (NSD)

- Degrades mRNAs without a stop codon

C. No-go mRNA decay (NGD)

- Degrades mRNAs that have a stalled ribosome

D. Ribosome extension-mediated decay (REMD)

- Degrades mRNAs where ribosome translates past

the stop codon and into the 3’ UTR

slide29

Nonsense-Mediated mRNA Decay

- Specialized pathway that degrades mRNAs thatcontain premature translation

termination signals

- Protects the cell from translating mRNAs that might produce truncated peptides that

could lead to harmful dominant negative effects

- Occurs in all eukaryotes.

- 30% of disease-generating mutations result in premature stop codons

- Up to 10-20% of the transcriptome is regulated by NMD

- PTC-containing transcripts caused by point mutations, frameshift mutations, mRNAs

with faulty alternative splicing, pre-mRNAs that escape nuclear retention, mRNAs that

contain upstream open reading frames, mRNAs that carry introns in 3´ untranslated

regions, or mRNAs with long 3´ untranslated regions

Czapllinski et al. (1999)

Bioeassay 21:685

slide30

Splicing-dependent NMD

Normal Termination

Aberrant Termination

Current NMD Models

Splicing-independent NMD

Wen & Brogna (2008) Biochem Soc Trans 36:514

slide32

core NMD

components

NMD Factors Associate With the EJC

Core NMD Components:

UPF3: associates with the EJC in the nucleus

UPF2: perinuclear and binds to Upf3 as the mRNA is exported

UPF1: associates at the stop codons in mRNAs during translation

slide33

Regulation of NMD by

Upf1p Phosphorylation

1. The SURF complex composed of

SMG1, UPF1, and the eRF1 and eRF3

release factors forms. This complex

recognizes stop codons.

2. If the SURF complex interacts with a

downstream EJC complex that

includes UPF2, UPF3, and Y14 (the

DECID complex), then SMG-1

phosphorylates UPF1. This marks

the stop codon as premature.

3. UPF1 phosphorylation induces a

change in the mRNP structure that

recruits SMG5, SMG6, SMG7, and the

PP2A phosphatase to dephosphorylate

UPF1. This signals for the mRNA to be

degraded.

Kashima et al. (2006) Gene Dev 20:355

slide34

Aberrant Translation Termination Mediates NMD

Muhlemann (2008) Biochem Soc Trans 36:497

slide35

Nonstop mRNA Decay

- Pathway to degrade mRNAs

thatdo not contain any stop

codons

- Typically caused by the

presence of cryptic poly(A)

addition sites that leads to

polyadenylation of transcripts

upstream of the termination

signal

- Utilizes Ski7p, the SKI

complex, and the exosome

to degrade nonstop messages

Maquat (2002) Science 295:2221

slide36

No Go mRNA Decay

- Degrades messages with translation

elongation stalls (i.e. hairpin structures,

pseudoknots, rare codons)

- Requires two factors that bind to the A

site of the stalled ribosome

-- Dom34p (eRF1-like homologue)

-- Hbs1p (eRF3-like homologue)

- Promotes ribosomal subunit dissociation

and peptidyl tRNAs to remove them from

translation elongation stalls and recycle

them

- mRNA cleaved at stalled position by

Dom34p which has endonuclease

activity and then degraded by the

exosome and/or Xrn1p

Tollervey (2006) Nature 440:425

slide37

Pathway

A site binder

GTPase

NMD eRF1 eRF3

NGD DOM34 HSB1

NSD ? SKI7

Many of the Specialized mRNA Decay Pathways Recruit Similar Proteins to the Ribosomal A Site to Trigger different mRNA Decay Mechanisms.

Clement & Lykke-Anderson (2006) Nat Struct Mol Biol 13:299

Chen et al. (2010) Nat Struct Mol Biol 17:1233

slide38

Ribosome Extension-Mediated mRNA Decay

- Constant Spring (CS) mutation in -globin

changes the UAA stop codon at the end of

the mRNA into a CAA (glutamine) codon

- Mutation is the most prevalent non-deletion

mutation that causes -thalassemia

- Ribosome continues to translate 31 codons

in the 3’ UTR until a stop codon is

encountered (UAA)

- Level of mRNA is severely decreased due

to this mutation

- CS mutation causes a decrease in the mRNA

half-life due to rapid induction of

deadenylation

- Mechanism behind this rapid deadenylation

is still unknown

- REMD is cell-type restricted

Kong & Liebhaber (2007) NSMB 14:670

slide39

Summary of mRNA Surveillance Pathways

Doma & Parker (2007) Cell 131:660

slide40

3) Specialized mRNA turnover pathways

- ARE-mediated mRNA decay

slide41

ARE-Mediated mRNA Turnover

Goldstrohm & Wickens (2008)

Nat Rev Mol CellBiol 9:337

ARE

- AU rich elements (50-150nts)

AUUUA; UUAUUUA(U/A)(U/A); or U-rich

- cis-acting element located in 3' UTRs of

mRNAs

- transcripts that encode proteins that

require rapid changes in response to stimuli

such changes in the cell cycle, growth

factors, response to microorganisms,

inflammatory stimuli, and environmental

factors

- 10% of mammalian mRNAs contain AREs

- A diverse set of trans-acting proteins

bind to AREs. These proteins can

mediate other protein interactions that

modulate mRNA stability. Various

ARE-associated proteins can promote

rapid mRNA turnover by promoting

enhanced decapping, deadenylation,

exosome recruitment, endonucleolytic

cleavage or combinations of these.

