Altered Chromosomal Positioning, Compaction, and Gene Expression with a Lamin A/C Gene Mutation

Lamins A and C, encoded by the LMNA gene, are filamentous proteins that form the core scaffold of the nuclear lamina. Dominant LMNA gene mutations cause multiple human diseases including cardiac and skeletal myopathies. The nuclear lamina is thought to regulate gene expression by its direct interaction with chromatin. LMNA gene mutations may mediate disease by disrupting normal gene expression.To investigate the hypothesis that mutant lamin A/C changes the lamina's ability to interact with chromatin, we studied gene misexpression resulting from the cardiomyopathic LMNA E161K mutation and correlated this with changes in chromosome positioning. We identified clusters of misexpressed genes and examined the nuclear positioning of two such genomic clusters, each harboring genes relevant to striated muscle disease including LMO7 and MBNL2. Both gene clusters were found to be more centrally positioned in LMNA-mutant nuclei. Additionally, these loci were less compacted. In LMNA mutant heart and fibroblasts, we found that chromosome 13 had a disproportionately high fraction of misexpressed genes. Using three-dimensional fluorescence in situ hybridization we found that the entire territory of chromosome 13 was displaced towards the center of the nucleus in LMNA mutant fibroblasts. Additional cardiomyopathic LMNA gene mutations were also shown to have abnormal positioning of chromosome 13, although in the opposite direction.These data support a model in which LMNA mutations perturb the intranuclear positioning and compaction of chromosomal domains and provide a mechanism by which gene expression may be altered

Comparative genomics of proteins involved in RNA nucleocytoplasmic export

Background: The establishment of the nuclear membrane resulted in the physical separation of transcription and translation, and presented early eukaryotes with a formidable challenge: how to shuttle RNA from the nucleus to the locus of protein synthesis. In prokaryotes, mRNA is translated as it is being synthesized, whereas in eukaryotes mRNA is synthesized and processed in the nucleus, and it is then exported to the cytoplasm. In metazoa and fungi, the different RNA species are exported from the nucleus by specialized pathways. For example, tRNA is exported by exportin-t in a RanGTP-dependent fashion. By contrast, mRNAs are associated to ribonucleoproteins (RNPs) and exported by an essential shuttling complex (TAP-p15 in human, Mex67-mtr2 in yeast) that transports them through the nuclear pore. The different RNA export pathways appear to be well conserved among members of Opisthokonta, the eukaryotic supergroup that includes Fungi and Metazoa. However, it is not known whether RNA export in the other eukaryotic supergroups follows the same export routes as in opisthokonts. Methods: Our objective was to reconstruct the evolutionary history of the different RNA export pathways across eukaryotes. To do so, we screened an array of eukaryotic genomes for the presence of homologs of the proteins involved in RNA export in Metazoa and Fungi, using human and yeast proteins as queries. Results: Our genomic comparisons indicate that the basic components of the RanGTP-dependent RNA pathways are conserved across eukaryotes, and thus we infer that these are traceable to the last eukaryotic common ancestor (LECA). On the other hand, several of the proteins involved in RanGTP-independent mRNA export pathways are less conserved, which would suggest that they represent innovations that appeared later in the evolution of eukaryotes. Conclusions: Our analyses suggest that the LECA possessed the basic components of the different RNA export mechanisms found today in opisthokonts, and that these mechanisms became more specialized throughout eukaryotic evolution

Involvement in surface antigen expression by a moonlighting FG-repeat nucleoporin in trypanosomes

Components of the nuclear periphery coordinate a multitude of activities, including macromolecular transport, cell-cycle progression, and chromatin organization. Nuclear pore complexes (NPCs) mediate nucleocytoplasmic transport, mRNA processing, and transcriptional regulation, and NPC components can define regions of high transcriptional activity in some organisms at the nuclear periphery and nucleoplasm. Lineage-specific features underpin several core nuclear functions and in trypanosomatids, which branched very early from other eukaryotes, unique protein components constitute the lamina, kinetochores, and parts of the NPCs. Here we describe a phenylalanine-glycine (FG)-repeat nucleoporin, TbNup53b, that has dual localizations within the nucleoplasm and NPC. In addition to association with nucleoporins, TbNup53b interacts with a known trans-splicing component, TSR1, and has a role in controlling expression of surface proteins including the nucleolar periphery-located, procyclin genes. Significantly, while several nucleoporins are implicated in intranuclear transcriptional regulation in metazoa, TbNup53b appears orthologous to components of the yeast/human Nup49/Nup58 complex, for which no transcriptional functions are known. These data suggest that FG-Nups are frequently co-opted to transcriptional functions during evolution and extend the presence of FG-repeat nucleoporin control of gene expression to trypanosomes, suggesting that this is a widespread and ancient eukaryotic feature, as well as underscoring once more flexibility within nucleoporin function

