LncRNA Jpx induces Xist expression in mice using both trans and cis mechanisms.

Mammalian X chromosome dosage compensation balances X-linked gene products between sexes and is coordinated by the long noncoding RNA (lncRNA) Xist. Multiple cis and trans-acting factors modulate Xist expression; however, the primary competence factor responsible for activating Xist remains a subject of dispute. The lncRNA Jpx is a proposed competence factor, yet it remains unknown if Jpx is sufficient to activate Xist expression in mice. Here, we utilize a novel transgenic mouse system to demonstrate a dose-dependent relationship between Jpx copy number and ensuing Jpx and Xist expression. By localizing transcripts of Jpx and Xist using RNA Fluorescence in situ Hybridization (FISH) in mouse embryonic cells, we provide evidence of Jpx acting in both trans and cis to activate Xist. Our data contribute functional and mechanistic insight for lncRNA activity in mice, and argue that Jpx is a competence factor for Xist activation in vivo.

Linking Chromosomal Silencing With Xist Expression From Autosomal Integrated Transgenes.

Xist is the master regulator of X-Chromosome Inactivation (XCI), the mammalian dosage compensation mechanism that silences one of the two X chromosomes in a female cell. XCI is established during early embryonic development. Xist transgene (Tg) integrated into an autosome can induce transcriptional silencing of flanking genes; however, the effect and mechanism of Xist RNA on autosomal sequence silencing remain elusive. In this study, we investigate an autosomal integration of Xist Tg that is compatible with mouse viability but causes male sterility in homozygous transgenic mice. We observed ectopic Xist expression in the transgenic male cells along with a transcriptional reduction of genes clustered in four segments on the mouse chromosome 1 (Chr 1). RNA/DNA Fluorescent in situ Hybridization (FISH) and chromosome painting confirmed that Xist Tg is associated with chromosome 1. To determine the spreading mechanism of autosomal silencing induced by Xist Tg on Chr 1, we analyzed the positions of the transcriptionally repressed chromosomal sequences relative to the Xist Tg location inside the cell nucleus. Our results show that the transcriptionally repressed chromosomal segments are closely proximal to Xist Tg in the three-dimensional nucleus space. Our findings therefore support a model that Xist directs and maintains long-range transcriptional silencing facilitated by the three-dimensional chromosome organization.

Exportins can inhibit major mitotic assembly events in vitro: membrane fusion, nuclear pore formation, and spindle assembly.

XENOPUS: egg extracts are a powerful in vitro tool for studying complex biological processes, including nuclear reconstitution, nuclear membrane and pore assembly, and spindle assembly. Extracts have been further used to demonstrate a moonlighting regulatory role for nuclear import receptors or importins on these cell cycle assembly events. Here we show that exportins can also play a role in these events. Addition of Crm1, Exportin-t, or Exportin-5 decreased nuclear pore assembly in vitro. RanQ69L-GTP, a constitutively active form of RanGTP, ameliorated inhibition. Both Crm1 and Exportin-t inhibited fusion of nuclear membranes, again counteracted by RanQ69L-GTP. In mitotic extracts, Crm1 and Exportin-t negatively impacted spindle assembly. Pulldowns from the extracts using Crm1- or Exportin-t-beads revealed nucleoporins known to be essential for both nuclear pore and spindle assembly, with RanQ69L-GTP decreasing a subset of these target interactions. This study suggests a model where exportins, like importins, can regulate major mitotic assembly events.

Functional Conservation of LncRNA JPX Despite Sequence and Structural Divergence.

Long noncoding RNAs (lncRNAs) have been identified in all eukaryotes and are most abundant in the human genome. However, the functional importance and mechanisms of action for human lncRNAs are largely unknown. Using comparative sequence, structural, and functional analyses, we characterize the evolution and molecular function of human lncRNA JPX. We find that human JPX and its mouse homolog, lncRNA Jpx, have deep divergence in their nucleotide sequences and RNA secondary structures. Despite such differences, both lncRNAs demonstrate robust binding to CTCF, a protein that is central to Jpx's role in X chromosome inactivation. In addition, our functional rescue experiment using Jpx-deletion mutant cells shows that human JPX can functionally complement the loss of Jpx in mouse embryonic stem cells. Our findings support a model for functional conservation of lncRNAs independent from sequence and structural divergence. This study provides mechanistic insight into the evolution of lncRNA function.

Transportin acts to regulate mitotic assembly events by target binding rather than Ran sequestration.

The nuclear import receptors importin ß and transportin play a different role in mitosis: both act phenotypically as spatial regulators to ensure that mitotic spindle, nuclear membrane, and nuclear pore assembly occur exclusively around chromatin. Importin ß is known to act by repressing assembly factors in regions distant from chromatin, whereas RanGTP produced on chromatin frees factors from importin ß for localized assembly. The mechanism of transportin regulation was unknown. Diametrically opposed models for transportin action are as follows: 1) indirect action by RanGTP sequestration, thus down-regulating release of assembly factors from importin ß, and 2) direct action by transportin binding and inhibiting assembly factors. Experiments in Xenopus assembly extracts with M9M, a superaffinity nuclear localization sequence that displaces cargoes bound by transportin, or TLB, a mutant transportin that can bind cargo and RanGTP simultaneously, support direct inhibition. Consistently, simple addition of M9M to mitotic cytosol induces microtubule aster assembly. ELYS and the nucleoporin 107-160 complex, components of mitotic kinetochores and nuclear pores, are blocked from binding to kinetochores in vitro by transportin, a block reversible by M9M. In vivo, 30% of M9M-transfected cells have spindle/cytokinesis defects. We conclude that the cell contains importin ß and transportin "global positioning system"or "GPS" pathways that are mechanistically parallel.