FRG1 is critical for development of the vertebrate musculature and vasculature and has been implicated in mediating FSHD pathophysiology5–7
. Still, very little is known about FRG1, hindering our understanding of how changes in its expression levels might lead to disease. In this study we further investigated human FRG1 by characterizing its subcellular localizations and biological activities, and identifying new interacting proteins.
Focusing on the nuclear aspects of FRG1, we have identified and characterized functional NLS and NoLS domains in FRG1. In silico
analysis of FRG1 revealed two amino terminal NLSs and a carboxyl bipartite NLS sequence. Our data demonstrate a major role for NLS2 in the nuclear localization of FRG1, which can partly be taken over by NLS1 in a phosphorylation-dependent manner, demonstrating that both are able to function independently in nuclear transport. Similar to FRG1, other proteins including transcription factors, enzymes, and structural proteins consist of multiple NLS sequences 46–49
. This redundancy ensures their efficient trafficking to the nucleus and likely indicates an essential nuclear function. Although proteins such as FRG1 (29 kb) are small enough to pass through the nuclear pore by diffusion, active nuclear transport via the nuclear import receptor KPNA2 results in rapid and efficient nuclear accumulation. This is the case for FRG1. We previously identified KPNA2 as a prominent and direct binding partner of FRG1 by yeast-two-hybrid screens 9
, and here confirmed the direct interaction by GST pull-down and co-IP experiments. KPNA2 is known to be involved in nuclear import of cargo-proteins through binding to the NLS sequence by means of its armadillo repeats located in the central part of the protein (reviewed in 33
). In agreement, all our yeast-two-hybrid clones of KPNA2 contained armadillo repeats, supporting the assumption that KPNA2 binds FRG1 by its armadillo repeats.
Phosphorylation can influence the nuclear transport of proteins by several mechanisms; it can regulate the binding or release of proteins masking a NLS, cause conformational changes in the NLS-containing protein, or alter the affinity of the NLS for nuclear import proteins 35
. The loss of functionality of NLS1 in nuclear import by mimicking the negatively charged phosphorylation status of this domain is in agreement with previous reports in which the affinity of the basic NLS sequence for the import receptor KPNA2 is decreased by addition of negative charges 35, 46
. These charges might disturb electrostatic interactions between the NLS-containing protein and KPNA2, thereby preventing the interaction between the two proteins and resulting in a cytoplasmic localization of the hyper-phosphorylated protein 35
. The balance between phosphorylation and dephosphorylation close to NLS sequences can thereby regulate the subcellular localization of a protein in a spatio-temporal manner 35, 46, 50
. This strictly regulated system is used by several important regulatory proteins such as the acryl hydrocarbon receptor, adenomatous polyposis coli protein, and Swi6, all showing classic NLS sequences flanked by phosphorylation sites 46, 51, 52
. Complementary to the direct interaction between FRG1 and KPNA2, we therefore suggest a phosphorylation-dependent localization of FRG1 through a KPNA2-mediated nuclear import mechanism.
In addition to nuclear localization, NLS1 and NLS2 showed synergistic roles in the nucleolar localization of FRG1, however, NLS1 is the major NoLS and its function in nucleolar import is dependent on the phosphorylation status of its flanking serine residues. Although the NoLS motifs are generally not well defined, they are composed mostly of Arg or Lys residues, like FRG1 53
. Although further experiments are needed to identify the in vivo phosphorylation of FRG1, the kinase(s) and phosphatase(s) involved in (de)phosphorylation of FRG1 and their roles in its function, this study provides more insight in the mechanism of nuclear and nucleolar localization of FRG1.
Experiments were also performed to determine the biological function of nuclear FRG1. The nuclear fraction of endogenous FRG1 is concentrated in Cajal bodies (CBs) and the granular component (GC) of nucleoli, as well as associated with nascent mRNA transcripts and the actively transcribed regions of the euchromatin. The CBs are nuclear bodies rich in factors involved in transcription and RNA processing and are sites of snRNP biogenesis 54, 55
. Thus, FRG1’s presence in CBs is consistent with its proposed role in RNA biogenesis and provides additional similarity with RNPs 8, 9, 11
. Similarly, FRG1’s nucleolar localization supports these functional roles. Nucleoli, the dynamic structures containing tandem chromosomal repeats encoding rRNAs, are sites where pre-ribosomal particles are transcribed and assembled; however, nucleoli have additional roles including maturation of some RNP particles, assembly of the RNA splicing machinery, and sequestration of certain nuclear regulatory factors 55, 56
. FRG1’s nucleolar localization to the GC and not with the fibrillar components suggests FRG1 is involved in later stages of rRNA processing and not directly with rDNA transcription 57
LBC spreads and RNA-IPs indicate the endogenous FRG1 associates with numerous nascent RNAPII generated mRNAs in vivo
. FRG1 has recently been biochemically isolated as a part of the activated human spliceosome, specifically as a component of the Bact
complex and is lost during the transition to the catalytic C complex 10
. Thus, FRG1 is one of the few Bact
complex proteins only transiently associated with the spliceosome during activation, indicating it is not part of the first catalytic splicing step and suggesting that FRG1 plays a specific role in spliceosome activation 10
. This spliceosome activation role is supported by the FRG1 RIP experiments () that did not find FRG1 associated with unspliced transcripts. Interestingly in FSHD-derived muscle cells, the muscle-specific isoforms of FXR1
, a target mRNA of FRG1 by RIP and REMSA, show an aberrant expression pattern due to decreased mRNA stability 38
while the mRNAs from fast skeletal muscle troponin T (TNNT3
) and myotubularin related protein 1 (MTMR1
) show aberrant alternative mRNA splicing 5
. However, due to the lack of detectable FRG1 overexpression in FSHD muscle it is not clear that FRG1 would be mediating this effect. Whether FRG1 overexpressing cells or FSHD-derived cells exhibit global aberrant alternative splicing has yet to be addressed.
