Phosphorylation of nucleoporin Tpr governs its differential localization and is required for its mitotic function

A major constituent of the nuclear basket region of the nuclear pore complex (NPC), nucleoporin Tpr, plays roles in regulating multiple important processes. We have previously established that Tpr is phosphorylated in both a MAP-kinase-dependent and MAP-kinase-independent manner, and that Tpr acts as both a substrate and as a scaffold for ERK2 (also known as MAPK1). Here, we report the identification of S2059 and S2094 as the major novel ERK-independent phosphorylation sites and T1677, S2020, S2023 and S2034 as additional ERK-independent phosphorylation sites found in the Tpr protein in vivo. Our results suggest that protein kinase A phosphorylates the S2094 residue and that the site is hyperphosphorylated during mitosis. Furthermore, we find that Tpr is phosphorylated at the S2059 residue by CDK1 and the phosphorylated form distinctly localizes with chromatin during telophase. Abrogation of S2059 phosphorylation abolishes the interaction of Tpr with Mad1, thus compromising the localization of both Mad1 and Mad2 proteins, resulting in cell cycle defects. The identification of novel phosphorylation sites on Tpr and the observations presented in this study allow better understanding of Tpr functions

Characterization of Leishmania donovani MCM4: Expression Patterns and Interaction with PCNA

Events leading to origin firing and fork elongation in eukaryotes involve several proteins which are mostly conserved across the various eukaryotic species. Nuclear DNA replication in trypanosomatids has thus far remained a largely uninvestigated area. While several eukaryotic replication protein orthologs have been annotated, many are missing, suggesting that novel replication mechanisms may apply in this group of organisms. Here, we characterize the expression of Leishmania donovani MCM4, and find that while it broadly resembles other eukaryotes, noteworthy differences exist. MCM4 is constitutively nuclear, signifying that, unlike what is seen in S.cerevisiae, varying subcellular localization of MCM4 is not a mode of replication regulation in Leishmania. Overexpression of MCM4 in Leishmania promastigotes causes progress through S phase faster than usual, implicating a role for MCM4 in the modulation of cell cycle progression. We find for the first time in eukaryotes, an interaction between any of the proteins of the MCM2-7 (MCM4) and PCNA. MCM4 colocalizes with PCNA in S phase cells, in keeping with the MCM2-7 complex being involved not only in replication initiation, but fork elongation as well. Analysis of a LdMCM4 mutant indicates that MCM4 interacts with PCNA via the PIP box motif of MCM4 - perhaps as an integral component of the MCM2-7 complex, although we have no direct evidence that MCM4 harboring a PIP box mutation can still functionally associate with the other members of the MCM2-7 complex- and the PIP box motif is important for cell survival and viability. In Leishmania, MCM4 may possibly help in recruiting PCNA to chromatin, a role assigned to MCM10 in other eukaryotes

Epigenetic Switches in Retinal Homeostasis and Target for Drug Development

Retinal homeostasis, a tightly regulated process maintaining the functional integrity of the retina, is vital for visual function. Emerging research has unveiled the critical role of epigenetic regulation in controlling gene expression patterns during retinal development, maintenance, and response to mutational loads and injuries. Epigenetic switches, including DNA methylation, histone modifications, and non-coding RNAs, play pivotal roles in orchestrating retinal gene expression and cellular responses through various intracellular, extracellular, and environmental modulators. This review compiles the current knowledge on epigenetic switches in retinal homeostasis, providing a deeper understanding of their impact on retinal structural integrity and function and using them as potential targets for therapeutic interventions

Localization of Nucleoporin Tpr to the Nuclear Pore Complex Is Essential for Tpr Mediated Regulation of the Export of Unspliced RNA

Nucleoporin Tpr is a component of the nuclear pore complex (NPC) that localizes exclusively to intranuclear filaments. Tpr functions as a scaffolding element in the nuclear phase of the NPC and plays a role in mitotic spindle checkpoint signalling. Export of intron-containing mRNA in Mason Pfizer Monkey Virus is regulated by direct interaction of cellular proteins with the cis-acting Constitutive Transport Element (CTE). In mammalian cells, the transport of Gag/Pol-CTE reporter construct is not very efficient, suggesting a regulatory mechanism to retain this unspliced RNA. Here we report that the knockdown of Tpr in mammalian cells leads to a drastic enhancement in the levels of Gag proteins (p24) in the cytoplasm, which is rescued by siRNA resistant Tpr. Tpr’s role in the retention of unspliced RNA is independent of the functions of Sam68 and Tap/Nxf1 proteins, which are reported to promote CTE dependent export. Further, we investigated the possible role for nucleoporins that are known to function in nucleocytoplasmic transport in modulating unspliced RNA export. Results show that depletion of Nup153, a nucleoporin required for NPC anchoring of Tpr, plays a role in regulating the export, while depletion of other FG repeat-containing nucleoporins did not alter the unspliced RNA export. Results suggest that Tpr and Nup153 both regulate the export of unspliced RNA and they are most likely functioning through the same pathway. Importantly, we find that localization of Tpr to the NPC is necessary for Tpr mediated regulation of unspliced RNA export. Collectively, the data indicates that perinuclear localization of Tpr at the nucleopore complex is crucial for regulating intron containin

Nucleoporin Nup153, a Tpr anchoring protein is also involved in regulating unspliced RNA export.

