Not1 mediates recruitment of the deadenylase Caf1 to mRNAs targeted for degradation by tristetraprolin.

7.1 MB·English

by  Sandler, Heike

Published online 29 January 2011 Nucleic Acids Research, 2011, Vol. 39, No. 10 4373–4386
doi:10.1093/nar/gkr011
Not1 mediates recruitment of the deadenylase Caf1
to mRNAs targeted for degradation by tristetraprolin
Heike Sandler1, Jochen Kreth1, H. Th. Marc Timmers2 and Georg Stoecklin1,*
1Helmholtz Junior Research Group Posttranscriptional Control of Gene Expression, German Cancer Research
Center, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany and 2Department of
Molecular Cancer Research and Netherlands Proteomics Centre, University Medical Center Utrecht,
Universiteitsweg 100, 3584 CG Utrecht, The Netherlands
Received August 26, 2010; Revised December 22, 2010; Accepted January 3, 2011
ABSTRACT
is located in the 30-untranslated region (UTR) of many
The carbon catabolite repressor protein 4 (Ccr4)– short-lived mRNAs and mediates rapid mRNA degrad-
Negative on TATA (Not) complex controls gene ation (3,4). Examples of mRNAs containing potent
expression at two levels. In the nucleus, it regulates AREs include many cytokine transcripts such as tumor
the basal transcription machinery, nuclear receptor- necrosis factor-a (TNF-a), granulocyte-macrophage
mediated transcription and histone modifications. In colony-stimulating-factor (GM-CSF) and interleukin
(IL)-3mRNAaswellasmRNAsencodingfortranscription
thecytoplasm,thecomplexisrequiredformessenger
factorssuchasc-myc,c-fosandIer3(5).
RNA (mRNA) turnover through its two associated
Tristetraprolin (TTP) is an RNA binding zinc-finger
deadenylases, Ccr4 and Caf1. Not1 is the largest
protein required for ARE-mediated mRNA decay
protein of the Ccr4–Not complex and serves as a
(AMD). The physiological role of TTP as an inhibitor
scaffold for other subunits of the complex. Here, we
of TNF-a expression was discovered by the analysis of
provide evidence that human Not1 in the cytoplasm TTP knock out mice (6). TTP was found to act at the
associates with the C-terminal domain of tristetra- post-transcriptional level by binding to the ARE of
prolin (TTP), an RNA binding protein that mediates TNF-a mRNA and accelerating its degradation (7). TTP
rapid degradation of mRNAs containing AU-rich recognizestheAREviaitstwocysteine–cysteine–cysteine–
elements (AREs). Not1 shows extensive interaction histidine (CCCH) zinc-finger domains. In addition to
through its central region with TTP, whereas binding TNF-a, TTP was shown to mediate rapid degradation
of the mRNAs encoding GM-CSF (8), IL-2 (9), IL-3
ofCaf1isrestrictedtoasmallercentraldomainwithin
(10), IL-10 (11) and Ier3 (12). The TTP family of
Not1.Importantly,Not1isrequiredfortherapiddecay
proteins comprises two additional paralogs, BRF1 and
of ARE-mRNAs, and TTP can recruit the Caf1 dead-
BRF2, which also promote AMD (13,14).
enylase only in presence of Not1. Thus, cytoplasmic
AREs destabilize mRNA by inducing a rapid short-
Not1 provides a platform that allows a specific RNA
ening of the poly(A) tail (15). Subsequent degradation
binding protein to recruit the Caf1 deadenylase and of the mRNA body occurs either from the 30-end
thereby trigger decay of its target mRNAs. through the exosome (16–18) or from the 50-end through
decapping followed by Xrn1-mediated mRNA decay
(19–21). Importantly, deadenylation is the first and
INTRODUCTION
rate-limiting step for both decay pathways. Several
Cells maintain tight control of gene expression by poly(A)-specific exoribonucleases in the cytoplasm share
regulating rates of transcription, messenger RNA the job of shortening poly(A) tails. Whereas the Pan2–
(mRNA) stability, protein translation and protein stabil- Pan3 complex was found to hydrolyse poly(A) in a dis-
ity. Genes that are rapidly turned on and off frequently tributivemanner,thecarboncataboliterepressorprotein4
achieve such dynamic expression patterns by synthesizing (Ccr4)–Negative on TATA (Not) complex hydrolyses
short-livedmRNAs.Genome-widestudiesinstimulatedT poly(A) in a processive manner and is responsible for
cells, macrophages and fibroblasts revealed that mRNA rapid deadenylation (22). According to an early yet still
half-life is a major determinant of gene expression valid classification, the highly potent class II AREs of
patterns (1,2). An adenosine/uridine-rich element (ARE) cytokine mRNAs cause asynchronous deadenylation
*To whom correspondence should be addressed. Tel:[phone]; Fax:[phone]; Email: [email protected]
(cid:2)TheAuthor(s)2011.PublishedbyOxfordUniversityPress.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://creativecommons.org/licenses/
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4374 NucleicAcidsResearch,2011,Vol.39,No.10
that reflects the activity of a processive deadenylase, MATERIALS AND METHODS
whereas class I AREs, found e.g. in the 30-UTR of the
Plasmid construction
c-fos and c-myc transcription factors, cause synchronous
deadenylation as the result of a distributive deadenylase PlasmidspcDNA3-TTP-mycHis(p2115),pTet-7B(p2254)
activity. TTP typically interacts with class II AREs of andpTet-7B-ARE(p2260)havebeendescribedpreviously
TNFa, GM-CSF and IL-3 mRNA (7,8,23), and may (40). For pTet-7B-MS2bs (p2255), six repeats of the
thus recruit a processive deadenylase. In vitro decay improved MS2-binding site were amplified by PCR with
studies suggested involvement of the poly-A ribonuclease primers G37 and G38 (Supplementary Table S1) from
(PARN) since recombinant TTP was found to stimulate plasmid b-6bs (41) and ligated as a BamHI—BglII
PARN-induced deadenylation of an ARE-mRNA (24). fragment into the BglII site of pTet-7B.
