ИСТИНА

Aim. The majority of known nucleolar proteins examined thus far are highly mobile: these proteins diffuse rapidly in the nucleoplasm and typically exchange quickly with their binding sites. The key questions are how macromolecules find their target sites inside the nucleus, and how they accumulate in these target sites. One way of protein retention in the nucleolus is associated with the presence of specific short amino acid sequences – nucleolar localization signals (NoLSs). The aim of the present study was to investigate mechanism by which NoLSs may lead to the nucleolar accumulation of the proteins. Methods. There are several ways for NoLS identification and investigation. We have used the approach based of analysis of tested sequence as factor for nucleolar accumulation of marker fluorescent protein (EGFP). To quantitatively estimate the effect of tested sequence on the nucleolar accumulation of GFP, the NoLS activity (concentration of EGFP in the nucleoli/concentration of EGFP in the nucleoplasm) was measured for all fusions obtained. Results. Three groups of observations are in agreement with the hypothesis of that charge-dependent (electrostatic) interactions of NoLSs with nucleolar components lead to nucleolar accumulation. (i) Known NoLSs are enriched in positively charged amino acids, but the structure of NoLSs is highly heterogeneous, and it is not possible to identify a consensus sequence for this type of signal. (ii) In two analyzed proteins (NF-κB inducing kinase and HIV Tat), the NoLS coincide with the boundaries of the region enriched with positively charged amino acids. Using the method of quantitative estimation of nucleolar accumulation, after substitution of charged amino acids to non-charged we were able to demonstrate that there is a strong correlation between the charge of protein fragment tested as a NoLS and its ability to accumulate marker protein (EGFP) in the nucleoli. (iii) The sequences containing only lysine or arginine (which were referred to as imitative NoLSs, iNoLSs) were accumulated in the nucleoli in a charge-dependent manner. We investigated localization of the strongest iNoLS, containing 19 agrinines (EGFP-R19), and found that the localization and some of properties were closely similar to that of B23, the protein of late rRNA processing. Using this iNoLS, we demonstrated that charge-dependent accumulation inside nucleoli was dependent on interaction with nucleolar RNAs. Conclusions. These results are in agreement with the hypothesis that the accumulation of proteins in the nucleolus by NoLSs can be determined by the electrostatic interaction of positively charged signals with nucleolar RNAs.