We identified and characterized a hitherto unknown protein, which we termed raver1. Raver1 is a multidomain hnRNP-like protein and forms complexes with other members of the hnRNP family and actin-binding proteins involved in microfilament membrane attachment.
In accordance with these properties, raver1 was seen resident in the nucleus and in various types of microfilament attachment sites. This dual compartment localization may be due to both bona fide NLS and NES motifs present in the raver1 sequence. Both motifs seem functional, since raver1 can leave the nucleus, traverse the cytoplasm, and even target to a second nucleus as seen in the heterokaryon assay.
For both the nuclear compartment and the cytoplasmic membrane attachment sites, we identified partner proteins for raver1. Vinculin/metavinculin and α-actinin, essential components of microfilament attachment sites, form complexes with raver1 in in vitro assays and under physiological conditions. As different parts of raver1 bind to these ligands, raver1 can bind simultaneously to both these proteins and may thus participate in the formation of adhesive complexes.
Immunofluorescence of various cell lines and transfection of HeLa cells with GFP raver1 (data not shown) displayed a prominent signal of raver1 in the nuclei of different cell types, arguing for an additional nuclear function. Raver1 colocalizes with PTB/hnRNPI in perinucleolar bodies that harbor several RNA-binding proteins, including hnRNPs, and are actively engaged in RNA metabolism (Huang, 2000
; Spector, 2001
). In accordance with this location, we found that raver1 contains three RRMs with homology to those of members of the hnRNP family (Shamoo et al., 1995
; Siomi and Dreyfuss, 1997
) such as PTB/hnRNPI (Valcarcel and Gebauer, 1997
), its homologue ROD1 (Yamamoto et al., 1999
), and the PTB/hnRNPI-associated splicing factor PSF (Patton et al., 1993
). All hnRNP proteins contain RNA-binding motifs (RRMs or KH domains), form heterooligomers with other members of the family, and some shuttle between the nucleus and the cytoplasm (Krecic and Swanson, 1999
). In all these criteria, raver1 is hnRNP like. However, raver1 is also unique in providing a direct link to microfilament proteins.
hnRNP proteins are thought to be involved in RNA splicing, trafficking, and translational control (Krecic and Swanson, 1999
; Shyu and Wilkinson, 2000
). For the prototype PTB/hnRNPI that participates in the RNA splicing and generation of tissue-specific isoforms of α-actinin (Southby et al., 1999
) and tropomyosin (Gooding et al., 1998
; Mulligan et al., 1992
) but is also involved in RNA splicing of nonmicrofilament proteins, it has been proposed that its differential activities require different protein ligands (Valcarcel and Gebauer, 1997
). With raver1, we identified a PTB/hnRNPI partner, which may be an important regulator of these processes. On the other hand, we have preliminary evidence that raver1, without PTB/hnRNPI, is able to direct RNA splicing of α-actinin (data not shown). Hence, again in analogy to PTB/hnRNPI and its relatives the RRMs in raver1 seem functional and bind to RNA, but the complex between raver1 and PTB/hnRNPI does not require the presence of RNA. RNA-independent complexes of PTB/hnRNPI-like and microfilament-associated proteins in the same hnRNP particle have also been observed recently for PSF and the focal adhesion adaptor protein paxillin (J.C. Norman and D.R. Critchley, personal communication).
The proteins PTB/hnRNPI, hnRNPA2, and ZBP-1 have also been implicated in mRNA trafficking within the cytoplasmic compartment (Ross et al., 1997
; Cote et al., 1999
; Munro et al., 1999
; Zhang et al., 2001
). Bearing this in mind, one could speculate that raver1 might bind to mature mRNA of α-actinin or vinculin, mediating their transport and anchorage to microfilament attachment sites as a prerequisite for site-specific translation and junction assembly. We tested a putative association of raver1 with α-actinin and vinculin mRNA in several experiments (data not shown) but failed to obtain positive results. However, even if raver1 does not bind directly to these or other mature mRNA species it may still contribute to mRNA trafficking as a member of a PTB/hnRNPI–mRNA complex.
Raver1 mRNA and protein were found widely expressed in different tissues and cultured cell lines but to different levels. High levels of raver1 mRNA were found in striated muscle and in organs consisting largely of epithelial tissue (kidney and liver), and raver1 protein was strongly expressed in epithelial cells (MDCK), whereas transformed fibroblasts (SV80) contained little, and HeLa cells no detectable raver1. In addition, although focal contact or cell–cell contact staining was observed in SV80 or MDCK cells, raver1 was found almost exclusively in the nuclear compartment of undifferentiated C2C12 and the smooth muscle–derived PAC-1 cells (data not shown). These variations in mRNA and protein levels indicate that raver1 expression and/or subcellular localization depend on the degree of polarization or differentiation of a particular cell. For murine-striated muscle, we could show that raver1 localization is linked to differentiation. As seen by immunofluorescence, the raver1 signal moves from an exclusively nuclear (C2C12 myoblasts) to a cytoplasmic (early myotubes) position and finally to Z-lines (cross-striated myotubes) where it colocalizes with α-actinin and vinculin in costameres (mature muscle). It will be interesting to investigate raver1 mobility during smooth or heart muscle differentiation, since in these muscle types the membrane attachment sites (costameres, intercalated discs, and dense plaques, respectively) contain metavinculin in addition to vinculin, which we identified as a raver1 ligand with an apparently higher affinity.
In conclusion, we suggest that raver1 communicates between a PTB/hnRNPI-guided splicing machinery and sites of cytoskeletal assembly. In concert with its nuclear and cytoskeletal ligands, raver1 may operate in a multifunctional complex essentially involved in tissue- and differentiation-specific gene expression and/or mRNA trafficking. Additional functions can of course not be excluded. At present, we cannot predict whether raver1 will exert all of these functions simultaneously or how its proposed activities may be regulated. Further studies are required to solve these questions.