Given that a direct interaction of TTP with PARN The Tet-inducible TOPuro (p2433) vector was created
could not be observed, it is not clear how TTP activates by cloning a PvuII—BamHI fragment from pPUR
(Clontech) containing a puromycine resistence gene into
or recruits PARN. In a different study, TTP was found
the PvuII/BamHI sites of pcDNA4/TO (Invitrogen). For
to interact through its N-terminal domain with the Ccr4
plasmid TOPuroGS (p2434) containing a protein-G and
deadenylase (20), and more recently, TTP was shown
streptavidin-binding peptide tag for N-terminal fusions,
to interact with Caf1 (25). Functional studies on the
theGStagwasamplifiedbyPCRfromplasmidpCeMM-
relative importance of Ccr4 and Caf1 deadenylases
NTAP(GS)-Gw (42) using primers G1050/G1051 and
are challenging since multiple paralogs exist for both
inserted into the KpnI/BamHI sites of TOPuro. For
proteins in the human genome. Studies with dominant-
TOPuroGS-TTP-wt (p2436), the mouse TTP cDNA was
negative mutants of Ccr4 and Caf1 suggested that
amplified by PCR using primers G1052/G1053 from
Ccr4 may have a predominant role in the regular
pcDNA3-TTPwt-mycHis (p2115) and ligated into the
turnover of mRNAs lacking a dedicated destabilizing
EcoRI/XhoI sites of TOPuroGS (p2434). To generate
element (22,26). By knocking down simultaneously
TOPuro-mycSG (p2484) for C-terminal fusions of a
Ccr4a (CNOT6) and Ccr4b (CNOT6L) or Caf1a
mycSG- tag, mycSG was amplified by PCR from
(CNOT7) and Caf1b (CNOT8, also CALIF or POP2)
pCeMM-CTAP(SG)-Gw (p2432) (42) using primers
in human HT1080 cells, we could previously show that
G1120/G1121thatcontainaSalIandXhoIlinker,respect-
Caf1a/b are required for the degradation of a reporter
ively, and cloned into the XhoI site of TOPuro (p2433).
mRNA containing the ARE of GM-CSF (a typical
Based on TOPuro-mycSG, TOPuro-Caf1a-mycSG
class II ARE), whereas the knock down of Ccr4a/b had
(p2485) encoding human wt Caf1a and TOPuro-Caf1a-
no effect (27). In human, Ccr4 and Caf1 together are
AA-mycSG(p2737)containingtheD40A/E42Amutation
part of the large, 1.2 MDa Ccr4–Not complex that
wereclonedasdescribedpreviously(43).
comprises Not1 (CNOT1), Not2 (CNOT2), Not3
For pcDNA3-YFP-TTP (p2789), pcDNA3-YFP-TTP-
(CNOT3), Caf40 (CNOT9 or Rcd1), CNOT10, TAB182
M1,2 (p2712), pcDNA3-YFP-TTP-N (p2790), pcDNA3-
and C2ORF29 (28,29). In the nucleus, the Not1, Not2
YFP-TTP-NZ (p2791) and pcDNA3-YFP-TTP-C
and Caf40 subunits of the human Ccr4–Not complex
(p2792), the TTP cDNA fragments were taken out of
play a role as regulators of transcription (30,31). Not1,
the corresponding pcDNA3-TTP-mycHis plasmids (40)
e.g. binds to oestrogen receptor and thereby represses
and cloned into the EcoRI/XbaI sites of pcDNA3-YFP
ligand-dependent transcriptional activation (31). In
(p2168) described previously (43). The TTP-M1,2 mutant
addition, studies in Saccharomyces cerevisiae showed
was kindly provided by Keith Blackwell (Joslin Diabetes
that the Not subunits and Not4, in particular, are
Center, Boston, USA).
requiredtomaintainnormallevelsofhistonemethylation
For pcDNA3-MS2cp-TTP-mycHis (p2200), the MS2cp
(32,33). As for the cytoplasmic function of the Ccr4–Not was excised from pcNMS2 (41) using HindIII and ligated
complex, early studies in yeast identified Ccr4 and Caf1 into the HindIII site of pcDNA3-mTTP-mycHis (p2115).
(also termed Pop2) as major cytoplasmic deadenylases pcDNA3-MS2cp-TTP-M1,2-mycHis (p2203) was
(34,35). In yeast, Ccr4 appears to be more active than generated in the same way using pcDNA3-TTP-M1,2-
Caf1 (36,37), whereas Caf1 seems to be the major mycHis (p2117) as the vector. To clone pcDNA3-MS2cp-
deadenylaseinDrosophila,trypanosomesandmammalian mycHis (p2205), pcDNA3-MS2cp-TTP-mycHis was
cells (27,38,39). Yeast studies further found that Not2 opened with KpnI/XbaI to release TTP, and the vector
and Not5 enhance the deadenylation rate of cytoplasmic was blunt-end religated. For pcDNA3-MS2cp-TTP-
mRNAs and may thus serve as activators of Ccr4 or C-mycHis (p2808) and pcDNA3-MS2cp-TTP-N-mycHis
Caf1 (36). A detailed analysis in Drosophila showed (p2806), TTP was replaced in the pcDNA3-MS2cp-TTP-
that in addition to Caf1, Not1, Not2 and Not3 are mycHisvectorwiththeC-orN-terminusofTTP,respect-
critical for rapid deadenylation of both bulk and Hsp70 ively, by swapping with a ClaI—XbaI fragment from
mRNA (38). pcDNA3-TTP-C-mycHis (p2122) or pcDNA3-TTP-N-
In this study, we purified proteins associated with TTP mycHis(p2123)(40).
and found that TTP interacts with Not1. We show that pCMV-Flag-Not1 (p2556) encoding full-length Not1
Not1 is required for TTP-mediated mRNA decay, map has been described previously (31). For pCMV-
the regions involved in the TTP–Not1 interaction and Flag-Not1:1-700 (p2594), pCMV-Flag-Not1 was digested
provide evidence that TTP recruits the Caf1 deadenylase with NheI/XbaI and religated, thereby ejecting the region
via its interaction with Not1. encoding amino acid 701–2376 of Not1. To generate
NucleicAcidsResearch,2011,Vol.39,No.10 4375
pCMV-Flag-Not1:1-997, pCMV-Flag-Not1 was digested In-gel tryptic digestion and LC-MS/MS analysis
with XbaI and religated, thereby ejecting the region
Massspecanalyseswerecarriedoutatthecorefacilityfor
encoding aa 998–2376 of Not1. The empty vector
mass spectrometry and proteomics of the Center for
pcDNA3-Flag (p2002) contains the sequence ACC ATG
Molecular Biology at the University of Heidelberg
GAC TAC AAG GAC GAT GAC GAC AAG between
(ZMBH). Proteins in the gel were stained with colloidal
the HindIII and BamHI sites of pcDNA3 (Invitrogen).
Coomassie, and the entire lane was cut into five gel slices
For pcDNA3-HA-Not1:727-1449 (p2642), pCMV-
withascalpel.Gelslicesweretransferredtoa96-wellplate
Flag-Not1 was digested with PvuII, and the 2172-bp
and reduced, alkylated and digested with trypsin (44)
fragment was ligated into the EcoRV site of pcDNA3-
using a Digest pro MS liquid handling system (Intavis).
