Active nuclear transport involves complex interactions between the transport machinery and protein cargo, mediated in part through NLSs. Typically composed of discrete patterns of basic residues, these sequences are recognized by the transport machinery and can vary in their affinity, rate, and efficiency of localization, which in turn can influence the function and biologic relevance of the cargo protein in different physiologic settings. Here, we have shown that NOL7 is targeted to the nucleus via an energy- and nucleoporin-dependent mechanism. This transport is mediated by three evolutionarily conserved but distinct NLSs. In addition, each NLS was found to be independently capable of directing the nuclear localization of the cytoplasmic protein PK or full length NOL7. Each NLS individually and additively contributed to the rate and efficiency of NOL7 nuclear targeting, suggesting that each of the NLSs has differential effects in driving the localization kinetics, likely reflecting differences in the regulation of import. Taken together, these data indicate that NOL7 localization is tightly regulated and may contribute to functions in various cellular compartments.
The transport of proteins and RNAs into the nucleus occurs through the NPC and is an important step in regulating the subcellular location of a number of different proteins, including transcription factors, signalling proteins, and various enzymes. Although alternative mechanisms exist, the classic nuclear import pathway appears to be the predominate method of transport into the nucleus. A recent survey of Saccharomyces cerevisiae screened over 5800 genomic sequences and found that 45% contained classic NLSs and nearly 60% of nuclear proteins contained monopartite or bipartite sequences . This is likely true across species, as a number of studies have found that the nuclear transport machinery for essential proteins is highly conserved between animals, yeast and plants [30–32]. This observation is certainly true for the three NLSs present in NOL7, where sequence alignment of the three NLSs demonstrated significant evolutionary conservation and aided in the identification of putative targeting sequences. It further suggested that targeting may play a significant role in the regulation and function of NOL7, as these sequences were highly conserved across species but demonstrated little similarity to other proteins or domains.
NOL7's three functional NLSs are located in the N terminus, middle, and C terminus of the protein. While a single functional NLS is sufficient for most proteins, the presence of multiple functional NLSs is seen frequently among proteins whose function is critically determined by its localization. Proteins such as p53 , E2F1 , c-Abl , p14ARF, HPV E6 , BRCA2 , most ribosomal proteins including RPS7 [84, 85], b-myb , ATF2/c-jun heterodimer , PAK-1  and others have been demonstrated to contain more than one NLS. Interestingly, many of these proteins are also implicated in cancer, and aberrant or mislocalized protein plays a significant role in the development and progression of the disease. As such, nuclear localization, and the rate and efficiency at which it occurs, has been shown to have many downstream functional consequences for proteins [89–92].
Terry, et al, have proposed a hierarchical regulation to classical nuclear transport via NLSs, with multiple mechanisms acting at the level of the cargo, receptors, and NPC . The existence of multiple NLSs within a single protein may therefore provide a mechanism to exploit these different targeting controls for proteins whose nuclear localization is critical for function . The first level of regulation involves the NPC, and the permeability, stability, and expression of the proteins that comprise this complex can affect the efficiency and targeting of cargo. The existence of multiple NLSs within NOL7 may therefore be used to achieve nuclear localization despite cellular conditions where NPC is less accessible. The next level of regulation involves the transport receptors. Here, differing accessibility, affinity, competition, and expression of the importins in various cell types and under different cellular conditions can affect transport [40, 94–99]. In this case, the existence of multiple NLSs can increase likelihood of transporter interaction regardless of environment, coordinate for better efficiency and rate of localization, or outcompete other NLS-bearing proteins for these receptors. Indeed, combined loss of NLS2 and NLS3 significantly impact both the rate and efficiency of NOL7 localization, and the presence of more than one NLS results in a statistically significant increase in NOL7 nuclear accumulation (Figure 7). Finally, at the level of the cargo, modifications and interactions of the cargo protein itself regulates its own localization. Inter- and intramolecular interactions can provide or preclude access to NLSs, and modifications within NLSs can also affect transport, either inhibiting or promoting import to the nucleus [79, 100]. The differential rate and efficiency of localization observed among NOL7 mutants, particularly in the N23(-) mutant, suggests each NLS may participate in different levels of this regulation. In addition, many NLSs have also been shown to harbor subnuclear targeting sequences such as NoLSs. NoLSs typically represent interaction motifs between nucleolar constituents, making nucleolar localization a dynamic, multidirectional process compared to nuclear targeting [44–50, 101, 102]. Our results have shown that NLS2 and NLS3 include four NoLSs. These sequences are composed of basic clusters and each is capable of individually driving nucleolar localization of NOL7. Whether these regions represent unique binding domains or are functionally redundant to ensure efficient interaction with nucleolar cofactors is unknown at this time.
Investigation of the nucleolar occupancy of proteins under various cellular conditions has demonstrated that the kinetics are often highly similar for functionally related proteins . In particular, FRAP analysis of the recovery and mobility of proteins within this compartment has been shown to reflect their functional roles. The nucleolar mobility is typically viewed as a reflection of the stability of the interactions and size of the interaction complex within that compartment, while the recovery reflects the shuttling characteristics of a protein between the nucleoplasm and nucleolus. While many ribosomal proteins are highly immobile within the nucleolus, proteins such as NCL, UBF, and NPM have higher mobility and rapid recovery, due to their multiple functional roles in the nucleus and nucleolus [60, 62, 71, 72]. FRAP analysis of the nucleolar occupancy of NOL7 demonstrates that a large fraction of nucleolar NOL7 is involved in a relatively stable complex, as evidenced by its small Mf. Interestingly, free NOL7 protein rapidly shuttles between compartments. These dynamics, with low Mf and high t1/2, have been demonstrated in the literature to be unique to proteins that functionally interact with ribonucleoproteins (RNPs) in both the nucleus and nucleolus such as NPM [60–62, 68–72]. Together, this suggests that NOL7 may interact in RNP complexes in both compartments. Further support for the potential nuclear and nucleolar interactions of NOL7 can be observed by the changes in localization for NOL7 upon specific depletion of nucleic acid species. The pattern of NOL7 expression is significantly altered by loss of RNA but not loss of DNA, suggesting that NOL7 is an RNA-associated protein, either directly or through RNP complexes. Further, changes in rRNA and mRNA abundance affected the abundance of NOL7 in the nucleolus and nucleoplasm, respectively, suggesting that NOL7 may be participating in distinct functional complexes within each compartment. Whether this is a direct effect of rRNA and mRNA interaction, or an indirect consequence of changes in the transcriptome of the cell remains to be investigated. However, together these observations indicate that the RNA abundance within the cell can influence the localization of NOL7 protein, and the dynamics of this localization is similar to the kinetics of proteins that play functional roles in nuclear and nucleolar RNP complexes. While it is unknown what, if any, function NOL7 may have in either compartment, it suggests that its localization is actively regulated and this differential targeting may influence its role in cancer development and progression.
Localization and function within multiple cellular compartments has previously been observed for many proteins. In addition, regulation of protein function through localization mechanisms is known to be employed in multiple cancer signaling pathways, including the Wnt, TGFβ, and Hh pathways. Oncogenes and tumor suppressors such as Rb, c-Myc, p53, VHL, and p14Arf have multiple, different functions depending on their localization or sequestration [103–117]. Our evidence suggests that like many of these oncogenes and tumor suppressors, NOL7 may have be regulated through its subcellular localization, and its targeting may be critically linked to its tumor suppressive activity.