Ya-Hui Chi

Requirement of DDX3 DEAD Box RNA Helicase for HIV-1 Rev-RRE Export Function

A single transcript in its unspliced and spliced forms directs the synthesis of all HIV-1 proteins. Although nuclear export of intron-containing cellular transcripts is restricted in mammalian cells, HIV-1 has evolved the viral Rev protein to overcome this restriction for viral transcripts. Previously, CRM1 was identified as a cellular cofactor for Rev-dependent export of intron-containing HIV-1 RNA. Here, we present evidence that Rev/CRM1 activity utilizes the ATP-dependent DEAD box RNA helicase, DDX3. We show that DDX3 is a nucleo-cytoplasmic shuttling protein, which binds CRM1 and localizes to nuclear membrane pores. Knockdown of DDX3 using either antisense vector or dominant-negative mutants suppressed Rev-RRE-function in the export of incompletely spliced HIV-1 RNAs. Plausibly, DDX3 is the human RNA helicase which functions in the CRM1 RNA export pathway analogously to the postulated role for Dbp5p in yeast mRNA export.

Heterozygous Deletion of Mitotic Arrest–Deficient Protein 1 (MAD1) Increases the Incidence of Tumors in Mice

Mitotic arrest-deficient protein 1 (MAD1) is a component of the mitotic spindle assembly checkpoint. We have created a knockout mouse model to examine the physiologic consequence of reduced MAD1 function. Mad1(+/-) mice were successfully generated, but repeated paired mating of Mad1(+/-) with Mad1(+/-) mice failed to produce a single Mad1(-/-) animal, suggesting that the latter genotype is embryonic lethal. In aging studies conducted for >18 months, Mad1(+/-) mice compared with control wild-type (wt) littermates showed a 2-fold higher incidence of constitutive tumors. Moreover, 42% of Mad1(+/-) (P < 0.03), but 0% of wt, mice developed neoplasia after treatment with vincristine, a microtubule depolymerization agent. Mad1(+/-) mouse embryonic fibroblasts (MEF) were found to be more prone than wt cells to become aneuploid; Mad1(+/-), but not wt, MEFs produced fibrosarcomas when explanted into nude mice. Our results indicate an essential MAD1 function in mouse development and correlate Mad1 haploinsufficiency with increased constitutive tumors.

Accumulation of the inner nuclear envelope protein Sun1 is pathogenic in progeric and dystrophic laminopathies.

Human LMNA gene mutations result in laminopathies that include Emery-Dreifuss muscular dystrophy (AD-EDMD) and Hutchinson-Gilford progeria, the premature aging syndrome (HGPS). The Lmna null (Lmna(-/-)) and progeroid LmnaΔ9 mutant mice are models for AD-EDMD and HGPS, respectively. Both animals develop severe tissue pathologies with abbreviated life spans. Like HGPS cells, Lmna(-/-) and LmnaΔ9 fibroblasts have typically misshapen nuclei. Unexpectedly, Lmna(-/-) or LmnaΔ9 mice that are also deficient for the inner nuclear membrane protein Sun1 show markedly reduced tissue pathologies and enhanced longevity. Concordantly, reduction of SUN1 overaccumulation in LMNA mutant fibroblasts and in cells derived from HGPS patients corrected nuclear defects and cellular senescence. Collectively, these findings implicate Sun1 protein accumulation as a common pathogenic event in Lmna(-/-), LmnaΔ9, and HGPS disorders.

Histone Acetyltransferase hALP and Nuclear Membrane Protein hsSUN1 Function in De-condensation of Mitotic Chromosomes

Replicated mammalian chromosomes condense to segregate during anaphase, and they de-condense at the conclusion of mitosis. Currently, it is not understood what the factors and events are that specify de-condensation. Here, we demonstrate that chromosome de-condensation needs the function of an inner nuclear membrane (INM) protein hsSUN1 and a membrane-associated histone acetyltransferase (HAT), hALP. We propose that nascently reforming nuclear envelope employs hsSUN1 and hALP to acetylate histones for de-compacting DNA at the end of mitosis.

