In this study, we have characterized structural and functional features of the β-actin mRNA binding protein ZBP1. These data present the first direct evidence for a role of ZBP1 in the localization of β-actin mRNA and consequently in directed fibroblast motility. They begin to elucidate a sequence of events involved in β-actin mRNA localization: (1) ZBP1 binds to the zipcode of β-actin mRNA via its COOH-terminal KH domains, (2) auxiliary factors (e.g., hnRNPs, motors, translational factors) assemble to form a fully functional transport complex (locasome), (3) the locasome associates with the cytoskeleton, (4) the locasome is transported by motors on the cytoskeleton, (5) the locasome anchors at the cell periphery, and (6) the mRNA is translated.
In vitro binding analyses identify the two most COOH-terminal KH domains (KH3-KH4) to be necessary and sufficient for binding the zipcode and the full-length β-actin mRNA. Other RNA binding modules identified within ZBP1 may contribute to protein–RNA complex formation, since slightly lower Kd
values were obtained for full-length ZBP1 compared with the KH domains alone. The affinities observed for either the RRM domains or the first and second KH domains were significantly lower, suggesting that these domains may contribute to nonspecific mRNA interactions or may bind other specific mRNA sequences. The latter possibility could explain why human ZBP1 (IMP-1) and mouse ZBP1 (CRD-BP) have distinct RNA sequence specificities from chicken ZBP1 (Doyle et al., 1998
; Nielsen et al., 1999
The RNA-binding specificity of KH3-4 is demonstrated by the two ACACCC repeats in the zipcode that are absolutely required for proper localization of the mRNA and for binding to ZBP1 (Kislauskis et al., 1993
; Ross et al., 1997
). The selection of an RCACCC consensus element by the KH3-KH4 domains in the SELEX experiment strongly agrees with this data. Secondary structure predictions of the zipcode suggest that it forms a stem-loop with (ACACCC)2
in the loop (Ross et al., 1997
). These findings indicate that, at least in the context of ZBP1 and the zipcode, the ACACCC region is an important RNA element for binding by KH domains. Such a phenomenon has been reported previously in the case of the KH domain–containing protein Nova, whose third KH domain was shown by biochemical and x-ray crystallographic studies to bind primarily to a UCAY tetranucleotide element in the loop region of a stem-loop RNA target identified by SELEX (Jensen et al., 2000
; Lewis et al., 2000
). Our results do not rule out the possibility that there are additional RNA sequences, either within the zipcode itself or within the full-length β-actin mRNA, that are involved in the interaction between ZBP1 and β-actin mRNA.
Full-length ZBP1 and all fragments that contained intact KH3-4 domains were capable of forming granules that were resistant to detergent extraction before fixation, suggesting a direct or indirect link to the cytoskeleton. Based on the mRNA binding properties of ZBP1, these findings strongly suggest that ZBP1 bound to a specific mRNA target assembles into mRNP complexes that associate with the cytoskeleton. This was confirmed by the fact that all COOH-terminal deletion fragments, in which KH3 and KH4 were either completely or partially deleted, showed a diffuse distribution and were extractable with nonionic detergent. Further analysis demonstrated the importance of the structural integrity of the last KH domain. ΔZBP1(1–522), which contains a partially truncated KH4 domain, showed a diffuse distribution, whereas ΔZBP1(1–556) in which the KH4 remained intact formed detergent resistant granules. Together these findings demonstrate that the last two KH domains are sufficient for binding the β-actin mRNA and for assembly into mRNPs that associate with the cytoskeleton. Recently, it has been shown that the third and fourth KH domains of the Xenopus
homologue of ZBP1, Vg1RBP, mediate self association (Git and Standart, 2002
). ZBP1 has also been shown to oligomerize via its KH domains (unpublished data), and this property may be a factor in our observations.
Cytoskeleton disruption experiments showed that in primary fibroblasts ZBP1 granules associate mainly with the actin cytoskeleton. Furthermore, the KH domains of ZBP1 were sufficient to maintain this interaction. These findings are consistent with previous observations that β-actin mRNA localization in fibroblasts is a predominantly actin-based process (Sundell and Singer, 1991
; Kislauskis et al., 1993
). Although there have been other observations of actin-based RNA localization (Hill and Gunning, 1993
; Broadus and Doe, 1997
; Long et al., 2000
), the majority of localized RNAs described to date are transported via the microtubule cytoskeleton (Elisha et al., 1995
; Carson et al., 1997
; Bassell et al., 1998
; Havin et al., 1998
; Wilkie and Davis, 2001
). Long distance transport of β-actin mRNA, such as that in neurons, seems to be more dependent on microtubules (Zhang et al., 2001a
), whereas short distance transport and anchoring is believed to rely on microfilaments. Since both systems use ZBP1, it may be a universal linker to either myosin or microtubule-based motors.
