In mammals, RNA interference (RNAi) was historically studied as a cytoplasmic event; however, in the last decade, a growing number of reports convincingly show the nuclear localization of the Argonaute (AGO) proteins. Nevertheless, the extent of nuclear RNAi and its implication in biological mechanisms remain to be elucidated. We found that reduced Lamin A levels significantly induce nuclear influx of AGO2 in SHSY5Y neuroblastoma and A375 melanoma cancer cell lines, which normally have no nuclear AGO2. Lamin A KO manifested a more pronounced effect in SHSY5Y cells compared to A375 cells, evident by changes in cell morphology, increased cell proliferation, and oncogenic miRNA expression. Moreover, AGO fPAR-CLIP in Lamin A KO SHSY5Y cells revealed significantly reduced RNAi activity. Further exploration of the nuclear AGO interactome by mass spectrometry identified FAM120A, an RNA-binding protein and known interactor of AGO2. Subsequent FAM120A fPAR-CLIP, revealed that FAM120A co-binds AGO targets and that this competition reduces the RNAi activity. Therefore, loss of Lamin A triggers nuclear AGO2 translocation, FAM120A mediated RNAi impairment, and upregulation of oncogenic miRNAs, facilitating cancer cell proliferation.
- MeSH
- Active Transport, Cell Nucleus MeSH
- Argonaute Proteins * metabolism genetics MeSH
- Cell Nucleus * metabolism MeSH
- Lamin Type A * metabolism genetics MeSH
- Humans MeSH
- Melanoma genetics metabolism pathology MeSH
- MicroRNAs * metabolism genetics MeSH
- Cell Line, Tumor MeSH
- Cell Proliferation * genetics MeSH
- RNA-Binding Proteins metabolism genetics MeSH
- RNA Interference * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Lamins, the nuclear intermediate filaments, are important regulators of nuclear structural integrity as well as nuclear functional processes such as DNA transcription, replication and repair, and epigenetic regulations. A portion of phosphorylated lamin A/C localizes to the nuclear interior in interphase, forming a lamin A/C pool with specific properties and distinct functions. Nucleoplasmic lamin A/C molecular functions are mainly dependent on its binding partners; therefore, revealing new interactions could give us new clues on the lamin A/C mechanism of action. In the present study, we show that lamin A/C interacts with nuclear phosphoinositides (PIPs), and with nuclear myosin I (NM1). Both NM1 and nuclear PIPs have been previously reported as important regulators of gene expression and DNA damage/repair. Furthermore, phosphorylated lamin A/C forms a complex with NM1 in a phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent manner in the nuclear interior. Taken together, our study reveals a previously unidentified interaction between phosphorylated lamin A/C, NM1, and PI(4,5)P2 and suggests new possible ways of nucleoplasmic lamin A/C regulation, function, and importance for the formation of functional nuclear microdomains.
- MeSH
- Cell Nucleus * metabolism MeSH
- Interphase MeSH
- Intermediate Filaments metabolism MeSH
- Lamin Type A * metabolism MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
The nuclear lamina is the main component of the nuclear cytoskeleton that maintains the integrity of the nucleus. However, it represents a natural barrier for viruses replicating in the cell nucleus. The lamina blocks viruses from being trafficked to the nucleus for replication, but it also impedes the nuclear egress of the progeny of viral particles. Thus, viruses have evolved mechanisms to overcome this obstacle. Large viruses induce the assembly of multiprotein complexes that are anchored to the inner nuclear membrane. Important components of these complexes are the viral and cellular kinases phosphorylating the lamina and promoting its disaggregation, therefore allowing virus egress. Small viruses also use cellular kinases to induce lamina phosphorylation and the subsequent disruption in order to facilitate the import of viral particles during the early stages of infection or during their nuclear egress. Another component of the nuclear cytoskeleton, nuclear actin, is exploited by viruses for the intranuclear movement of their particles from the replication sites to the nuclear periphery. This study focuses on exploitation of the nuclear cytoskeleton by viruses, although this is just the beginning for many viruses, and promises to reveal the mechanisms and dynamic of physiological and pathological processes in the nucleus.