Requirement for Sun1 in the expression of meiotic reproductive genes and piRNA

The inner nuclear envelope (NE) proteins interact with the nuclear lamina and participate in the architectural compartmentalization of chromosomes. The association of NE proteins with DNA contributes to the spatial rearrangement of chromosomes and their gene expression. Sun1 is an inner nuclear membrane (INM) protein that locates to telomeres and anchors chromosome movement in the prophase of meiosis. Here, we have created Sun1–/– mice and have found that these mice are born and grow normally but are reproductively infertile. Detailed molecular analyses showed that Sun1–/– P14 testes are repressed for the expression of reproductive genes and have no detectable piRNA. These findings raise a heretofore unrecognized role of Sun1 in the selective gene expression of coding and non-coding RNAs needed for gametogenesis.

The nuclear envelopathies and human diseases

The nuclear envelope (NE) consists of two membrane layers that segregate the nuclear from the cytoplasmic contents. Recent progress in our understanding of nuclear-lamina associated diseases has revealed intriguing connections between the envelope components and nuclear processes. Here, we review the functions of the nuclear envelope in chromosome organization, gene expression, DNA repair and cell cycle progression, and correlate deficiencies in envelope function with human pathologies.

Requirements for protein phosphorylation and the kinase activity of polo-like kinase 1 (Plk1) for the kinetochore function of mitotic arrest deficiency protein 1 (Mad1).

Mitotic arrest deficiency protein 1 (Mad1) is associated with microtubule-unattached kinetochores in mitotic cells and is a component of the spindle assembly checkpoint (SAC). Here, we have studied the phosphorylation of Mad1 and mapped using liquid chromatography-tandem mass spectrometry several phosphorylated amino acids in this protein. One phosphorylated residue, Thr680, was characterized to be important for the kinetochore localization of Mad1 and its SAC function. We also found that in mitotic cells Mad1 co-immunoprecipitated with Plk1. Depletion of cellular Plk1 using small interfering RNAs and inhibition of the kinase activity of Plk1 using a kinase-dead mutant or a small molecule inhibitor attenuated Mad1 phosphorylation and its association with kinetochores. Collectively, these findings indicate mechanistic roles contributed by protein phosphorylation and Plk1 to the SAC activity of Mad1.

Evidence for Cooperative Transforming Activity of the Human Pituitary Tumor Transforming Gene and Human T-Cell Leukemia Virus Type 1 Tax

ABSTRACT Aneuploidy is frequent in cancers. Recently it was found that pituitary tumor transforming gene (PTTG; also called Pds1p or securin) is overexpressed in many different tumors. Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that primarily infects CD4+ T lymphocytes and causes adult T-cell leukemia. Here, we report that overexpression of human PTTG cooperated with the HTLV-I Tax oncoprotein in cellular transformation. Coexpression of Tax and PTTG enhanced chromosomal instability and neoplastic changes to levels greater than overexpression of either factor singularly. Cells that overexpressed both PTTG and Tax induced tumors more robustly in nude mice than cells that expressed either PTTG alone or Tax alone.

Reversal of laminopathies: the curious case of SUN1.

Mutations in the LMNA gene are associated with a spectrum of human dystrophic diseases termed the “nuclear laminopathies.” We recently found that the accumulation of the inner nuclear envelope proteins SUN1 is pathogenic in progeric and dystrophic laminopathies. This conclusion arose from the unexpected observation that the deletion of Sun1, instead of accelerating aging, actually ameliorated the progeric and dystrophic phenotypes in Lmna-deficient mice. In human cells, knocking down SUN1 corrected the nuclear aberrancies and the senescent tendencies of HGPS (Hutchinson-Gilford progeria syndrome) skin fibroblasts. Here we offer additional comments on the contributions of SUN1 and the process of normal protein turnover to cellular aging.