Although the motor proteins involved in ZBP1 granule transport remain to be identified, the data presented provide insight as to the mechanism by which ZBP1 connects to the transport machinery. Efficient ZBP1 granule localization to the cell periphery depends on the NH2
-terminal part of ZBP1 containing the RRM domains. Granules formed by most constructs lacking the NH2
terminus were evenly distributed throughout the cytoplasm. We suggest that nonlocalized ZBP1 granules represent localization “incompetent” granules. Presumably, the ZBP1 constructs present in these granules are capable of interacting with the RNA but lack key components necessary for distributing the locasome to the cell periphery. Mislocalization of these granules may be due to defects in targeting, transport, or peripheral anchoring. Similar observations were made for the COOH-terminal KH domains of the human ortholog of ZBP1, IMP1, which formed “immobile aggregates” in NIH-3T3 cells (Nielsen et al., 2002
). Similarly, overexpression of the RNA binding domain of Staufen (Stau-RBD) alone resulted in inefficient dendritic localization of Stau-RBD granules (Tang et al., 2001
). Together these findings point to a common mechanism where the protein–RNA complex provides a scaffold for mRNP transport. A finer analysis of the NH2
terminus of ZBP1 indicated that 10 amino acids that lie between the two RRM domains (ΔZBP1 [74–576] versus ΔZBP1 [84–576]) enable ZBP1 granule localization. Whether this 10 amino acid region contributes to the overall stability of a critical secondary structure or whether a specific element in that sequence is responsible for granule localization remains to be determined.
ZBP1 and its homologues associate with a variety of localized transcripts (Deshler et al., 1997
; Mueller-Pillasch et al., 1997
; Ross et al., 1997
; Doyle et al., 1998
; Havin et al., 1998
; Nielsen et al., 1999
; Zhang et al., 1999a
). However, conclusive evidence indicating that RNA regulation is a direct result of the function of ZBP1 and its family members is not well documented. A recent publication provides evidence of the role of Vg1RBP in the localization of Vg1
mRNA (Kwon et al., 2002
). However, experimental limitations have made it difficult to prove that the function of these RNA binding proteins is actually required for RNA localization to occur. The data presented here demonstrates that overexpression of the zipcode binding region of ZBP1 (KH1-KH4) and (KH3-KH4) significantly delocalizes β-actin mRNA. Based on the high affinity binding of the KH domains to β-actin mRNA, these overexpressed deletion fragments likely suppress mRNA localization by competing with the endogenous ZBP1 for β-actin. Since the COOH-terminal fragments do not localize, mRNA is sequestered within localization “incompetent” granules.
Interestingly, one NH2-terminal fragment, ΔZBP1 (1–289), delocalizes β-actin mRNA. Although this construct does not bind the zipcode, since it lacks KH3 and KH4, it might still bind to auxiliary factors that are essential for proper localization of ZBP1-containing granules. Thus, overexpression of this construct may suppress proper mRNA localization by competing with endogenous ZBP1 for these essential factors preventing β-actin transcripts bound to endogenous ZBP1 from being properly localized.
Together these findings confirm ZBP1 as an essential factor for localizing β-actin mRNA to the cell periphery. The data presented suggest that ZBP1 not only binds the mRNA via its COOH-terminal KH domains but also tethers the RNA to auxiliary factors via its NH2 terminus that are required for proper localization of the ZBP1 containing mRNPs.
This model also provides an explanation for the finding that ZBP1 is involved not only in β-actin mRNA localization but also contributes to the regulation of fibroblast motility. Overexpression of either ZBP1 or the COOH-terminal β-actin delocalizing construct ZBP1 (aa 189–576) decreased total path length, cell speed, and protrusion velocity of CEFs. Shestakova et al. (2001)
demonstrated that CEFs treated with antisense oligonucleotides, which disrupt β-actin mRNA localization, moved with reduced net path length, directionality, and persistence. Furthermore, this motility phenotype was attributed to delocalized actin nucleation sites resulting from antisense treatment.
These results may seem to be discordant with the current study. However, antisense oligonucleotides were specific for the zipcode of β-actin mRNA and specifically disrupt ZBP1–β-actin mRNA interactions (Zhang et al., 2001a
; Oleynikov and Singer, 2003
). Therefore, the RNA was freed from any spatial or regulatory constraints due to its interaction with ZBP1. In contrast, in the experiments reported here the interaction of the mRNA with ZBP1 is maintained. This may result in different physiological consequences. For example, localized RNAs are believed to be translationally repressed during transport; the human ortholog of ZBP1, IMP-1, translationally represses insulin-like growth factor II mRNA during fetal development (Nielsen et al., 1999
). In contrast to β-actin mRNA delocalized by antisense (Kislauskis et al., 1997
; Shestakova et al., 2001
), which is free to translate in an unregulated manner, β-actin mRNA bound to ZBP1 may be translationally repressed. Therefore, random actin nucleation may occur as a result of free cytoplasmic β-actin mRNA but not as a result of translationally repressed, ZBP1-bound RNA. Consequently, motility phenotypes described in our experiments fit with a model where there is decreased β-actin protein synthesis at the leading edge, resulting in decreased motility and protrusion rates.
It is important to consider that the physiological effects of both β-actin mRNA localization and ZBP1 function and the mechanism of β-actin mRNA localization are not completely understood at this time. Furthermore, it has not been established that β-actin mRNA is the only RNA target of ZBP1. Thus, a dominant-negative ZBP1 might affect additional mRNAs, causing a broader impact on cell polarity and motility than the results seen by antisense for β-actin mRNA. Additionally, ZBP1 has been implicated in several processes of RNA metabolism besides localization. Therefore, it is difficult to distinguish which aspects of ZBP1, motility regulation, and mRNA localization are being targeted in these experiments. As a result, although β-actin mRNA localization and ZBP1 function are involved in the regulation of cell motility, predictions about how these effects are mediated await further experiments.