HIV Integration Targeting: A Pathway Involving Transportin-3 and the Nuclear Pore Protein RanBP2

Genome-wide siRNA screens have identified host cell factors important for efficient HIV infection, among which are nuclear pore proteins such as RanBP2/Nup358 and the karyopherin Transportin-3/TNPO3. Analysis of the roles of these proteins in the HIV replication cycle suggested that correct trafficking through the pore may facilitate the subsequent integration step. Here we present data for coupling between these steps by demonstrating that depletion of Transportin-3 or RanBP2 altered the terminal step in early HIV replication, the selection of chromosomal sites for integration. We found that depletion of Transportin-3 and RanBP2 altered integration targeting for HIV. These knockdowns reduced HIV integration frequency in gene-dense regions and near gene-associated features, a pattern that differed from that reported for depletion of the HIV integrase binding cofactor Psip1/Ledgf/p75. MLV integration was not affected by the Transportin-3 knockdown. Using siRNA knockdowns and integration targeting analysis, we also implicated several additional nuclear proteins in proper target site selection. To map viral determinants of integration targeting, we analyzed a chimeric HIV derivative containing MLV gag, and found that the gag replacement phenocopied the Transportin-3 and RanBP2 knockdowns. Thus, our data support a model in which Gag-dependent engagement of the proper transport and nuclear pore machinery mediate trafficking of HIV complexes to sites of integration

Drosophila dosage compensation: Males are from Mars, females are from Venus

Dosage compensation of X-linked genes is a phenomenon of concerted, chromosome-wide regulation of gene expression underpinned by sustained and tightly regulated histone modifications and chromatin remodeling, coupled with constrains of nuclear architecture. This elaborate process allows the accomplishment of regulated expression of genes on the single male X chromosome to levels comparable to those expressed from the two X chromosomes in females. The ribonucleoprotein Male Specific Lethal (MSL) complex is enriched on the male X chromosome and is intricately involved in this process in Drosophila melanogaster. In this review we discuss the recent advances that highlight the complexity lying behind regulation of gene expression by just 2-fold

Regulation of survival gene hsp70

Rapid expression of the survival gene, inducible heat shock protein 70 (hsp70), is critical for mounting cytoprotection against severe cellular stress, like elevated temperature. Hsp70 protein chaperones the refolding of heat-denatured peptides to minimize proteolytic degradation as a part of an eukaryotically conserved phenomenon referred to as the heat shock response. The physiologic stress associated with exercise, which can include elevated temperature, mechanical damage, hypoxia, lowered pH, and reactive oxygen species generation, may promote protein unfolding, leading to hsp70 gene expression in skeletal myofibers. Although the pre-transcriptional activation of hsp70 gene expression has been thoroughly reviewed, discussion of downstream hsp70 gene regulation is less extensive. The purpose of this brief review was to examine all levels of hsp70 gene regulation in response to heat stress and exercise with a special focus on skeletal myofibers where data are available. In general, while heat stress represses bulk gene expression, hsp70 mRNA expression is enhanced. Post-transcriptionally, intronless hsp70 mRNA circumvents a host of decay pathways, as well as heat stress-repressed pre-mRNA splicing and nuclear export. Pre-translationally, hsp70 mRNA is excluded from stress granules and preferentially translated during heat stress-repressed global cap-dependent translation. Post-translationally, nascent Hsp70 protein is thermodynamically stable at elevated temperatures, allowing for the commencement of chaperoning activity early after synthesis to attenuate the heat shock response and protect against subsequent injury. This review demonstrates that hsp70 mRNA expression is closely coupled with functional protein translation