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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.

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., Author summary Long noncoding RNA (lncRNA) have been identified in all eukaryotes but mechanisms of lncRNA function remain challenging to study in vivo. A classic model of lncRNA function and mechanism is X-Chromosome Inactivation (XCI): an essential process which balances X-linked gene expression between male and female mammals. The “master regulator” of XCI is lncRNA Xist, which is responsible for silencing one of the two X chromosomes in females. Another lncRNA, Jpx, has been proposed to activate Xist gene expression in mouse embryonic stem cells; however, no mouse models exist to address Jpx function in vivo. In this study, we developed a novel transgenic mouse system to demonstrate the regulatory mechanisms of lncRNA Jpx. We observed a dose-dependent relationship between Jpx copy number and Xist expression in transgenic mice, suggesting that Jpx is sufficient to activate Xist expression in vivo. In addition, we analyzed Jpx’s allelic origin and have provided evidence for Jpx inducing Xist transcription using both trans and cis mechanisms. Our work provides a framework for lncRNA functional studies in mice, which will help us understand how lncRNA regulate eukaryotic gene expression.

Transportin-specific molecular tools are used to show that the mitotic cell contains importin β and transportin “global positioning system” pathways that are mechanistically parallel. Transportin works to control where the spindle, nuclear membrane, and nuclear pores are formed by directly affecting assembly factor function., The nuclear import receptors importin β 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 β is known to act by repressing assembly factors in regions distant from chromatin, whereas RanGTP produced on chromatin frees factors from importin β 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 β, 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 β and transportin “global positioning system”or “GPS” pathways that are mechanistically parallel.

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