- MeSH
- Actins metabolism MeSH
- Cell Nucleus metabolism MeSH
- Cytoskeleton genetics metabolism MeSH
- Species Specificity MeSH
- Host-Pathogen Interactions * MeSH
- Nuclear Lamina metabolism MeSH
- Nuclear Envelope metabolism MeSH
- Lamins metabolism MeSH
- Humans MeSH
- Disease Susceptibility * MeSH
- Gene Expression Regulation, Viral MeSH
- Virus Replication MeSH
- Virus Diseases etiology metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
The nuclear lamina supports many functions, including maintaining nuclear structure and gene expression control, and correct spatio-temporal assembly is vital to meet these activities. Recently, multiple lamina systems have been described that, despite independent evolutionary origins, share analogous functions. In trypanosomatids the two known lamina proteins, NUP-1 and NUP-2, have molecular masses of 450 and 170 kDa, respectively, which demands a distinct architecture from the ∼60 kDa lamin-based system of metazoa and other lineages. To uncover organizational principles for the trypanosome lamina we generated NUP-1 deletion mutants to identify domains and their arrangements responsible for oligomerization. We found that both the N- and C-termini act as interaction hubs, and that perturbation of these interactions impacts additional components of the lamina and nuclear envelope. Furthermore, the assembly of NUP-1 terminal domains suggests intrinsic organizational capacity. Remarkably, there is little impact on silencing of telomeric variant surface glycoprotein genes. We suggest that both terminal domains of NUP-1 have roles in assembling the trypanosome lamina and propose a novel architecture based on a hub-and-spoke configuration.
The essential components of splicing are the splicing factors accumulated in nuclear speckles; thus, we studied how DNA damaging agents and A-type lamin depletion affect the properties of these regions, positive on the SC-35 protein. We observed that inhibitor of PARP (poly (ADP-ribose) polymerase), and more pronouncedly inhibitors of RNA polymerases, caused DNA damage and increased the SC35 protein level. Interestingly, nuclear blebs, induced by PARP inhibitor and observed in A-type lamin-depleted or senescent cells, were positive on both the SC-35 protein and another component of the spliceosome, SRRM2. In the interphase cell nuclei, SC-35 interacted with the phosphorylated form of RNAP II, which was A-type lamin-dependent. In mitotic cells, especially in telophase, the SC35 protein formed a well-visible ring in the cytoplasmic fraction and colocalized with β-catenin, associated with the plasma membrane. The antibody against the SRRM2 protein showed that nuclear speckles are already established in the cytoplasm of the late telophase and at the stage of early cytokinesis. In addition, we observed the occurrence of splicing factors in the nuclear blebs and micronuclei, which are also sites of both transcription and splicing. This conclusion supports the fact that splicing proceeds transcriptionally. According to our data, this process is A-type lamin-dependent. Lamin depletion also reduces the interaction between SC35 and β-catenin in mitotic cells.
- MeSH
- HeLa Cells MeSH
- Lamins metabolism MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- Poly(ADP-ribose) Polymerase Inhibitors therapeutic use MeSH
- Poly (ADP-Ribose) Polymerase-1 MeSH
- RNA Polymerase II metabolism MeSH
- RNA Splicing Factors metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder caused by a mutation of lamin A, which contributes to nuclear architecture and the spatial organization of chromatin in the nucleus. The expression of a lamin A mutant, named progerin, leads to functional and structural disruption of nuclear organization. Since progerin lacks a part of the actin-binding site of lamin A, we hypothesized that nuclear actin dynamics and function are altered in HGPS cells. Nuclear F-actin is required for the organization of nuclear shape, transcriptional regulation, DNA damage repair, and activation of Wnt/β-catenin signaling. Here we show that the expression of progerin decreases nuclear F-actin and impairs F-actin-regulated transcription. When nuclear F-actin levels are increased by overexpression of nuclear-targeted actin or by using jasplakinolide, a compound that stabilizes F-actin, the irregularity of nuclear shape and defects in gene expression can be reversed. These observations provide evidence for a novel relationship between nuclear actin and the etiology of HGPS.
- MeSH
- Actins genetics metabolism MeSH
- Cell Nucleus genetics metabolism pathology MeSH
- NIH 3T3 Cells MeSH
- Lamin Type A genetics metabolism MeSH
- Humans MeSH
- Mice MeSH
- DNA Repair * MeSH
- Progeria genetics metabolism pathology MeSH
- Wnt Signaling Pathway * MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Video-Audio Media MeSH
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Intramural MeSH
The molecular architecture and assembly mechanism of intermediate filaments have been enigmatic for decades. Among those, lamin filaments are of particular interest due to their universal role in cell nucleus and numerous disease-related mutations. Filament assembly is driven by specific interactions of the elementary dimers, which consist of the central coiled-coil rod domain flanked by non-helical head and tail domains. We aimed to investigate the longitudinal 'head-to-tail' interaction of lamin dimers (the so-called ACN interaction), which is crucial for filament assembly. To this end, we prepared a series of recombinant fragments of human lamin A centred around the N- and C-termini of the rod. The fragments were stabilized by fusions to heterologous capping motifs which provide for a correct formation of parallel, in-register coiled-coil dimers. As a result, we established crystal structures of two N-terminal fragments one of which highlights the propensity of the coiled-coil to open up, and one C-terminal rod fragment. Additional studies highlighted the capacity of such N- and C-terminal fragments to form specific complexes in solution, which were further characterized using chemical cross-linking. These data yielded a molecular model of the ACN complex which features a 6.5 nm overlap of the rod ends.
