Differential nuclear import regulates nuclear RNA inheritance following mitosis.

Mitosis results in a dramatic reorganization of chromatin structure to promote chromosome compaction and segregation to daughter cells. Consequently, mitotic entry is accompanied by transcriptional silencing and removal of most chromatin-bound RNA from chromosomes. As cells exit mitosis, chromatin rapidly decondenses and transcription restarts as waves of differential gene expression. However, little is known about the fate of chromatin-bound RNAs following cell division. Here we explored whether nuclear RNA from the previous cell cycle is present in G1 nuclei following mitosis. We found that half of all nuclear RNA is inherited in a transcription-independent manner following mitosis. Interestingly, the snRNA U2 is efficiently inherited by G1 nuclei, while the lncRNAs NEAT1 and MALAT1 show no inheritance following mitosis. We found that the nuclear protein SAF-A, which is hypothesized to tether RNA to DNA, did not play a prominent role in nuclear RNA inheritance, indicating that the mechanism for RNA inheritance may not involve RNA chaperones that have chromatin-binding activity. Instead, we observe that the timing of RNA inheritance indicates that a select group of nuclear RNAs are reimported into the nucleus after the nuclear envelope has reassembled. Our work demonstrates that there is a fraction of nuclear RNA from the previous cell cycle that is reimported following mitosis and suggests that mitosis may serve as a time to reset the interaction of lncRNAs with chromatin.

Cell division requires RNA eviction from condensing chromosomes.

During mitosis, the genome is transformed from a decondensed, transcriptionally active state to a highly condensed, transcriptionally inactive state. Mitotic chromosome reorganization is marked by the general attenuation of transcription on chromosome arms, yet how the cell regulates nuclear and chromatin-associated RNAs after chromosome condensation and nuclear envelope breakdown is unknown. SAF-A/hnRNPU is an abundant nuclear protein with RNA-to-DNA tethering activity, coordinated by two spatially distinct nucleic acid-binding domains. Here we show that RNA is evicted from prophase chromosomes through Aurora-B-dependent phosphorylation of the SAF-A DNA-binding domain; failure to execute this pathway leads to accumulation of SAF-A-RNA complexes on mitotic chromosomes, defects in metaphase chromosome alignment, and elevated rates of chromosome missegregation in anaphase. This work reveals a role for Aurora-B in removing chromatin-associated RNAs during prophase and demonstrates that Aurora-B-dependent relocalization of SAF-A during cell division contributes to the fidelity of chromosome segregation.

Production of Xenopus tropicalis egg extracts to identify microtubule-associated RNAs.

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Functional analysis of the microtubule-interacting transcriptome.

RNA localization is an important mechanism for achieving precise control of posttranscriptional gene expression. Previously, we demonstrated that a subset of cellular mRNAs copurify with mitotic microtubules in egg extracts of Xenopus laevis. Due to limited genomic sequence information available for X. laevis, we used RNA-seq to comprehensively identify the microtubule-interacting transcriptome of the related frog Xenopus tropicalis. We identified ~450 mRNAs that showed significant enrichment on microtubules (MT-RNAs). In addition, we demonstrated that the MT-RNAs incenp, xrhamm, and tpx2 associate with spindle microtubules in vivo. MT-RNAs are enriched with transcripts associated with cell division, spindle formation, and chromosome function, demonstrating an overrepresentation of genes involved in mitotic regulation. To test whether uncharacterized MT-RNAs have a functional role in mitosis, we performed RNA interference and discovered that several MT-RNAs are required for normal spindle pole organization and γ-tubulin distribution. Together, these data demonstrate that microtubule association is one mechanism for compartmentalizing functionally related mRNAs within the nucleocytoplasmic space of mitotic cells and suggest that MT-RNAs are likely to contribute to spindle-localized mitotic translation.

Heterogeneity in the treatment of moderately severe scalp psoriasis in Scotland - results of a survey of Scottish health professionals.

