A role for eIF4E and eIF4E-transporter in targeting mRNPs
to mammalian processing bodies
MARIA ALEXANDRA ANDREI,1,5DIERK INGELFINGER,1,5RAINER HEINTZMANN,2TILMANN ACHSEL,3
ROLANDO RIVERA-POMAR,2,4and REINHARD LÜHRMANN1
1Department of Cellular Biochemistry and2Department of Molecular Biology, Max-Planck-Institute of Biophysical Chemistry, D-37077
3IRCCS Fondazione Santa Lucia, Neurobiologia, 00179 Rome, Italy
4Centro Regional de Estudios Genómicos (CREG), 1888-Florencio Varela, Argentina
mRNP remodeling events required for the transition of an mRNA from active translation to degradation are currently poorly
understood. We identified protein factors potentially involved in this transition, which are present in mammalian P bodies,
cytoplasmic foci enriched in 5? → 3? mRNA degrading enzymes. We demonstrate that human P bodies contain the cap-binding
protein eIF4E and the related factor eIF4E-transporter (eIF4E-T), suggesting novel roles for these proteins in targeting mRNAs
for 5? → 3? degradation. Furthermore, fluorescence resonance energy transfer (FRET) studies indicate that eIF4E interacts with
eIF4E-T and the putative DEAD box helicase rck/p54 in the P bodies in vivo. RNAi-mediated knockdowns revealed that a subset
of P body factors, including eIF4E-T, LSm1, rck/p54, and Ccr4 are required for the accumulation of each other and eIF4E in P
bodies. In addition, treatment of HeLa cells with cycloheximide, which inhibits translation, revealed that mRNA is also required
for accumulation of mRNA degradation factors in P bodies. In contrast, knockdown of the decapping enzyme Dcp2, which
initiates the actual 5? → 3? mRNA degradation did not abolish P body formation, indicating it first functions after mRNPs have
been targeted to these cytoplasmic foci. These data support a model in which mRNPs undergo several successive steps of
remodeling and/or 3? trimming until their composition or structural organization promotes their accumulation in P bodies.
Keywords: mRNA degradation; translation; P bodies; eIF4E transporter
mRNA degradation is an important step in the regulation of
gene expression in eukaryotic cells. Most mRNAs are either
degraded in the 3? → 5? direction by the exosome, a mul-
ticomponent protein complex, or in the 5? → 3? direction
by other degradation factors. In yeast, the major mRNA
degradation pathway initiates with the deadenylation of the
mRNA by Ccr4p, followed by decapping at its 5? end by
Dcp1p/Dcp2p, and concludes with 5? → 3? digestion by the
exonuclease Xrn1p (Meyer et al. 2004). In addition to de-
grading enzymes, auxiliary factors such as Pat1p/Mrt1p and
the Sm-like proteins Lsm1p to Lsm7p, which bind sub-
sequent to deadenylation, are also required (Tharun and
Parker 2001). Mammalian homologs of most of these pro-
teins, which are structurally and functionally similar to the
yeast proteins, have been identified indicating that this
mRNA degradation pathway is conserved in mammals
(Chen et al. 2002; Ingelfinger et al. 2002; Lykke-Andersen
2002; van Dijk et al. 2002; Wang et al. 2002). Several of
these factors are not evenly distributed in the cytoplasm of
mammalian cells, but rather are found in discrete foci.
These include the 3? deadenylase Ccr4, the 5? decapping
factors Dcp1 and Dcp2, the exonuclease Xrn1, and LSm
proteins (Bashkirov et al. 1997; Ingelfinger et al. 2002;
Lykke-Andersen 2002; van Dijk et al. 2002; Cougot et al.
2004). In addition, the mRNA-binding protein GW182 also
accumulates in these foci (Eystathioy et al. 2003). Several
factors required for mRNA degradation in yeast have also
been reported to be enriched in similar cytoplasmic foci.