HA (p2003) described previously (43). Subsequently the
Following digestion, tryptic peptides were extracted from
HA tag was replaced by the YFP tag from plasmid
the gel pieces with 50% acetonitrile/0.1% trifluoroacetic
pcDNA3-YFP by a HindIII/KpnI digest. From the same
acid (TFA), concentrated nearly to dryness in a vacuum
PvuII digest of pCMV-Flag-Not1, the 1911-bp fragment
centrifugeanddilutedtoatotalvolumeof30mlwith0.1%
was cloned into the EcoRV site of pcDNA3-HA
TFA; 25ml of the sample was analyzed by a nano-HPLC
to generate pcDNA3-HA-Not1:1880-2376 (p2657). For
system(Eksigent1Dplus)coupledtoaESILTQOrbitrap
pcDNA3-YFP-Not1:1880-2376 (p2693), the HA tag mass spectrometer (Thermo Fisher). Sample was loaded
was then replaced by YFP via an SpeI/BamHI digest. on a C18 trapping column (Inertsil, LC Packings) with a
For pcDNA3-HA-Not1:1330-1601 (p2673), the Not1 flow rate of 10ml/min 0.1% TFA. Peptides were eluted
fragment 1330–1601 was amplified by PCR using primers and separated on an analytical column (75mm(cid:4)150mm)
G1653/G1654 and cloned into the BamHI/XhoI sites packed with Inertsil 3mm C18 material (LC Packings)
of pcDNA3-HA. Subsequently the HA tag was replaced with a flow rate of 200 nl/min in a gradient of buffer A
by YFP via an SpeI/BamHI digest to generate pcDNA3- (0.1%formicacid)andbufferB(0.1%formicacid,aceto-
YFP-Not1:1330-1601 (p2694). For HA-tagged Ccr4a, nitrile): 0–6min: 3% B; 6–60min: 3–40% B; 60–65min:
the CNOT6 cDNA was cloned into the pMT2MS-HA 60–90% B. The column was connected with a nano-ESI
vector. emitter (New Objectives), and 1500 V was applied via
liquid junction. One survey scan (resolution: 60000) was
followedbyfiveinformation-dependentproductionscans
Cell culture and transfection
in the LTQ. Only doubly and triply charged ions were
HeLa and HEK293T cells were cultured in Dulbecco’s selected for fragmentation. All MS/MS samples were
modified Eagle’s medium (DMEM; Gibco) supplemented analyzed using Mascot (Matrix Science; version 2.2.03).
with10%fetalcalfserum(FCS)(Gibco),2mML-glutam- The NCBInr database (taxonomy: homo sapiens,
ine, 100 U/ml penicillin and 0.1mg/ml streptomycin 204932 entries) was used with the following search par-
(all PAN). T-REx-HeLa- cells (Invitrogen) were cultured ameters: cleavage with trypsin, parent ion tolerance of
inthesamemediumcontaining10%tetracycline-negative 4.0ppm and fragment ion mass tolerance of 0.20 Da.
FCS (PAA). All cells were grown at 37(cid:2)C and 5% CO . Iodoacetamide derivative of cysteine was specified in
2
Cells were transfected with DNA using polyethyl- Mascot as a fixed modification. Deamidation of aspara-
eneimine (PEI) (Polysciences Europe; 1mg/ml, pH 7.0) gine and oxidation of methionine were specified as
at a ratio of 1:2 (DNA:PEI) in serum-free DMEM variable modifications.
without antibiotics. For transfection of siRNAs,
Lipofectamine 2000 (Invitrogen) and Optimem (Gibco) Immunoprecipitation and western blot analysis
were used according to the manufacturer’s protocol.
Cells were transiently transfected 24–48h prior to lysis.
Medium was changed to regular DMEM 4h after
When Tet-inducible expression vectors were used, cells
transfection of DNA or siRNA.
were treated over night with 1mg/ml doxycycline
(Sigma). Typically, cells from a 10-cm dish were
solubilized in 400ml RNP lysis buffer and nuclei were
TTP purification
removed by centrifugation. YFP-tagged proteins were
T-REx-HeLa cells stably transfected with TOPuroGS purified using the GFP-binder as described in Ref. (45).
or TOPuroGS-TTP-wt were treated with doxycycline Cytoplasmiclysateswereallowedtobindaffinitymatrices
(1mg/ml) for 16h to induce expression of the proteins. for 2–4h at 4(cid:2)C prior to washing. For IPs with specific
Cytoplasmic lysates were prepared from (cid:3)2(cid:4)108 cells antibodies, cytoplasmic lysates were pre-cleared by the
using RNP lysis buffer (1% NP40, 150mM NaCl, addition of 30ml protein A/G agarose beads (Thermo
50mM Tris–HCl, pH 8.0, 1mM MgCl , 10% glycerol, Scientific) for 1h at 4(cid:2)C, incubated with 1mg of
2
1mM dithiothreitol, 1mM Na VO , 50mM NaF and antibody for 2h and 30ml of protein A/G beads for add-
2 3
40nM okadaic acid), and tagged proteins were bound to itional 2h before washing. Three to five washes were
streptavidin sepharose (GE Healthcare). The beads were carried out using RNP lysis buffer. From the GFP-
washed five times with RNP buffer and eluted by the binder and the protein A/G agarose beads, proteins were
addition of biotin (1mM, pH 5.0). The eluate was eluted with 1% SDS-containing sample buffer. Where
concentrated by vacuum centrifugation, resolved on a indicated, RNase A (Genomed) was added during the IP
5–20% gradient polyacrylamide gel, and proteins were ataconcentrationof1mg/ml.RNaseI(Ambion)wasused
visualized by colloidal Coomassie staining. ataconcentrationof500U/ml. RNA wasextracted from
4376 NucleicAcidsResearch,2011,Vol.39,No.10
theunbound fraction using TriFast (Peqlab) according to siRNAs
manufacturer’s protocol.
CellsweretransfectedtwicewithsiRNAatafinalconcen-
Proteins were resolved on 5–20% gradient polyacryl-
tration of 100nM over a period of 4 days using
amide gels and transferred onto 0.2mm pore size nitrocel-
Lipofectamine 2000 (Invitrogen). The siRNA concen-
lulose membranes (Peqlab) for western blotting.
tration was 50 nm each in cases where two siRNAs were
Horseradish peroxidase-coupled secondary antibodies
transfected simultaneously. Where indicated, plasmid
(Jackson Immunoresearch) in combination with Western
DNA was added to the second siRNA transfection.
