Nuclear bodies: multifunctional companions of the genome

It has become increasingly apparent that gene expression is regulated by the functional interplay between spatial genome organization and nuclear architecture. Within the nuclear environment a variety of distinct nuclear bodies exist. They are dynamic, self-organizing structures that do not assemble as pre-formed entities but rather emerge as a direct reflection of specific activities associated with gene expression and genome maintenance. Here I summarize recent findings on functions of some of the most prominent nuclear bodies, including the nucleolus, Cajal body, PML nuclear body, Polycomb group body and the 53BP1 nuclear body. The emerging view is that their organization is orchestrated by similar principles, and they function in fundamental cellular processes involved in homeostasis, differentiation, development and disease.     

Gene expression and nuclear architecture during development and differentiation

Development requires a precise program of gene expression to be carried out. Much work has focussed on the regulatory networks that control gene expression, for example in response to external cues. However, it is important to recognize that these regulatory events take place within the physical context of the nucleus, and that the physical position of a gene within the nuclear volume can have strong influences on its regulation and interactions. The first part of this review will summarize what is currently known about nuclear architecture, that is, the large-scale three-dimensional arrangement of chromosome loci within the nucleus. The remainder of the review will examine developmental processes from the point of view of the nucleus.     

Identification of the binding sites for potential regulatory proteins in the upstream enhancer element of the Drosophila fushi tarazu gene.

With a view to identifying proteins that regulate the expression of the Drosophila ftz gene we have sequenced its enhancer-like upstream element (USE) and determined the binding sites for embryonic nuclear proteins within this region by in vitro DNAaseI footprinting. We find that greater than 50% of this element is bound by nuclear protein. By footprinting and gel-retardation studies in embryonic extracts from different developmental stages, we have characterised a number of USE/protein complexes whose nature alters in concert with changes in the ftz expression pattern, suggesting that these USE-binding proteins may be involved in the regulation of gene activity. In some cases this suggestion is substantiated by the observation that the protected DNA sequences show homology to the binding sites for ftz regulating DNA-binding proteins such as the pair-rule gene product even-skipped.     

The contribution of nuclear compartmentalization to gene regulation

Recent developments in live-cell imaging are challenging our stereotyped view of the fixed cell nucleus. The emerging picture is that nuclear processes may rely on a constant flow of molecules between dynamic compartments created by relatively immobile binding or assembly sites. This article discusses current views on the origins of nuclear compartments and their roles in gene expression.     

The rules and roles of nucleocytoplasmic shuttling proteins.

The spatial separation of mRNA synthesis from translation, while providing eukaryotes with the possibility to achieve higher complexity through a more elaborate regulation of gene expression, has set the need for transport mechanisms through the nuclear envelope. In a simplistic view of nucleocytoplasmic transport, nuclear proteins are imported into the nucleus while RNAs are exported to the cytoplasm. The reality is, however, that transport of either proteins or RNAs across the nuclear envelope can be bi-directional. During the past years, an increasing number of proteins have been identified that shuttle continuously back and forth between the nucleus and the cytoplasm. The emerging picture is that shuttling proteins are key factors in conveying information on nuclear and cytoplasmic activities within the cell.     

Microarray analysis of cytoplasmic versus whole cell RNA reveals a considerable number of missed and false positive mRNAs

With no known exceptions, every published microarray study to determine differential mRNA levels in eukaryotes used RNA extracted from whole cells. It is assumed that the use of whole cell RNA in microarray gene expression analysis provides a legitimate profile of steady-state mRNA. Standard labeling methods and the prevailing dogma that mRNA resides almost exclusively in the cytoplasm has led to the long-standing belief that the nuclear RNA contribution is negligible. We report that unadulterated cytoplasmic RNA uncovers differentially expressed mRNAs that otherwise would not have been detected when using whole cell RNA and that the inclusion of nuclear RNA has a large impact on whole cell gene expression microarray results by distorting the mRNA profile to the extent that a substantial number of false positives are generated. We conclude that to produce a valid profile of the steady-state mRNA population, the nuclear component must be excluded, and to arrive at a more realistic view of a cell''s gene expression profile, the nuclear and cytoplasmic RNA fractions should be analyzed separately.     