The latter have been shown to contain mRNA degradation
intermediates suggesting that they are mRNA processing
centers (Sheth and Parker 2003). In mammals, RNAi-me-
diated knockdown of Xrn1 leads to the accumulation of
5These authors (listed alphabetically) contributed equally to this work.
Reprint requests to: Reinhard Lührmann, Department of Cellular Bio-
chemistry, Max-Planck-Institute of Biophysical Chemistry, D-37077 Göt-
tingen, Germany; e-mail: firstname.lastname@example.org; fax: 49
551 201 1197; or Rolando Rivera-Pomar, Centro Regional de Estudios
Genómicos (CREG), Av. Calchaqui km 35, 500, 1888-Florencio Varela,
Argentina; e-mail: email@example.com; fax: 541142758100.
Article and publication are at http://www.rnajournal.org/cgi/doi/
RNA (2005), 11:717–727. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 RNA Society.
poly(A)+containing mRNAs in these foci indicating that,
like in yeast, the mammalian structures are likely mRNA
processing bodies (Cougot et al. 2004). Thus, we henceforth
refer to them as mammalian P bodies.
While several factors involved in mRNA degradation are
known to be present in P bodies, many unanswered ques-
tions concerning the assembly and function of these foci
remain. For example, what are the factor requirements for
the assembly of P bodies and how are degradation factors
targeted to these sites? Are they imported independently of
each other or as part of protein or mRNP complexes? Fi-
nally, what targets a translating mRNA to these structures
and which factors are required to initiate the transition
from translation to degradation within P bodies? The tran-
sition of an mRNA from active translation to being com-
mitted for degradation has been proposed to involve one or
more mRNP remodeling events (Tharun and Parker 2001).
Deadenylation is clearly a crucial determinant for initiating
mRNA degradation, and thus factors involved in 3? end
trimming may play an important role. In addition, factors
interacting at the 5? end of the mRNA could also be in-
volved. For example, a block in translation initiation could
potentially trigger the degradation process and thus, trans-
lation initiation factors and proteins that repress their ac-
tivity might also play a decisive role. However, whether
translation factors are present in P bodies, and more im-
portantly, whether they are required for P body formation,
is currently unknown.
The cap-binding, translation initiation factor eIF4E is
present in P bodies
To address these questions, we first checked for the pres-
ence of the translation initiation factor eIF4E in P bodies of
HeLa cells by performing immunofluorescence studies with
a mouse monoclonal anti-eIF4E antibody (see Materials
and Methods). Cells were counterstained with antibodies
against LSm1, a marker of P bodies (Ingelfinger et al. 2002).
eIF4E was distributed throughout the cytoplasm, but not
the nucleus, of HeLa cells and accumulated in discrete cy-
toplasmic foci (Fig. 1a–c). A similar distribution of eIF4E in
HeLa cells was observed with a rabbit anti-eIF4E serum
(data not shown). The latter represent P bodies as evidenced
by the colocalization of eIF4E with LSm1 in these foci (Fig.
1c). eIF4E was previously detected by immunofluorescence
FIGURE 1. eIF4E and eIF4E-T colocalize with LSm1 in distinct cytoplasmic foci. HeLa SS6 cells were grown on coverslips, fixed, and stained with
antibodies specific for eIF4E (monoclonal anti-eIF4E, Santa Cruz Biotechnology) (A,N), LSm1 (B,E,H,K,Q,T), eIF4G (G,J,M), or eIF4E-T (P).
Alternatively, cells were transfected with plasmids encoding YFP-eIF4E (D) or YFP-eIF4E-T (S). Panels C,F,I,L,O,R,U show the merged picture
of the proceeding two panels, with overlaying signals appearing yellow. In panels J–O, cells were treated with 100 µM arsenite for 45 min at 37°C
to induce stress granules (indicated by arrows). Cells were analyzed by confocal fluorescence microscopy. Scale bars = 10 µm.
Andrei et al.
RNA, Vol. 11, No. 5
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mRNA targeting to P bodies