Lightning-enhanced chemiluminescence substrate (Perkin
siRNAs were synthesized by Ambion and correspond to
Elmer)wereusedfordetectiononchemiluminescencefilm
the following sequences (sense strand):
from GE Healthcare.
D0/S015 (control), 50-GCAUUCACUUGGAUAGUA
AdTdT-30; C2/S014 (control), 50-GCAUUCACUUGGA
Antibodies
UAGUAAdTdT-30; U0 (control), 50-GAAUGCUCAUG
The following antibodies were used for IP and western UUGAAUCAdTdT-30 S021 (Not1), 50-GGAACUUGU
blot analysis: Mouse monoclonal anti-HA (HA.11, UUGAAGAAUAdTdT-30; S034 (Not1), 50-GAGGAUG
Covance), anti-myc 9E10 (MMS-150P, Covance), ACAAUCGAGAAAdTdT-30.
anti-Flag (M2, Sigma Aldrich), rabbit polyclonal
anti-GFP (Abcam ab290) and anti-14-3-3 K-19 (Sant
Cruz, sc-629). Anti-Caf1a antibody was kindly provided RESULTS
by Ann-Bin Shyu (University of Texas, Houston, TX,
Not1 interacts with TTP
USA).
In order to identify proteins that associate with TTP, we
Northern blot analysis generated a Tet-inducible vector expressing GS-tagged
TTP. This tag encodes for protein G and a streptavidin-
For RNA decay experiments, HeLa cells were transiently
bindingpeptide(42).StablytransfectedHeLaT-RExcells
transfected with pTet-Off (Clontech) and a pTet-7B
weregeneratedwithinducibleexpressionofGS-TTP,and
reporter plasmid; 1mg/ml doxycycline (Sigma) was added
the fusion protein was affinity-purified on streptavidin
to cell cultures for indicated time periods and total RNA
sepharose. Due to difficulties with TEV cleavage, we
was extracted using the Genematrix RNA purification kit
could not perform a two-step purification as intended ini-
(Eurx, Roboklon). To generate deadenylated mRNA, the
tially. Upon elution with biotin, co-purifying proteins
20mg of total RNA was annealed with 1nmol/ml oligo
were visualized by Coomassie staining and compared to
dT in presence of 1mM EDTA for 15min at 26(cid:2)C.
18 proteins co-purifying with the GS tag alone (Figure 1A).
RNA:DNA hybrids were digested using 5 U/ml RNase
Proteins from both purifications were identified by mass
H (NEB) for additional 15min at 37(cid:2)C. RNA was ex-
spectrometry. If a protein was identified by three or more
tracted with phenol:chloroform:isoamylalcohol (50:50:1)
peptides, and if the peptide number was (cid:5)3-fold higher
and precipitated; 5–15mg of RNA was resolved by 1.1%
in the GS-TTP purification compared with the GS purifi-
agarose/2% formaldehyde/MOPS gel electrophoresis and
cation, it was considered a potential candidate associated
blotted over night with 8(cid:4)saline-sodium citrate (SSC)
with TTP (Supplemental Table S2). Among previ-
buffer onto Hybond-N+ Nylon membranes (GE
ously known interacting partners (46), we found 14-3-3
Healthcare). Membranes were hybridized overnight at
adaptor proteins (40,47) to co-purify with wild-type TTP
55(cid:2)C with digoxigenin-labeled RNA probes synthesized
(Figure 1A), but not with a non-phosphorylatable S52A/
in vitro using Sp6 polymerase (Fermentas). Membranes
S178AmutantofTTP(datanotshown).Interestingly,five
were washed twice with 2(cid:4) SSC/0.1% SDS for 5min,
of the seven isoforms of 14-3-3 could be identified in our
and twice with 0.5(cid:4) SSC/0.1% SDS for 20min at 65(cid:2)C.
mass spec analysis, suggesting that the interaction of
Alkaline phosphatase-coupled anti-digoxigenin Fab frag-
phospho-TTP with 14-3-3 is not isoform-specific.
mentsandCDP-Starsubstrate(bothRoche)wereusedfor
Wefurthernoticedthatalargeprotein>200kDainsize
detection according to the manufacturer’s instructions.
co-purified with GS-TTP (Figure 1A), and this protein
The following primers were used to generate templates
was identified as Not1 (Supplementary Table S2). Not1
for Sp6 probes by PCR: G1000 and G1001 for the
isthescaffoldproteinoftheCcr4–Notcomplexthatregu-
probe against exons 1 and 2 of rabbit b-globin; G83 and
lates transcription in the nucleus and contains two major
G1009 for the probe against human nucleolin.
cytoplasmicdeadenylases,Ccr4andCaf1(48).SinceCcr4
andCaf1arerequiredforturnoverofeukaryoticmRNAs
Real-time PCR
(27,34,35,39,49), we further explored the interaction of
cDNA was synthesized from 5mg of total RNA using Not1 with TTP. By transient transfection in HEK293
oligo-dT (Invitrogen) and M-MLV H(–) reverse tran- cells, we found that yellow fluorescent protein (YFP)-
18
scriptase (Promega). PCRs were carried out with 1:40 of tagged TTP co-IPs with Flag-tagged Not1, whereas YFP
a cDNA reaction, 200nM target-specific primers and alonedidnot(Figure1B,lanes3and4).ByinverseIPwe
SYBR Green Mastermix (Roche) in a total volume of confirmed that Flag-Not1 co-IPs with myc-tagged TTP,
10ml on a Lightcycler 480 (Roche). The following whereas the Flag tag alone does not (Figure 1C, lanes 3
primers were used: G1578/G1579 for Not1, and G1576/ and 4). We then expressed the TTP mutant M1,2 (50),
G1577 for nucleolin as internal control. which does not bind RNA because of CCCH to SSCH
NucleicAcidsResearch,2011,Vol.39,No.10 4377
A B Input IP: YFP C Input IP: Flag
kDa TTP P P-TTP P P-TTP g g-Not1 g g-Not1
210500 GS GS- YF YF YF YF Fla Fla Fla Fla
110200 Not1 Flag-Not1 Flag-Not1 TTP-myc TTP-myc
85 kDa kDa
657000 5750 YFP-TTP 250 Flag-Not1
432005 14-3-3 3257 YFP 5355 TTP-myc
15+1200 1 2 250 Flag-Not1 1 2 3 4
1 2 3 4
D Input IP:YFP E Input IP: YFP
