ZBP1 recognition of b-actin zipcode
induces RNA looping
Jeffrey A. Chao,1Yury Patskovsky,2Vivek Patel,1Matthew Levy,2Steven C. Almo,2
and Robert H. Singer1,3
1Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA;
2Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA
ZBP1 (zipcode-binding protein 1) was originally discovered as a trans-acting factor for the ‘‘zipcode’’ in the 39
untranslated region (UTR) of the b-actin mRNA that is important for its localization and translational regulation.
Subsequently, ZBP1 has been found to be a multifunctional regulator of RNA metabolism that controls aspects of
localization, stability, and translation for many mRNAs. To reveal how ZBP1 recognizes its RNA targets, we
biochemically characterized the interaction between ZBP1 and the b-actin zipcode. The third and fourth KH
(hnRNP K homology) domains of ZBP1 specifically recognize a bipartite RNA element located within the first 28
nucleotides of the zipcode. The spacing between the RNA sequences is consistent with the structure of IMP1
KH34, the human ortholog of ZBP1, that we solved by X-ray crystallography. The tandem KH domains are
arranged in an intramolecular anti-parallel pseudodimer conformation with the canonical RNA-binding surfaces at
opposite ends of the molecule. This orientation of the KH domains requires that the RNA backbone must undergo
an ~180° change in direction in order for both KH domains to contact the RNA simultaneously. The RNA looping
induced by ZBP1 binding provides a mechanism for specific recognition and may facilitate the assembly of post-
transcriptional regulatory complexes by remodeling the bound transcript.
[Keywords: ZBP1; RNA-binding protein; KH domain; RNA localization]
Supplemental material is available at http://www.genesdev.org.
Received September 10, 2009; revised version accepted November 23, 2009.
Localization of messenger RNA (mRNA) into distinct
subcellular compartments allows for the spatial regula-
tion of gene expression that is required for the establish-
ment and maintenance of cell polarity (for review, see
Martin and Ephrussi 2009). A global study using Dro-
sophila embryos found that >71% of all the mRNAs
characterized (3370 genes) were localized and, further-
more, these mRNA could be grouped into 35 unique
localization patterns (Lecuyer et al. 2007). A second
genome-wide study that isolated mRNAs from fibroblast
cell protrusions identified >50 mRNAs that were specif-
ically localized to pseudopodia (Mili et al. 2008). These
recent studies underscore the fundamental role of mRNA
localization in diverse cellular and developmental pro-
cesses and, while technological advances have increased
the number of mRNAs that been shown to localize, the
underlying mechanisms that give rise to these asymmet-
ric distributions have remained elusive.
The localization of b-actin mRNA to the leading edge
of chicken embryo fibroblasts was one of the earliest
transcripts identified to be subcellularly localized and has
served as a model system for understanding the process
(Lawrence and Singer 1986). Asymmetric sorting of the
b-actin transcript is achieved by transport along both
microtubule and actin microfilaments, and is delocal-
ized in myosin II-B knockout fibroblasts (Latham et al.
1994; Fusco et al. 2003; Oleynikov and Singer 2003). A
54-nucleotide (nt) cis-acting element, termed the zipcode,
positioned directly following the termination codon in
the 39 untranslated region (UTR) of b-actin mRNA was
shownto benecessary andsufficient for targetingreporter
RNA constructs to thecellular periphery (Kislauskis et al.
1994). The trans-acting factor Zipcode-binding protein
1 (ZBP1) was identified based on its ability to interact
with the zipcode, and its knockdown results in impaired
invadopodia formation, cytoplasmic spreading, and cell
adhesion (Ross et al. 1997; Vikesaa et al. 2006).
ZBP1 is the founding member of a highly conserved
family (termed VICKZ in reference to the founding
members:Vg1RBP/Vera, IMP1-3, CRD-BP, KOC, and
ZBP1) of RNA-binding proteins that have been impli-
cated in the post-transcriptional regulation of several
different RNAs (Yisraeli 2005). In Xenopus laevis,
Vg1RBP/Vera is required for the localization of Vg1
mRNA to the vegetal cortex of oocytes and also the
localization of b-actin mRNA in axons (Deshler et al.