Many RNA-binding proteins and mRNPs are involved in pre-mRNA splicing and processing as well as the transportation, localization, translation and the stability of mRNAs 40, 58, 59
. We have shown that FRG1 can bind RNA directly in vitro
and FRG1 is in a complex with mRNA in vivo
, however, it is still not clear that the in vivo
RNA association is mediated directly or indirectly through the identified noncanonical FRG1 RBD 60–62
. Regardless of the nature of the in vivo
mRNA association, FRG1 interacts directly with TAP supporting a role in mRNA nuclear export 41
. TAP:NXT1 heterodimers associate with RNA-binding adaptor proteins, similar to FRG1, in the nucleus to help mediate the nuclear export of the majority of mature mRNAs through the nuclear pore complex. In addition, many adaptor proteins for mRNA export play multiple roles during mRNA biogenesis 59, 63
. This is likely the case for FRG1. Despite being involved in spliceosome activation, the RIP for FRG1-associated RNAs revealed an association with the spliced FXR
mRNA supporting FRG1 being involved downstream of mRNA splicing as well. In this light, the abundance of frg1 on the numerous actively transcribing chromatin loops in the LBCs, and in the nucleoli and Cajal bodies () is not surprising. Together, these data support a role for FRG1 in multiple aspects of RNA biogenesis, including RNA splicing and mRNA transport.
The nuclear localization of FRG1 seems to be essential considering its double NLS and bipartite sequences; yet despite these signals, a consistent cytoplasmic pool of FRG1 remains ( and )3, 4
. This suggests that FRG1 is actively being retained in the cytoplasm and that the nuclear function of FRG1 may only be part of the story. We have previously proposed that FRG1 has a role beyond the nucleus as well. The C. elegans
FRG-1 bundles F-actin in vitro
and localizes to the body wall muscle dense body, a structure analogous to the vertebrate muscle Z-disk and costameres 4
. Here we show that human FRG1 retains the conserved actin binding activity, binding to actin in a ratio of 2:1, similar to its C. elegans
homolog, and forms dimers capable of bundling F-actin 4
. Thus, FRG1 may have a structural role in stabilizing the actin cytoskeleton or may be mediating other cellular functions by associating with the actin cytoskeleton. In support of the latter situation, we showed that FRG1 requires the intact actin cytoskeleton for maintaining its particular punctate cytoplasmic localization suggesting that actin filaments actively anchor FRG1 in the cytoplasm. This may also suggest a mechanism by which overexpressed FRG1 preferentially accumulates in the nucleus; the cytoplasmic binding sites for retaining FRG1 may be occupied with endogenous FRG1.
Considering the associations of FRG1 with mRNA biogenesis and trafficking, we propose a model whereby FRG1’s nuclear and cytoplasmic functions are linked through RNA. If FRG1 is functioning as a TAP-mRNA adaptor protein, it would be the first such protein with actin binding activity. FRG1 may associate with nascent mRNA transcripts in the nucleus, which are then exported together to the cytoplasm through the TAP:NXT1 pathway, and the actin cytoskeleton anchors the FRG1-mRNA complex at its designated location. One remaining question, however, revolves around specificity of the FRG1-RNA interaction. How would a protein such as FRG1, which seemingly interacts with most mRNAs in the nucleus based on lampbrush, RIP and spliceosome studies, selectively transport specific mRNAs to locations in the cytoplasm as we propose? Just such a mechanism for regulating cytoplasmic mRNA localization was recently identified in yeast 64
. In yeast, the nuclear mRNA transport proteins, She2p and Puf6p, bind their cargo mRNAs in the nucleus with low affinity; however, specific mRNA recognition takes place after nuclear export when the cytoplasmic protein She3p associates with the complex and provides sequence binding specificity such that only specific mRNAs form a transport complex resulting in those cargo mRNAs being targeted and transported to a specific subcellular localization 64
. Thus, FRG1’s mRNA specificity in the nucleus may be similarly non-sequence specific but a cytoplasmic FRG1 complex could provide the high affinity binding for specific mRNAs required for a role in cytoplasmic mRNA transport and localized translation.
FRG1 is clearly an important multifunctional protein involved in muscle development, however a role in FSHD pathophysiology is controversial due to the failure to consistently find any significant changes in FRG1 levels between patients and unaffected individuals. Still, recent data towards understanding FRG1 is compelling in respect to FSHD. Overexpression of FRG1 specifically disrupts development of the vertebrate musculature and vasculature, the two tissues most affected in FSHD 6, 7
. Cytoplasmic FRG1 localizes to the skeletal muscle Z-disc in mouse and humans, the subcellular localization of numerous proteins related to myriad myopathies 3, 65, 66
. Here we show FRG1 is a dynamic RNA-associated actin binding protein. In addition, FRG1’s interactions with both a nuclear importer (KPNA2) and a nuclear exporter (TAP) indicate its subcellular localization is highly regulated. Interestingly, in all systems tested including mammalian cell culture, C. elegans, Xenopus
, and Drosophila
, overexpressed FRG1 preferentially accumulates in the nucleus 4, 9, 11
). Alterations of FRG1 protein levels could change the subcellular distribution of FRG1, subsequently dysregulating its function. Increasing nuclear levels of FRG1 may ultimately result in mis-spliced mRNA transcripts, altered mRNA stability, or affect mRNA transport and thus translation, any of which could adversely affect the maintenance of muscle integrity.