Nucleolar localization/retention signal is responsible for transient accumulation of histone H2B in the nucleolus through electrostatic interactions

Abstract

The majority of known nuclear proteins are highly mobile. The molecular mechanisms by which they accumulate inside stable compartments that are not separated from the nucleoplasm by membranes are obscure. The compartmental retention of some proteins is associated with their biological function; however, some protein interactions within distinct nuclear structures may be non-specific. The non-specific retention may lead to the accumulation of proteins in distinct structural domains, even if the protein does not function inside this domain. In this study, we have shown that histone H2B-EGFP initially accumulated in the nucleolus after ectopic expression, and then gradually incorporated into the chromatin to leave only a small amount of nucleolus-bound histone that was revealed by removing chromatin-bound proteins with DNase I treatment. Nucleolar histone H2B had several characteristics: (i) it preferentially bound to granular component of the nucleolus and interacted with RNA or RNA-containing nucleolar components; (ii) it freely exchanged between the nucleolus and nucleoplasm; (iii) it associated with the nuclear matrix; and (iv) it bound to interphase prenuclear bodies that formed after hypotonic treatment. The region in histone H2B that acts as a nucleolar localization/retention signal (NoRS) was identified. This signal overlapped with a nuclear localization signal (NLS), which appears to be the primary function of this region. The NoRS activity of this region was non-specific, but the molecular mechanism was probably similar to the NoRSs of other nucleolar proteins. All known NoRSs are enriched with basic amino acids, and we demonstrated that positively charged motifs (nona-arginine (R9) and nona-lysine (K9)) were sufficient for the nucleolar accumulation of EGFP. Also, the correlation between measured NoRS activity and the predicted charge was observed. Thus, NoRSs appear to achieve their function through electrostatic interactions with the negatively charged components of the nucleolus. Though these interactions are non-specific, the functionally unrelated retention of a protein can increase the probability of its interaction with specific and functionally related binding sites.

A Simple Method for the Immunocytochemical Detection of Proteins Inside Nuclear Structures That Are Inaccessible to Specific Antibodies

“…The antibodies to several proteins (including anti-GFP and anti-FLAG antibodies) properly label their antigens inside the nucleoli, but when the antigen is overexpressed (such as EGFP-FLAG-B23 in the present work), the same antigens cannot be labeled by the same antibodies inside the nucleoli. Similar difficulties have been found with such abundant proteins as B23 (Zatsepina et al 1997;Misteli 2008;Musinova et al 2011; this study). One can speculate that the antibodies that were bound to abundant antigens at the nucleolar periphery created a mechanical barrier against the further penetration into the inner region of the nucleoli.…”

“…However, in cells with a high level of expression of exogenous fibrillarin, the protein cannot be detected in the intranucleolar regions and the antibodies labeled antigens only in the periphery of the nucleoli. Importantly, such strange peripheral staining was observed after the overexpression of fibrillarin (Sheval et al 2005) and also in normal, non-transfected cells after the immunocytochemical detection of nucleophosmin/B23 (Zatsepina et al 1997;Misteli 2008;Musinova et al 2011). Furthermore, using postembedding immunoelectron microscopy in HeLa cells with specific antibodies, it was demonstrated that B23 was not localized to the nucleolar periphery but to the dense fibrillar component and the granular component (Biggiogera et al 1989).…”

“…Even if the fluorescent protein that is fused does not influence the functional characteristics of the protein of interest, the expression of an exogenous protein usually increases the concentration of the protein investigated, which can lead to artifacts. For example, it was shown that (1) exogenous proteins can be localized differently from the endogenous protein (Maeshima and Laemmli 2003;Musinova et al 2011) and that (2) the protein overexpression can significantly influence the formation of the structures in which it is distributed and even lead to the formation of de novo structures (Volkova et al 2011). Therefore, in many cases, it is preferable to detect the localization of proteins using immunocytochemistry.…”

Identification and Characterization of Nuclear and Nucleolar Localization Signals in the Adeno-Associated Virus Serotype 2 Assembly-Activating Protein

“…5E). It has previously been shown that adding a nonapeptide containing 9 basic amino acids to the C terminus of GFP allows nucleolar accumulation of the protein, not through a specific NoLS motif, but presumably through nonspecific, electrostatic interactions with negatively charged nucleolar components (9). There are 8 basic amino acids within the AAP2BR1-AAP2BR2 segment, and there are 10 basic residues in AAP2mt22 (Fig.…”

“…Importin-␤ mediates nuclear entry of the heterotrimer through the nuclear pores by its increasing affinity for nucleoporins along the inside of the nuclear pore complex (8). If the cargo protein also contains an NoLS, it can then be targeted to the nucleolus through charge-based interactions (9) or interactions with nucleolar proteins (10,11), although the specific requirements defining nucleolar localization are not as well understood as those for nuclear import.…”