Musinova Yana R, Lisitsyna Olga M, Golyshev Sergey A, Tuzhikov Alexander I, Polyakov Vladimir Y, Sheval Eugene V
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
Biochim Biophys Acta. 2011 Jan;1813(1):27-38. doi: 10.1016/j.bbamcr.2010.11.003. Epub 2010 Nov 21.
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.
大多数已知的核蛋白具有高度的流动性。它们在不被膜与核质分隔的稳定区室中积累的分子机制尚不清楚。一些蛋白质的区室化保留与其生物学功能相关；然而，不同核结构内的一些蛋白质相互作用可能是非特异性的。即使蛋白质在该结构域内不起作用，非特异性保留也可能导致蛋白质在不同的结构域中积累。在本研究中，我们发现组蛋白H2B-EGFP在异位表达后最初在核仁中积累，然后逐渐整合到染色质中，通过用DNase I处理去除与染色质结合的蛋白质后，仅留下少量与核仁结合的组蛋白。核仁组蛋白H2B具有几个特征：（i）它优先结合核仁的颗粒成分并与RNA或含RNA的核仁成分相互作用；（ii）它在核仁和核质之间自由交换；（iii）它与核基质相关；（iv）它与低渗处理后形成的间期核前体结合。确定了组蛋白H2B中作为核仁定位/保留信号（NoRS）的区域。该信号与核定位信号（NLS）重叠，这似乎是该区域的主要功能。该区域的NoRS活性是非特异性的，但分子机制可能与其他核仁蛋白的NoRS相似。所有已知的NoRS都富含碱性氨基酸，我们证明带正电荷的基序（九聚精氨酸（R9）和九聚赖氨酸（K9））足以使EGFP在核仁中积累。此外，还观察到测量的NoRS活性与预测电荷之间的相关性。因此，NoRS似乎通过与核仁带负电荷的成分的静电相互作用来实现其功能。尽管这些相互作用是非特异性的，但蛋白质的功能无关保留可以增加其与特定和功能相关结合位点相互作用的概率。