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148 GENES & DEVELOPMENT 24:148–158 ? 2010 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/10; www.genesdev.org
1998; Havin et al. 1998; Leung et al. 2006; Yao et al. 2006).
Humans contain three paralogs (IMP1–3) that were orig-
inally identified because of their ability to regulate in-
sulin-like growth factor II (Igf-II) mRNA translation, but
have since been found to promote the localization of H19
and tau mRNAs as well as stabilize CD44 and b-TrCP1
mRNAs (Nielsen et al. 1999; Runge et al. 2000; Atlas
et al. 2004; Vikesaa et al. 2006; Elcheva et al. 2009). In
was shown to protect the c-myc mRNA from endonu-
cleolytic cleavage, thereby stabilizing the transcript
(Doyle et al. 1998). This family’s ability to broadly
regulate RNA metabolism can lead to adverse cellular
effects, as evidenced by their overexpression and correla-
tion with poor prognosis in several types of cancers
(Hammer et al. 2005; Dimitriadis et al. 2007; Jiang et al.
2008; Kobel et al. 2009).
VICKZ family members share a characteristic arrange-
ment of six canonical RNA-binding modules with two
RNA recognition motifs (RRM) followed by four hnRNP-
K homology (KH) domains. Sequence alignments of the
proteins show that conserved residues are clustered into
three didomains (RRM12, KH12, and KH34), which sug-
gests that these evolutionarily conserved regions may
function in concert (Fig. 1A; Git and Standart 2002).
Interestingly, ZBP1 and IMP1 share considerable se-
quence identity (>94%), yet studies of their interactions
with their respective RNA targets have failed to produce
a unified understanding of the requirements for specific
RNA recognition (Runge et al. 2000; Farina et al. 2003;
Nielsen et al. 2004; Patel and Bag 2006; Atlas et al. 2007;
Jonson et al. 2007). Models for RNA recognition differ
with regard to both the domains and oligomerization
state of ZBP1 required for binding as well as the pro-
posed RNA determinants, which range from a minimal
59-ACACCC-39 sequence to RNA binding being entirely
sequence-independent (Farina et al. 2003; Nielsen et al.
2004; Atlas et al. 2007; Oberman et al. 2007).
Here we present a biochemical characterization of
ZBP1 recognition of the b-actin zipcode RNA. The
ZBP1 KH34 monomer binds to two nonsequential
stretches of RNA located within the proximal portion
of the zipcode. This bipartite recognition element is
consistent with our crystal structure of IMP KH34 (98%
sequence identity with ZBP1 KH34), the first structural
data for this family of proteins, that positions the KH
domains in an anti-parallel arrangement with their puta-
tive RNA-binding surfaces located at opposite ends of the
molecule. This orientation of the KH domains explains
both the sequence and distance dependence of RNA
binding that were determined biochemically. The resi-
dues that link KH3 to KH4 were also shown to play a role
in RNA binding, demonstrating that the KH34 domain
functions as a single unit whose precisegeometry dictates
its interaction with RNA.
ZBP1 recognition of zipcode RNA
We took the interaction between ZBP1 and the first 54 nt
of the 39 UTR of b-actin mRNA (zipcode[1–54]) as a start-
ing point to further investigate both the protein and RNA
contributions to specific recognition. A polyacrylamide
gel electrophoretic mobility shift assay (EMSA) was used
to resolve fluorescein-labeled zipcode[1–54] in complex
responsible for recognition of zipcode[1–54]
RNA. (A) Schematic diagram of ZBP1
showing conserved didomain organization.
(B) Representative EMSA results for full-
length ZBP1, RRM12, KH12, and KH34
binding to zipcode[1–54] RNA. The filled
triangle represents a 1:1 serial dilution of
recombinant protein. Free RNA (*) and
RNA–protein complexes (**) are labeled.
(C) Quantification of the fraction of RNA
bound in EMSA data for ZBP1 and KH34
were fit to the Hill equation to measure
the Kd, appand Hill coefficient.
The KH34 didomain of ZBP1 is
ZBP1 recognition of zipcode
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