2 22 2 2
1, 1,1, 1, 1,
M MM M M
PP- PPP-P- P P- P P-
TT TTTT T T T T
TT TTTT T T T T
PP-P- PPP-P-P-P- P P- P- P P- P-
FFF FFFFFF F F F F F F
YYY YYYYYY Y Y Y Y Y Y kDa
RNase A – – – – + – + – + 70
Flag-Not1 YFP-TTP 55
35
kDa YFP 27
YFP-TTP 70
55
globin-ARE mRNA
35
YFP 27
nucleolin mRNA
Flag-Not1 250
1 2 3 4 5 6 7 8 9 1 2 3 4 5 6
Figure 1. TTP interacts with Not1 independently of RNA. (A) T-REx-Hela cells were stably transfected with either the GS tag alone or GS-TTP,
andtreatedwithdoxycyclinefor16htoinduceexpressionofthetaggedproteins.Cytoplasmiclysateswerepreparedfrom(cid:3)2(cid:4)108cells,andtagged
proteinswerepurifiedusingstreptavidinsepharose.Proteinswereelutedwithbiotin,resolvedona5–20%gradientpolyacrylamidegelandvisualized
bycolloidalCoomassiestaining.(B)HEK293cellsweretransientlytransfectedwithFlag-Not1togetherwitheitherYFPorYFP–TTP.After1day,
cytoplasmiclysates(input)werepreparedforIPwithGFP-binder.WesternblotanalysiswascarriedoutwithantibodiesagainsttheFlagandYFP
tags.(C)HEK293cellsweretransientlytransfectedwithTTP-myctogetherwitheitherFlagaloneorFlag-Not1.CytoplasmiclysateswereusedforIP
with Flag antibody, and western blots analysis was carried out with antibodies against the myc and Flag tags. (D) HEK293 cells were transiently
transfectedwithFlag-Not1togetherwithYFP,YFP–TTPorthezinc-fingermutantYFP–TTP-M1,2.IPandwesternblotanalysiswascarriedoutas
in panel B, except that RNase A was added during IP where indicated. (E) HeLa cells were transiently transfected with a b-globin reporter gene
containing the ARE of TNFa in its 30-UTR. In addition, cells were transfected with YFP, YFP–TTP or the zinc-finger mutant YFP–TTP–M1,2.
RNA and protein were extracted from both the input and IP samples. Western blot analysis was carried out with an antibody against YFP, and
globin-ARE as well as nucleolin mRNA were detected by northern blot analysis.
mutations in both of its zinc fingers. IP analysis showed transcriptionwithdoxycycline.Inthepresenceoftwodif-
that Flag-Not1 was efficiently co-immunoprecipitated ferent control siRNAs, the globin-ARE mRNA showed
with both wt and mutant TTP (Figure 1D, lanes 6 and very rapid deadenylation and subsequent degradation
8). Likewise, addition of RNase A during the IP did not (Figure 2A, left panels). In contrast, both siRNAs
abolish the interaction between YFP-TTP and Flag-Not1 directed against Not1 caused a strong stabilization of
(Figure1D,lanes7and9),suggestingthattheinteraction the globin-ARE mRNA (right panels). By analyzing the
between TTP and Not1 is RNA-independent. Lack of reporter mRNA size, we noticed that the globin-ARE
RNA binding by TTP-M1,2 was confirmed by RNA-IP: mRNA remained above the 800nt mark after Not1
whereaswtTTPefficientlyco-purifiedwithaglobin-ARE knock down, whereas it was shortened to (cid:3)700nt in the
reporter mRNA (Figure 1E, lane 5) and the zinc-finger control conditions. A lower exposure of the globin-ARE
mutant TTP-M1,2 did not (lane 6). mRNA signal in the second panel of Figure 2A clearly
shows the deadenylated mRNA species that is present in
Not1 is required for TTP-mediated mRNA deadenylation
the control knock downs, but absent in the Not1 knock
and decay
downs.Thedifferencewasvisualizedbyplottingthesignal
Next, we determined whether Not1 is required for AMD. intensity as a function of mRNA size in the panels below
We first reduced Not1 expression by transfection of two of the northern blots of Figure 2A. This result indicated
different siRNAs in HeLa cells, and subsequently trans- that Not1 is primarily required for full deadenylation of
fected TTP together with a b-globin reporter gene that thereportermRNA. QuantificationoftheoverallmRNA
contains the ARE of TNFa in its 30-UTR and is driven signals in three biological repeat experiments showed that
by a tetracycline-sensitive promoter. Degradation of the knockingdownNot1increasesthehalf-lifeofglobin-ARE
reporter mRNA was measured after blocking its mRNA from 0.5 to 1.2h (Figure 2B). By quantitative
4378 NucleicAcidsResearch,2011,Vol.39,No.10
A TTP
si-control si-control si-Not1 si-Not1
(U0) (C2) (S021) (S034)
nt 0 1 2 0 1 2 0 1 2 0 1 2 dox. (hrs)
1000
globin-ARE
800
(high exp.)
600
1000
globin-ARE
800
(low exp.)
600
nucleolin
1000
e
z
si
A 800 dox.
N
R 0
m 1 hrs
2 hrs
600
signal intensity (A.U.) signal intensity (A.U.) signal intensity (A.U.) signal intensity (A.U.)
B si-control t = 0.5 hrs ± 0.1 C D
1/2
(combined) 2) 1) 4)
C 2 3
%)120 s(cio-Nmobitn1e d ) t1/2 = 1.2 hrs ± 0.3 A (%)110200 si-contsri-olN(ots1i-(NS0ot1(S0
A (100 RN 80
N m
mR 80 ncl 4600 Flag-Not1
ncl 60 1 / 20 14-3-3
E / Not 0
R 40 O) 2) 1) 4)
globin-A 200 contrsiol-c(oHns2tir-olNo(tsCi1-(NSo0t21(S03
0 0.5 1 1.5 2
dox.(hours)
Figure 2. Not1 is required for ARE-mediated mRNA deadenylation and decay. (A) HeLa cells were transfected with two independent control
siRNAs or two siRNAs targeting Not1. After 48h, cells were transfected again with the same siRNAs together with a plasmid encoding TTP, a
pTet-Off-drivenb-globinreportergenecontainingtheAREofTNF-ainits30 UTR,andtheTet-Offtransactivator.Reportergenetranscriptionwas
blocked specifically by addition of doxycycline 24h after the second transfection, and RNA was isolated after the time intervals indicated.