- MeSH
- Mass Spectrometry MeSH
- Crystallography, X-Ray MeSH
- Lamin Type A chemistry MeSH
- Humans MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Replication stress (RS) is a major driver of genomic instability and tumorigenesis. Here, we investigated whether RS induced by the nucleotide analog fludarabine and specific kinase inhibitors [e.g. targeting checkpoint kinase 1 (Chk1) or ataxia telangiectasia and Rad3-related (ATR)] led to apoptosis or senescence in four cancer cell lines differing in TP53 mutation status and expression of lamin A/C (LA/C). RS resulted in uneven chromatin condensation in all cell types, as evidenced by the presence of metaphasic chromosomes with unrepaired DNA damage, as well as detection of less condensed chromatin in the same nucleus, frequent ultrafine anaphase bridges, and micronuclei. We observed that responses to these chromatin changes may be distinct in individual cell types, suggesting that expression of lamin A/C and lamin B1 (LB1) may play an important role in the transition of damaged cells to senescence. MCF7 mammary carcinoma cells harboring wild-type p53 (WT-p53) and LA/C responded to RS by transition to senescence with a significant reduction of lamin B receptor and LB1 proteins. In contrast, a lymphoid cancer cell line WSU-NHL (WT-p53) lacking LA/C and expressing low levels of LB1 died after several hours, while lines MEC-1 and SU-DHL-4, both with mutated p53, and SU-DHL-4 with mutations in LA/C, died at different rates by apoptosis. Our results show that, in addition to being influenced by p53 mutation status, the response to RS (apoptosis or senescence) may also be influenced by lamin A/C and LB1 status.
- MeSH
- Apoptosis physiology MeSH
- Lamin Type A metabolism MeSH
- Humans MeSH
- MCF-7 Cells MeSH
- Mutation MeSH
- Cell Line, Tumor MeSH
- Tumor Suppressor Protein p53 genetics MeSH
- DNA Replication physiology MeSH
- Cellular Senescence physiology MeSH
- Vidarabine analogs & derivatives pharmacology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The organization of the nuclear periphery is crucial for many nuclear functions. Nuclear lamins form dense network at the nuclear periphery and play a substantial role in chromatin organization, transcription regulation and in organization of nuclear pore complexes (NPCs). Here, we show that TPR, the protein located preferentially within the nuclear baskets of NPCs, associates with lamin B1. The depletion of TPR affects the organization of lamin B1 but not lamin A/C within the nuclear lamina as shown by stimulated emission depletion microscopy. Finally, reduction of TPR affects the distribution of NPCs within the nuclear envelope and the effect can be reversed by simultaneous knock-down of lamin A/C or the overexpression of lamin B1. Our work suggests a novel role for the TPR at the nuclear periphery: the TPR contributes to the organization of the nuclear lamina and in cooperation with lamins guards the interphase assembly of nuclear pore complexes.
- MeSH
- HeLa Cells MeSH
- Nuclear Lamina metabolism ultrastructure MeSH
- Nuclear Envelope metabolism ultrastructure MeSH
- Nuclear Pore Complex Proteins antagonists & inhibitors genetics metabolism MeSH
- Lamin Type A antagonists & inhibitors genetics metabolism MeSH
- Lamin Type B genetics metabolism MeSH
- Humans MeSH
- RNA, Small Interfering genetics metabolism MeSH
- Molecular Imaging MeSH
- Proto-Oncogene Proteins antagonists & inhibitors genetics metabolism MeSH
- Gene Expression Regulation MeSH
- Signal Transduction MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Background Hutchinson-Gilford progeria syndrome (HGPS) is a rare disease caused by pathogenic variants in the LMNA gene, which leads to premature aging. The median life expectancy is shortened to 13 years due to cardiovascular complications. Case report We present a boy born with a pathogenic LMNA variant c.433G > A, which causes atypical progeria syndrome (APS) and was previously described in one single patient. When investigated for poor growth prior to the diagnosis of APS, his laboratory tests revealed growth hormone (GH) deficiency and magnetic resonance imaging (MRI) of the midbrain showed partial empty sella. GH treatment had only a limited and transient effect. His first ischemic complication manifested at age 4.2 years; at the age of 7 years, he had a fatal haemorrhagic stroke. Conclusion To the best of our knowledge, this is the first patient with APS showing partial empty sella and GH deficiency that might have contributed to his poor growth. GH failed to improve long-term outcome.
- MeSH
- Fatal Outcome MeSH
- Infant MeSH
- Lamin Type A genetics MeSH
- Humans MeSH
- Human Growth Hormone administration & dosage deficiency MeSH
- Mutation * MeSH
- Dwarfism drug therapy etiology MeSH
- Child, Preschool MeSH
- Progeria complications genetics pathology MeSH
- Check Tag
- Infant MeSH
- Humans MeSH
- Male MeSH
- Child, Preschool MeSH
- Publication type
- Case Reports MeSH