Alternatively, some proteins that bind

to AREs can stabilize the mRNA.

slide42

Various Triggers Alter the Balance of Effectors

to Modulate ARE-mediated mRNA Decay

Eberhardt et al. (2007) Pharm Ther 114:56

slide43

Example of ARE-Mediated Change in mRNA

Levels Before and After the DNA Damage Response

Under non-damage conditions:

- AUF1 competes with the PABP for

poly(A) tail binding, exposing it to

PARN; TTP (tristetraproline) and KSRP

(KH-type splicing regulatory protein)

recruit PARN and CCR4 to deadenylate

prior to degradation by the exosome.

Under DNA damage conditions:

- Genes involved in the DNA damage

response pathway are up-regulated.

HuR is up-regulated and competes

with AUF1 for binding to the same

ARE region. Loss of AUF1 binding

stabilizes PABP association with the

poly(A) tail. HuR also competes with

TTP and KRSP to prevent recruitment

of the deadenylases and exosome.

Cevhar & Kleiman (2010) WIRE 1:193

slide44

4) Specialized sites of cytoplasmic mRNA turnover

- Processing bodies

- Exosome granules

slide45

RNA Processing Bodies (P bodies)

  • - Discrete cytoplasmic granular structures that contain a reservoir of 5’ 3’
  • mRNA decay factors; NMD factors; RNA-induced silencing complex
  • - Found in all eukaryotes
  • - Size and number of P bodies depend upon the amount of RNA to be degraded
  • Conditions that promote P body formation include: 
  • - Glucose deprivation
  • - Osmotic stress
  • - UV light
  • - Decreased translation initiation rates
  • - Non-translating mRNA
  • Note that all of these conditions involve moving mRNAs from a translatable

pool that is ribosome-associated to a non-translatable pool that is not ribosome-

associated

  • Conditions that reduce P bodies include:
  • - Inhibition of translation elongation (ribosome can’t dissociate from mRNA)
  • - RNase A treatment (RNA degraded)
  • - Increased rates of translation initiation (increase in ribosome-bound mRNA)
slide46

Identification of Factors That Reside in P Bodies

Factors Not Found in P Bodies

- Translation initiation factors

- Ribosomal subunits

- SKI proteins & exosome

Kulkarni et al. (2010) Biochem Soc Trans 38: 242

slide47

RNA Decay

Intermediates

Localize to

P Bodies

- PolyG tract (18) in 3’ UTR

blocks Xrn1p to create a

mRNA decay intermediate

- MS2 = bacteriophage coat

protein binding site

- MS2-GFP protein will bind

to MS2 sites in mRNA and

allows its localization by

visualization of the green

fluorescence protein (GFP)

Sheth & Parker (2003) Science 300:805

slide48

P Bodies Disassemble

After Translation

Restoration

- Polysome profile (ribosomes

fractionated through a sucrose

gradient)

- Active translation = (+) polysomes

- Inactive translation = (-) polysomes

- P body-associated mRNAs

can return to the translatable

pool

Brengues et al. (2005) Science 310:486.

slide49

Exosome Granules Are Distinct From P Bodies

and Contain ARE mRNAs and PARN

exosome

protein

merge

P body

protein

exosome

protein

PARN

merge

ARE

mRNA

Lin (2007) JBC 282:19958

slide50

Lecture Overview

1. General mRNA turnover pathways

- 5’3 & 3’5’

- Deadenylases

- Decapping complex

- Xrn1, exosome, DcpS

2. Aberrant RNA turnover pathways

- Premature stop codons: nonsense-mediated mRNA decay (NMD)

- No stop codons: non-stop mRNA decay (NSD)

- Elongation stall: no-go mRNA decay (NGD)

- Translation into the 3’ UTR: ribosome extension-mediated mRNA decay (REMD

3. Specialized mRNA turnover pathways

- ARE-mediated mRNA turnover: AU-rich elements in the 3’ UTR are bound by

proteins that modulate the stability of mRNAs in response to regulatory signals

4. Locale of mRNA turnover

- P bodies: contain 5’3’ mRNA turnover machinery and degrade mRNAs

that are no longer available for translation

- Exosome granules: contain PARN and exosome and participate in ARE-

mediated decay