Globin-ARE and nucleolin mRNA were detected by northern blot analysis; a high and low exposure is provided for the globin-ARE signal. The
sizeofthemRNAwasdeterminedbycomparisontoanRNAmarker.Inthebottompanel,deadenylationwasvisualizedbyquantifyingthesignal
intensity of globin-ARE mRNA along the length of the signal and plotting it as a function of mRNA size. (B) Quantification of the globin-ARE
reporter mRNA decay in panel A and repeat experiments. Signal intensities of globin-ARE mRNA were normalized to nucleolin mRNA and
representedas%oftheinitialvalue.ResultsfromthetwocontrolandthetwoNot1siRNAtransfectionswerecombined.Thegraphshowsaverage
values±SEfromeight(si-control)andsix(si-Not1)biologicalrepeatexperiments.(C)HeLacellsweretransfectedtwicewithsiRNAsasinpanelA.
Not1 mRNA levels were quantified by real-time PCR using nucleolin mRNA for normalization. (D) HEK293 cells were first transfected with
Flag-Not1,and1daylaterwiththesiRNAsindicated.Afteroneadditionalday,cytoplasmiclysateswerepreparedandanalyzedbywesternblotting
using antibodies against Flag and 14-3-3.
PCR, we determined that the two siRNAs reduced Not1 analysis (Figure 2D). Taken together, we concluded
mRNA expression levels to (cid:3)10% and 30%, respectively from these experiments that Not1 is required for AMD
(Figure2C).Knockdownefficiencywasalsoconfirmedat of a reporter mRNA containing a class II ARE.
the protein level, as both siRNAs reduced Flag-Not1 Since several ARE-binding proteins including TTP,
levels below the detection limit of our western blot BRF1, KSRP and AUF1 participate in AMD (51), we
NucleicAcidsResearch,2011,Vol.39,No.10 4379
thentestedwhetheranmRNAwhosedecayismediatedby predominant deadenylase (27,49). Previously, we have
TTP alone would also require Not1 for degradation. For shown that in human HT1080 fibrosarcoma cells, Caf1
thispurpose,wechoseatetheringapproachwherebyTTP is of particular importance for the rapid degradation of
is forced to bind an mRNA lacking an ARE through the an ARE-containing mRNA (27). To further establish the
tight interaction of the MS2 bacteriophage coat protein link between Caf1 and AMD, we examined whether TTP
(cp) with its cognate binding site (bs) in the MS2 RNA. could co-IP with endogenous Caf1a in HEK293 cells.
MS2cp-TTP fusion proteins were expressed in HeLa cells Indeed, we found that YFP-tagged TTP associates with
together with a b-globin reporter mRNA containing six Caf1a (Figure 4A, lane 6), whereas YFP alone did not
repeatsoftheMS2bsinits30-UTR.Figure3Ashowsthat (lane 4). Importantly, addition of RNase A to the IP did
tethering of MS2cp-TTP causes rapid degradation of the not reduce the interaction of YFP-TTP with Caf1a (lane
mRNA,whereasthemRNAremainsstableaftertethering 7). Given that YFP-TTP-M1,2 also co-IPs with Caf1a
of MS2cp alone. Tethering of the TTP-M1,2 mutant, (lanes 8 and 9), we concluded that TTP associates with
which does not bind RNA, was as efficient as tethering the Caf1 deadenylase in an RNA-independent manner.
of wt TTP(Figure 3A). Thisconfirms that TTP functions We then tested whether Caf1 is important for
byrecruitingcomponentsoftheRNAdecaymachineryto TTP-induced mRNA degradation. Since knocking down
the bound RNA (16,20). We then tested the effect of both Caf1a and Caf1b together is not very efficient (27),
knocking down Not1 by transfecting two siRNAs (S021 we made use of a dominant-negative mutant of Caf1a,
and S034) simultaneously. As seen with the Caf1a-AA (D40A/E42A), which can efficiently prevent
ARE-containing mRNA above, we observed that the deadenylation (43). TTP was expressed together with
mRNA tethered to MS2cp-TTP-M1,2 was stabilized by a globin-ARE reporter mRNA in order to accelerate
knock down of Not1 (Figure 3B). Quantification of four AMD (Figure 4B). Compared with vector control,
biological repeat experiments showed an increase in the co-expression of Caf1a-wt had only a small effect on the
mRNA half-life from 1.6h to >6h. Thus, mRNA decay deadenylation pattern and decay rate of globin-ARE
mediatedbybindingofTTPaloneisclearlydependenton mRNA. Conversely, co-expression of dominant-negative
the deadenylase scaffold protein Not1. Caf1a-AAstronglyreducedbothdeadenylationanddecay
oftheARE-containingreportermRNA.Figure4Bshows
Caf1 is required for TTP-induced mRNA deadenylation
that only about half of the poly(A) tail is removed in the
In yeast, Ccr4 and Caf1 were found tobe themajor cyto- presenceofCaf1a-AA. Thisisinline withthenotion that
plasmic deadenylases (35), whereas in trypanosomes, Caf1 takes part in the second, rapid phase of
Drosophila and human cells, Caf1 appears to be the deadenylation, as pointed out by Yamashita et al. (22).
Figure 3. Not1isrequiredformRNAdecayinducedbytetheringofTTP.(A)HeLacellsweretransientlytransfectedwithMS2cp,MS2cp-TTPor
thezinc-fingermutantMS2cp-TTP-M1,2thatdoesnotbindmRNAbyitself.Inaddition,apTet-Off-drivenb-globinreportergenethatcontainssix
MS2-bindingsites(bs)inits 30 UTRtogetherwiththeTet-Offtransactivator weretransfected intoallcells. Transcription ofthereportergenewas
turned offby treating cells with doxycycline. RNA was isolated after the indicated time intervals. Globin-MS2bs and nucleolin mRNA levels were
detectedbynorthernblotanalysis.Thebottomgraphshowsquantificationoftheglobin-MS2bsmRNA.Averagevalues±SEfromthreebiological
repeatexperimentsarerepresented.(B)HeLacells weretransfectedsimultaneously witheithertwocontrolsiRNAsortwosiRNAstargetingNot1.
After 48h, cells were transfected with the same siRNAs again together with MS2cp-TTP-M1,2, the pTet-Off-globin-MS2bs reporter gene and the
Tet-Off transactivator; 24h later, cells were treated with doxycycline and total RNA was isolated after the indicated time intervals. Globin-MS2bs
andnucleolinmRNAlevelsweredetectedbynorthernblotanalysis.Forquantification,signalintensitiesofglobin-MS2bsmRNAwerenormalized
to nucleolin mRNA and represented as percentage of the initial value. The bottom graph shows average values±SE from four biological repeat
experiments.
