Maria Carmo-Fonseca

To be or not to be in the nucleolus

Compartmentalization has long been known to have a key role in regulation of cellular processes. By keeping enzymes and regulatory complexes in compartments where the delivery of substrate or exit of product is controlled, competing reactions can occur simultaneously in different parts of the cell. Moreover, spatial confinement facilitates the working of molecules participating in reaction chains and is crucial for coupling unfavourable with energetically favourable chemical reactions. Although in many cases intracellular compartmentalization relies on boundaries imposed by membranes, several non-membrane-bounded compartments exist in eukaryotic cells. One of these, the nucleolus, has recently attracted much attention. The emerging view is that molecular confinement in the nucleolus actively contributes to the control of cellular survival and proliferation.

The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates.

Messenger RNAs are exported from the nucleus as large ribonucleoprotein complexes (mRNPs). To date, proteins implicated in this process include TAP/Mex67p and RAE1/Gle2p and are distinct from the nuclear transport receptors of the beta-related, Ran-binding protein family. Mex67p is essential for mRNA export in yeast. Its vertebrate homolog TAP has been implicated in the export of cellular mRNAs and of simian type D viral RNAs bearing the constitutive transport element (CTE). Here we show that TAP is predominantly localized in the nucleoplasm and at both the nucleoplasmic and cytoplasmic faces of the nuclear pore complex (NPC). TAP interacts with multiple components of the NPC including the nucleoporins CAN, Nup98, Nup153, p62, and with three major NPC subcomplexes. The nucleoporin-binding domain of TAP comprises residues 508-619. In HeLa cells, this domain is necessary and sufficient to target GFP-TAP fusions to the nuclear rim. Moreover, the isolated domain strongly competes multiple export pathways in vivo, probably by blocking binding sites on the NPC that are shared with other transport receptors. Microinjection experiments implicate this domain in the export of specific CTE-containing RNAs. Finally, we show that TAP interacts with transportin and with two proteins implicated in the export of cellular mRNAs: RAE1/hGle2 and E1B-AP5. The interaction of TAP with nucleoporins, its direct binding to the CTE RNA, and its association with two mRNP binding proteins suggest that TAP is an RNA export mediator that may bridge the interaction between specific RNP export substrates and the NPC.

Molecular mechanisms involved in cisplatin cytotoxicity.

Abstract.cis-diamminedichloroplatinum(II) or cisplatin is a DNA-damaging agent that is widely used in cancer chemotherapy. Cisplatin cross-links to DNA, forming intra- and interstrand adducts, which bend and unwind the duplex and attract high-mobility-group domain and other proteins. Presumably due to a shielding effect caused by these proteins, the cisplatin-modified DNA is poorly repaired. The resulting DNA damage triggers cell-cycle arrest and apoptosis. Although it is still debatable whether the clinical success of cisplatin relies primarily on its ability to trigger apoptosis, at least two distinct pathways have been proposed to contribute to cisplatin-induced apoptosis in vitro. One involves the tumour-suppressor protein p53, the other is mediated by the p53-related protein p73. Coupling cisplatin damage to apoptosis requires mismatch repair activity, and recent observations further suggest involvement of the homologous recombinatorial repair system. At present it is generally accepted that abortive attempts to repair the DNA lesions play a key role in the cytotoxicity of the drug, and loss of the mismatch repair activity is known to cause cisplatin resistance, a major problem in antineoplastic therapy. Clearly, a better understanding of the signalling networks involved in cisplatin toxicity should provide a rational basis for the development of new therapeutic strategies.

The small nucleolar RNP protein NOP1 (fibrillarin) is required for pre-rRNA processing in yeast.

The yeast snoRNP protein, NOP1, is structurally and functionally homologous to vertebrate fibrillarin and is essential for viability. A conditionally lethal allele was constructed by placing NOP1 expression under the control of a GAL promoter. Growth on glucose medium results in the depletion of NOP1 over several generations, during which cell growth is progressively impaired. Pulse labelling of proteins shows that NOP1 depleted strains are greatly impaired in the production of cytoplasmic ribosomes, and they have a reduced level of rRNA. Northern hybridization and pulse‐chase labelling of pre‐rRNA show a progressive impairment of all pre‐rRNA processing steps. The pathway leading to 18S rRNA is particularly affected. Methylation of pre‐rRNA is concomitantly impaired and unmethylated pre‐rRNA accumulates and is not processed over long periods. NOP1 depletion does not prevent the accumulation of seven snoRNAs tested including U3; the levels of two species, U14 and snR190, decline. The snoRNAs synthesized in the absence of NOP1 retain TMG cap structures. Subnuclear fractionation and immunocytochemistry indicate that they continue to be localized in the nucleolus.