A 38 kDa nuclear protein is involved in the retention of an antisense oligonucleotide directed against cytosolic phospholipase A2.

Recent studies suggest that antisense phosphorothioate oligonucleotides (APO) are useful tools not only to impair gene expression, but also to modify the splicing of pre-mRNA, as the classical view that they act by suppressing the translation of mature mRNA has been challenged by several examples showing their nuclear site of action. In this work we show that an APO directed against cytosolic phospholipase A2 (cPLA2) mRNA localises in the nucleus and interacts with a specific nuclear protein.     

The photoreceptor cell-specific nuclear receptor gene (PNR) accounts for retinitis pigmentosa in the Crypto-Jews from Portugal (Marranos), survivors from the Spanish Inquisition

The last Crypto-Jews (Marranos) are the survivors of Spanish Jews who were persecuted in the late fifteenth century, escaped to Portugal and were forced to convert to save their lives. Isolated groups still exist in mountainous areas such as Belmonte in the Beira-Baixa province of Portugal. We report here the genetic study of a highly consanguineous endogamic population of Crypto-Jews of Belmonte affected with autosomal recessive retinitis pigmentosa (RP). A genome-wide search for homozygosity allowed us to localize the disease gene to chromosome 15q22-q24 (Zmax=2.95 at theta=0 at the D15S131 locus). Interestingly, the photoreceptor cell-specific nuclear receptor (PNR) gene, the expression of which is restricted to the outer nuclear layer of retinal photoreceptor cells, was found to map to the YAC contig encompassing the disease locus. A search for mutations allowed us to ascribe the RP of Crypto-Jews of Belmonte to a homozygous missense mutation in the PNR gene. Preliminary haplotype studies support the view that this mutation is relatively ancient but probably occurred after the population settled in Belmonte.     

Myosin‐1C uses a novel phosphoinositide‐dependent pathway for nuclear localization

Accurate control of macromolecule transport between nucleus and cytoplasm underlines several essential biological processes, including gene expression. According to the canonical model, nuclear import of soluble proteins is based on nuclear localization signals and transport factors. We challenge this view by showing that nuclear localization of the actin‐dependent motor protein Myosin‐1C (Myo1C) resembles the diffusion–retention mechanism utilized by inner nuclear membrane proteins. We show that Myo1C constantly shuttles in and out of the nucleus and that its nuclear localization does not require soluble factors, but is dependent on phosphoinositide binding. Nuclear import of Myo1C is preceded by its interaction with the endoplasmic reticulum, and phosphoinositide binding is specifically required for nuclear import, but not nuclear retention, of Myo1C. Our results therefore demonstrate, for the first time, that membrane association and binding to nuclear partners is sufficient to drive nuclear localization of also soluble proteins, opening new perspectives to evolution of cellular protein sorting mechanisms.     

A-type lamin complexes and regenerative potential: a step towards understanding laminopathic diseases?

The lamins are nuclear intermediate filament-type proteins forming the nuclear lamina meshwork at the inner nuclear membrane as well as complexes in the nucleoplasm. The recent discoveries that mutated A-type lamins and lamin-binding nuclear membrane proteins can be linked to numerous rare human diseases (laminopathies) affecting a multitude of tissues has changed the cell biologist’s view of lamins as mere structural nuclear scaffold proteins. It is still unclear how mutations in these ubiquitously expressed proteins give rise to tissue-restricted pathological phenotypes. Potential disease models include mutation-caused defects in lamin structure and stability, the deregulation of gene expression, and impaired cell cycle control. This review brings together various previously proposed ideas and suggests a novel, more general, disease model based on an impairment of adult stem cell function and thus compromised tissue regeneration in laminopathic diseases.