4380 NucleicAcidsResearch,2011,Vol.39,No.10
A Input IP:YFP
2 2 2
1, 1, 1,
M M M
- - -
P P P P P P
T T T T T T
T T T T T T
- - - - - -
P P P P P P P P P
F F F F F F F F F
Y Y Y Y Y Y Y Y Y
– – – – + – + – + RNase A
70
YFP-TTP
55
35
27 YFP
35
Caf1a
27
1 2 3 4 5 6 7 8 9
B TTP
dT vector dT Caf1a-wt dT Caf1a-AA
A+
globin-
ARE
A–
nucleolin
0 0 1 2 4 0 0 1 2 4 0 0 1 2 4 dox. (hrs)
A+
e
A siz dox.0
N
R 1 hr
m
2 hrs
A– 4 hrs
signal intensity (A.U.) signal intensity (A.U.) signal intensity (A.U.)
t1/2 = 1.2 hrs ± 0.2 t1/2 = 0.9 hrs ± 0.05 t1/2 = 2.6 hrs ± 1.1
120
100
%) 80
NA ( 60
R 40
m
20
0
0 1 2 3 4 0 1 2 3 4 0 1 2 3 4
dox. (hrs) dox. (hrs) dox. (hrs)
Figure 4. Caf1interactswithTTPandmediatesdeadenylationofglobin-ARE-mRNA.(A)HeLacellsweretransientlytransfectedwithYFP,YFP–
TTPorTTP–M1,2.Cytoplasmiclysates(input)werepreparedafter24hforIPwithGFP-binder.Inputsweredividedandonehalfwastreatedwith
RNase A during IP. Western blot analysis was carried out with antibodies against YFP and endogenous Caf1a. (B) HeLa cells were transiently
transfected with TTP, the Tet-Off transactivator and a pTet-Off-driven b-globin reporter gene containing the ARE of TNF-a in its 30-UTR. Cells
were co-transfected with either empty vector, Caf1a-wt or dominant negative Caf1a-AA. Reporter gene transcription was blocked specifically by
addition of doxycycline 20h after transfection, and total RNA was isolated after the indicated time intervals. Globin-ARE and nucleolin mRNA
weredetectedbynorthernblotanalysis.dTindicatesthattheRNAwastreatedwitholigo-dTandRNaseHtogeneratedeadenylatedmRNA.Inthe
middlepanel,deadenylationwasvisualizedbyquantifyingthesignalintensityofglobin-AREmRNAalongthelengthofthesignalandplottingitas
a function of mRNA size. In the bottom panel, the overall signal intensity of globin-ARE mRNA was quantified and normalized to nucleolin
mRNA. Average values±SE were obtained from three biological repeat experiments and plotted as % of the initial time point.
The C-terminal domain of TTP is critical for recruitment
The effect on deadenylation was visualized by plotting
of Not1 and Caf1
the mRNA signal intensity along the length of the
mRNA signal (Figure 4B, middle panel). Quantification TTP contains in its central part a tandem CCCH
of the mRNA decay rate from three biological repeat zinc-finger domain that is required for binding of AREs
experiments (Figure 4B, bottom panel) showed that (13),whereastheN-terminaldomainofTTPwasfoundto
Caf1a-AA increased the mRNA half-life by >2-fold. stimulate mRNA decay by recruiting components of the
NucleicAcidsResearch,2011,Vol.39,No.10 4381
general mRNA decay machinery (20). To further under- importantfortheinteractionwithTTP,andthatTTPcan
stand the interaction of TTP with Not1 and Caf1, we ex- associate with Not1 independently of Caf1.
pressed the N- and C-terminal domains of TTP, Our previous yeast two-hybrid experiments mapping
schematically depicted in Figure 5A, in HEK293 cells. the interactions between human Ccr4-Not subunits
By co-IP analysis we found that the N-terminal domain indicated that Caf1a/CNOT7 interacts with amino acid
of TTP (amino acid 1–94) does not interact with either 648–2376 of Not1 (28). We further examined the middle
Caf1a or Flag-Not1 (Figure 5B, lane 10). The NZ region of Not1 using a fragment that spans amino acid
fragment (amino acid 1–176) comprising the N-terminus, 727–1449.Thisfragmentwasabletoco-IPwithbothTTP
the zing-finger domain and a small part of the C-terminal and Caf1a (Figure 6C, lane 6). Interestingly, a smaller
domain showed only a very weak interaction with Not1:1330–1601 fragment shifted slightly toward the
Flag-Not1 or Caf1a (lane 12). In contrast, the C-terminus interacted efficiently with TTP, but not with
C-terminal domain (amino acid 152–319) was able to Caf1a (lane 7). The last fragment we tested contains the
co-IP Flag-Not1 and Caf1a as efficiently as full-length C-terminus of Not1 (amino acid 1880–2373), and did not
TTP (compare lanes 8 and 14). Treatment of the interactwitheitherTTPorCaf1a(lane8).Takentogether,
samples during IP with RNase I, which also cleaves it is evident that TTP and Caf1a interact with different
RNA within a poly(A) tail, did not abrogate the inter- though overlapping regions of Not1. TTP shows a more
action with either Flag-Not1 or Caf1a (lanes 9 and 15). extensive interaction with Not1 in the region between
Efficient degradation of RNA is shown in the bottom amino acid 700 and 1601, whereas the interaction
panels of Figure 5B for rRNA and nucleolin as an with Caf1 is restricted to the region between amino acid
abundant mRNA. Since TTP–Not1/Caf1a interactions 727–1499 of human Not1.
are technically challenging to detect, a repeat IP is Finally, we wanted to test if the interaction between
shown in Supplementary Figure S1A and B. From these TTP and Caf1 is dependent on Not1. To this end, we
results we concluded that Not1 and Caf1a primarily knocked down Not1 expression in HeLa cells. Co-IP ex-
interact with the C-terminal domain of TTP in an RNA- periments clearly showed that the interaction between
independent manner. We further tested the interaction of YFP-TTP and endogenous Caf1a is lost after knock
TTP with Ccr4a and again found the C-terminal domain downofNot1,whereasinacontrolknockdowntheinter-
of TTP to associate with HA-Ccr4a (Supplementary action was unaffected (Figure 6D, lanes 6 and 8). Taken
Figure S1C). together, these results provide compelling evidence that
To functionally compare the N- and C-terminal Not1 provides a platform on which TTP recruits the
domains of TTP, we again made use of the tethering Caf1 deadenylase to its target mRNAs.
assay. TTP-N and TTP-C were fused to the MS2cp, and
both fragments were found to induce very rapid
deadenylation and decay of the globin-M2bs mRNA DISCUSSION
(Figure 5C). Co-expression of the dominant-negative
Initially, the Not proteins were identified by genetic
Caf1a-AA mutant efficiently prevented deadenylation of
studies in S. cerevisiae as general repressors of transcrip-
the tethered reporter mRNA and reduced its decay rate
tion that preferentially acton promoters lackinga canon-
(Figure 5C, bottom panels). Taken together, these results
ical TATA box (Negative on TATA, Not). This function
provide strong evidence that the interaction of the TTP
ismediatedthroughregulationoftheTFIIDtranscription
C-terminal domain with Not1–Caf1a is functionally
complex and histone modification by the Not proteins
relevant for the destabilizing activity of TTP. However,
(48,52). In parallel, catabolite repressor protein 4 (Ccr4)
it is interesting to note that the N-terminal domain can
was identified as a factor required for the expression of
also activate deadenylation and mRNA decay, although
genesthatareinducedundernon-fermentativeconditions.
we do not detect an interaction with Not1/Caf1a.