Dbp5, a DEAD‐box protein required for mRNA export, is recruited to the cytoplasmic fibrils of nuclear pore complex via a conserved interaction with CAN/Nup159p

Dbp5 is a DEAD‐box protein essential for mRNA export from the nucleus in yeast. Here we report the isolation of a cDNA encoding human Dbp5 (hDbp5) which is 46% identical to yDbp5p. Like its yeast homologue, hDbp5 is localized within the cytoplasm and at the nuclear rim. By immunoelectron microscopy, the nuclear envelope‐bound fraction of Dbp5 has been localized to the cytoplasmic fibrils of the nuclear pore complex (NPC). Consistent with this localization, we show that both the human and yeast proteins directly interact with an N‐terminal region of the nucleoporins CAN/Nup159p. In a conditional yeast strain in which Nup159p is degraded when shifted to the nonpermissive temperature, yDbp5p dissociates from the NPC and localizes to the cytoplasm. Thus, Dbp5 is recruited to the NPC via a conserved interaction with CAN/Nup159p. To investigate its function, we generated defective hDbp5 mutants and analysed their effects in RNA export by microinjection in Xenopus oocytes. A mutant protein containing a Glu→Gln change in the conserved DEAD‐box inhibited the nuclear exit of mRNAs. Together, our data indicate that Dbp5 is a conserved RNA‐dependent ATPase which is recruited to the cytoplasmic fibrils of the NPC where it participates in the export of mRNAs out of the nucleus.

Inefficient processing impairs release of RNA from the site of transcription

We describe here for the first time the site of retention within the nucleus of pre‐mRNA processing mutants unable to be exported to the cytoplasm. Fluorescence in situ hybridization was used to detect transcripts from human β‐globin genes that are either normal or defective in splicing or 3′ end formation. Nuclear transcripts of both wild‐type and mutant RNAs are detected only as intranuclear foci that colocalize with the template gene locus. The kinetics of transcript release from the site of transcription was assessed by treatment of cells with the transcriptional inhibitors actinomycin D, α‐amanitin and DRB. These drugs induce the rapid disappearance of nuclear foci corresponding to wild‐type human β‐globin RNA. In contrast, pre‐mRNA mutants defective in either splicing or 3′ end formation and which fail to be transported to the cytoplasm, are retained at the site of transcription. Therefore, 3′ end processing and splicing appear to be rate limiting for release of mRNA from the site of transcription.

Vesicular Stomatitis Virus Matrix Protein Inhibits Host Cell Gene Expression by Targeting the Nucleoporin Nup98

Vesicular stomatitis virus matrix protein (VSV M) has been shown to inhibit both transcription and nucleocytoplasmic transport. We have isolated a mutant form of M, termed M(D), lacking both inhibitory activities. HeLa cells expressing M, but not M(D), accumulate polyadenylated RNAs within the nucleus. Concomitantly, a fraction of M, but not of the M(D) mutant, localizes at the nuclear rim. Additionally, the nucleoporin Nup98 specifically interacts with M but not with M(D). In Nup98(-/-) cells, both the levels of M at the nuclear envelope and its inhibitory effects on host cell-directed expression of reporter genes were significantly reduced. Together, our data demonstrate that VSV M inhibits host cell gene expression by targeting a nucleoporin and primarily blocking nuclear export.

Mammalian nuclei contain foci which are highly enriched in components of the pre-mRNA splicing machinery.

The organization of the major snRNP particles in mammalian cell nuclei has been analysed by in situ labelling using snRNA‐specific antisense probes made of 2′‐OMe RNA. U3 snRNA is exclusively detected in the nucleolus while all the spliceosomal snRNAs are found in the nucleoplasm outside of nucleoli. Surprisingly, U2, U4, U5 and U6 snRNAs are predominantly observed in discrete nucleoplasmic foci. U1 snRNA is also present in foci but in addition is detected widely distributed throughout the nucleoplasm. An anti‐peptide antibody specific for the non‐snRNP splicing factor U2AF reveals it to have a similar distribution to U1 snRNA. Co‐localization studies using confocal fluorescence microscopy prove that U2AF is present in the snRNA‐containing foci. Antibody staining also shows the foci to contain snRNP‐specific proteins and m3G‐cap structures. The presence of major components of the nuclear splicing apparatus in foci suggests that these structures may play a role in pre‐mRNA processing.

Dynamic association of RNA-editing enzymes with the nucleolus

ADAR1 and ADAR2 are editing enzymes that deaminate adenosine to inosine in long double stranded RNA duplexes and specific pre-mRNA transcripts. Here, we show that full-length and N-terminally truncated forms of ADAR1 are simultaneously expressed in HeLa and COS7 cells owing to the usage of alternative starting methionines. Because the N-terminus of ADAR1 contains a nuclear export signal, the full-length protein localizes predominantly in the cytoplasm, whereas the N-terminally truncated forms are exclusively nuclear and accumulate in the nucleolus. ADAR2, which lacks a region homologous to the N-terminal domain of ADAR1, localizes exclusively to the nucleus and similarly accumulates in the nucleolus. Within the nucleolus, ADAR1 and ADAR2 co-localize in a novel compartment. Photobleaching experiments demonstrate that, in live cells, ADAR1 and ADAR2 are in constant flux in and out of the nucleolus. When cells express the editing-competent glutamate receptor GluR-B RNA, endogenous ADAR1 and ADAR2 de-localize from the nucleolus and accumulate at sites where the substrate transcripts accumulate. This suggests that ADAR1 and ADAR2 are constantly moving through the nucleolus and might be recruited onto specific editing substrates present elsewhere in the cell.

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.