<p>(A) HEK293T cells were transfected with siRNA's against different nucleoporins together with Gag/Pol-CTE and CMV- β-Gal constructs. Western blot of cell extracts confirming the depletion of various nucleoporins on treatment with respective siRNA's. (B) 48 hours post transfection, the amount of p24 and β-Gal expression was estimated in the lysates and the obtained values were normalized. (C) Cells were transfected with siRNA's against Nup153 or Tpr or Nup153+Tpr together with Gag/Pol-CTE and CMV- β-Gal constructs. Western blot of cell extracts confirming the depletion of Nup153 and Tpr on treatment with respective siRNA's. (D) The amount of normalized p24 expression was estimated in the lysates.</p

Tpr knockdown in HEK293T cells mediated by RNA interference.

<p>(A) Western blot analysis of whole cell extracts made from cells after 48 hours after treatment either with Non-specific siRNA (NS-Si) or three different Tpr siRNA's (TSi, TSi-1, TSi-2), probed with anti-Tpr and anti-ERK antibodies. (B) Immunofluorescence analysis of cells 48 hours after transfection with various Tpr siRNA oligonucleotides. Clear staining of nuclear membrane is seen in cells treated with NS-Si whereas only traces of staining is detected in cells where Tpr is knocked down.</p

Overexpression of Sam68 in Tpr depleted cells leads to synergistic increase in the unspliced RNA export.

<p>(A) 48 hours after transfection with NS-Si and TSi, HEK293T cells were re-transfected with the same siRNA oligos along with Gag/Pol-CTE and CMV- β-Gal plasmids and 2 µg of HA-Sam68 or HA-Tap/Nxf1constructs. The lysates thus obtained were analyzed by Immunoblot for knockdown of Tpr and overexpression of HA-Sam68 and HA-Tap (B) The amount of p24 and β-Gal expression was estimated in the lysates and the obtained values were normalized. (C) Real-Time PCR analysis of mRNA isolated from nuclear and cytoplasmic fractions of HEK293T cells co-transfected with either NS-Si or TSi along with HA-Sam68 or HA-Tap/Nxf1constructs and Gag/Pol-CTE reporter plasmid.</p

Ectopic and stable expression of siRNA resistant Tpr rescues CTE mediated unspliced RNA export.

<p>(A) Schematic representation of the silent point mutations that were introduced into the nucleotide sequence of Tpr, to protect it from being targeted by siRNA duplexes. (B) Immunoblot demonstrating the restoration of Tpr levels upon co- transfection of FLAG-Tpr-Si construct with TSi. 2 µg of Flag-Tpr or Flag-Tpr-Si were used for transfection. (C) Indirect immunofluorescence microscopy of HEK293T cells transfected with Wild-type Flag-Tpr clone or siRNA resistant clone of Tpr along with NS-Si or TSi using mouse monoclonal anti-Flag antibodies (D) p24 and β-Galactosidase levels in each of the samples were analyzed. The p24 values were then adjusted against variations in β-Gal readings. (E) HEK293T cells stably expressing Flag-Tpr or Flag-Tpr-Si were transfected with either NS-Si or TSi together with Gag/Pol-CTE and CMV- β-Gal plasmids. Western blot of whole cell extracts probed with anti-Tpr, anti-FLAG and anti-ERK antibodies depicting stable expression. (F) Immunofluorescence analysis of Flag-Tpr or Flag-Tpr-Si stable cells after 2 days of transfection with either NS-Si or TSi oligonucleotides. (G) The cell lysates were assayed for p24 and β-Gal expression and the p24 levels were normalized with β-Gal readings.</p

Tpr mediated regulation of unspliced RNA export is independent of Tap/Nxf1 and Sam68's functions.

<p>(A) Cells were transfected with 1 µg of siRNA against either Sam68 or Tap/Nxf1 along with NS-Si or TSi. The cells were replated the next day and 24 hours after replating, the cells were transfected once again with siRNA oligos and the reporter constructs. Western blot analysis of extracts of transfected HEK293T cells confirming the depletion of Tpr, Sam68 and Tap/Nxf1 proteins on treatment with respective siRNA's. (B) 48 hours after the second transfection, the amount of reporter gene expression from transfected Gag/Pol-CTE and CMV- β-Gal constructs was assayed. The p24 ELISA readings obtained from the samples were adjusted against variations in the β-Galactosidase readings. (C) Cells were transfected with 1 µg of different siRNA oligos against Tap/Nxf1 along with NS-Si or TSi. The cells were replated the next day and 24 hours after replating, the cells were transfected once again with siRNA oligos and the reporter constructs. Western blot of extracts of transfected HEK293T cells to analyze the depletion of Tpr and Tap/Nxf1 proteins. (D & E) The amount of reporter gene expression from transfected Gag/Pol-CTE and CMV- β-Gal constructs were assayed.</p

Depletion of Tpr has no significant effect on cellular protein transport and mRNA export.

<p>(A) Western blot analysis of extracts of HeLa cells made 48 hours after treatment with TSi. (B) HeLa cells which stably express hormone responsive GFP reporter construct (RGG) were transfected with NS-Si or TSi. Import of chimeric GFP was monitored by fixing the cells with 4% paraformaldehyde at different times post dexamethasone treatment. (C) Export of RGG construct was tracked at indicated time points after removal of the hormone. (D) Western blot depicting Tpr knockdown in HEK293T cells after 48 hours after siRNA transfection. (E) Fluorescence in situ hybridization with FITC-oligo(dT) probe to assess distribution of poly(A)⁺ mRNA in HEK293T cells transfected with TSi.</p