Biochemical studies then revealed that Ccr4 and Not
Similarly, a previous study by Lykke-Andersen and
proteins are actually part of the same complex (53). In
Wagner (2005) had found that both the N- and
addition to its functions in the nucleus, the yeast Ccr4–
C-terminal domains of TTP mediate mRNA degradation
Not complex was found to play an important role in the
(20).
cytoplasm where Ccr4 and Caf1 represent the two major
exoribonucleases responsible for cytoplasmic mRNA
Interaction of TTP with Not1 is extensive and required
deadenylation (34,35,37).
for Caf1 recruitment
In metazoa, the Ccr4–Not complex shares many of
To further examine the interactions between TTP, Not1 its characteristics with the yeast complex. In the nucleus,
and Caf1a, we divided the human Not1 cDNA, which the human complex serves as a negative regulator of
encodes for a 2376-amino acid long protein, into several transcription through its subunits Not1, Not2 and
fragments (Figure 6A). The most N-terminal fragment of Caf40 (30,31). Moreover, Not1 interacts with the ligand-
Not1 encompassing amino acid 1–700 did not co-IP with binding domain of estrogen receptor alpha and represses
TTP, whereas a longer N-terminal fragment covering estrogen receptor-mediated transcription (31). The gen-
amino acid 1–997 did interact with TTP (Figure 6B, eral role of the cytoplasmic Ccr4–Not complex in
lanes7and8).Importantly,neitherofthesetwofragments mRNA deadenylation is also conserved in metazoa
showed an interaction with Caf1a. This result suggests (22,49). In contrast to yeast, however, Caf1 was found
that the Not1 region between amino acid 700 and 997 is to be the major deadenylase in trypanosomes as well as
4382 NucleicAcidsResearch,2011,Vol.39,No.10
A 1 100 200 300 aa
TTP 2x CCCH 1-319
TTP-N 1-94
TTP-NZ 2x CCCH 1-176
TTP-C 152-319
B Input IP:YFP
Z ZZ
NNC NNNNCC
PP-P-P- PPP-P-P-P-P-P-
TTTT TTTTTTTT
TTTT TTTTTTTT
PP-P-P-P- PPP-P-P-P-P-P-P-P-
FFFFF FFFFFFFFFF
YYYYY YYYYYYYYYY
– – – – – – + – + – + – + – + RNase I
70 YFP-TTP
55
35
27 YFP
250 Flag-Not1
35 Caf1a
27
1 2 3 4 5 28S
18S
tRNA
nucleolin mRNA
6 7 8 9 10 11 12 13 14 15
C MS2cp-TTP-N MS2cp-TTP-C
dT vector dT Caf1a-AA dT vector dT Caf1a-AA
A+
globin-
MS2bs
A–
nucleolin
0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 dox. (hours)
A+ A+ A+ A+
A size dox0.
N 2 hrs
R
m 4 hrs
A– A– A– A– 6 hrs
signal intensity (A.U.) signal intensity (A.U.) signal intensity (A.U.) signal intensity (A.U.)
t1/2 = 1.6 hrs ± 0.4 t1/2 = 3.9 hrs ± 0.5 t1/2 = 1.4 hrs ± 0.3 t1/2 = 3.7 hrs ± 0.4
120
%)100
NA ( 6800
R 40
m 20
0
0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6
dox.(hrs) dox.(hrs) dox.(hrs) dox.(hrs)
Figure 5. TTP C-terminal domain interacts with Not1–Caf1a and induces mRNA deadenylation. (A) Schematic representation of the murine TTP
fragments used in this study. N, N-terminal domain; Z, zinc-finger domain; C, C-terminal domain; aa, amino acids. (B) HEK293 cells were
transiently transfected with YFP-tagged TTP constructs as indicated together with Flag-Not1. Cytoplasmic lysates (input) were prepared after
24h for IP with GFP-binder. Inputs were divided and one half was treated with RNase I during IP. Western blot analysis was carried out with
antibodies against YFP, Flag and endogenous Caf1a. To control RNase I efficiency, RNA was isolated from unbound fractions. Ribosomal and
tRNAwasvisualizedbyethidiumbromidestaining,nucleolinmRNAwasvisualizedonanorthernblot.(C)HeLacellsweretransientlytransfected
with MS2cp-TTP-C or MS2cp-TTP-N together with the Tet-Off transactivator and a pTet-Off-driven b-globin reporter gene that contains six
MS2-binding sites (bs) in its 30 UTR. In addition, empty vector or dominant negative Caf1a-AA was co-transfected. Reporter gene transcription
was blocked specifically by addition of doxycycline 20h after transfection, and total RNA was isolated after the time intervals indicated.
Globin-MS2bs and nucleolin mRNA were detected by northern blot analysis. dT indicates that the RNA was treated with oligo-dT and RNase
HtogeneratedeadenylatedmRNA. Inthe middlepanel,deadenylation wasvisualized byquantifying thesignalintensity ofglobin-MS2bsmRNA
alongthelengthofthesignalandplottingitasafunctionofmRNAsize.Inthebottompanel,theoverallsignalintensityofglobin-MS2bsmRNA
was quantified and normalized to nucleolin mRNA. Average values±SE were obtained from three biological repeat experiments and plotted as
percentage of the initial time point.
in Drosophila and human cells, whereas Ccr4 is either mRNA (38). The exact role of these proteins in mRNA
lacking (in trypanosomes) or of minor importance for deadenylation, however, is not known.
deadenylation (25,27,49). Aside from Caf1 that carries In this study, we provide evidence through biochemical
the exoribonucleolytic activity, a detailed analysis in purification and co-IP experiments that TTP associates
Drosophila cells showed that Not1, Not2 and Not3 are with Not1 (Figure 1). TTP binds to mRNAs
also essential for deadenylation of both bulk and Hsp70 containing AREs and mediates their rapid degradation,