BACKGROUND: Scalp psoriasis is a chronic recalcitrant condition. An aging literature for topical treatments used in clinical practice and no treatment guidelines means there is no current gold standard for its management in Scotland. There are no Scottish data on the resources and costs of treatment of the scalp psoriasis patient. OBJECTIVE: Conduct a survey of Scottish healthcare professionals to understand how patients are typically managed to support the development of a model estimating the cost-effectiveness of a new treatment for moderately severe scalp psoriasis in Scotland. RESEARCH DESIGN AND METHODS: Experts from primary and secondary care were invited to participate in an interview programme to collect information on the management of scalp psoriasis in Scotland. This was further informed by Scottish prescribing statistics. Simple descriptive statistics were performed. RESULTS: Forty-three healthcare professionals (33 from primary care and ten in secondary care) completed the survey which illuminated national prescribing statistics. While an overall 72% response rate was achieved, representation from five of 14 Health Boards was not available. There was significant variation in stated patient pathways but some common themes. Most patients were treated initially with coal tar preparations and shampoos, then often progressing to topical potent corticosteroids. There was no consensus on the order patients might receive topicals thereafter although if referred for specialist review they would typically have been treated with three topicals in primary care first. Treatment in secondary care comprised application of topicals available in primary care or alternative preparations with nurse assistance to improve compliance. Phototherapy and systemic agents were not given to patients with scalp psoriasis alone. Study limitations are not considered to impact on the study observations. CONCLUSIONS: There was a large variety in first-, second- and third-line agents in primary care in scalp psoriasis although our interview programme and prescribing data confirmed which treatments were most frequently prescribed. Treatment heterogeneity reflects the limitations in current therapies, paucity of evidence-based effectiveness data and lack of clinical guidelines. Experts agreed 'current standard practice' in Scotland was best described as an average across five plausible treatment pathways.

The histone domain of macroH2A1 contains several dispersed elements that are each sufficient to direct enrichment on the inactive X chromosome.

Histone variants replace the core histones in a substantial fraction of nucleosomes, affecting chromatin structure and impacting chromatin-templated processes. In many instances incorporation of histone variants results in formation of specialized regions of chromatin. Proper localization of histone variants to distinct regions of the genome is critical for their function, yet how this specific localization is achieved remains unclear. macroH2A1 is enriched on the inactive X chromosome in female mammalian cells, where it functions to maintain gene silencing. macroH2A1 consists of a histone H2A-like histone domain and a large, globular C-terminal macro domain that is not present in other histone proteins. The histone domain of macroH2A1 is alone sufficient to direct enrichment on the inactive X chromosome when expressed in female cells, indicating that sequences important for correct localization lie in this domain. Here we investigate whether divergent sequences of the H2A variant macroH2A1 contribute to its correct localization. We mapped the regions of the macroH2A1 histone domain that are sufficient for localization to the inactive X chromosome using chimeras between H2A and the histone domain of macroH2A1. Multiple short sequences dispersed along the macroH2A1 histone domain individually supported enrichment on the inactive X chromosome when introduced into H2A. These sequences map to the surface of the macroH2A1/H2B dimer, but are buried in the crystal structure of the macroH2A1 containing nucleosome, suggesting that they may contribute to recognition by macroH2A1/H2B deposition factors.

The XIST noncoding RNA functions independently of BRCA1 in X inactivation.

Females with germline mutations in BRCA1 are predisposed to develop breast and ovarian cancers. A previous report indicated that BRCA1 colocalizes with and is necessary for the correct localization of XIST, a noncoding RNA that coats the inactive X chromosome (Xi) to mediate formation of facultative heterochromatin. A model emerged from this study suggesting that loss of BRCA1 in female cells could reactivate genes on the Xi through loss of the XIST RNA. However, our independent studies of BRCA1 and XIST RNA revealed little evidence to support this model. We report that BRCA1 is not enriched on XIST RNA-coated chromatin of the Xi. Neither mutation nor depletion of BRCA1 causes significant changes in XIST RNA localization or X-linked gene expression. Together, these results do not support a role for BRCA1 in promoting XIST RNA localization to the Xi or regulating XIST-dependent functions in maintaining the stability of facultative heterochromatin.

Regulation of histone deposition proteins Asf1/Hir1 by multiple DNA damage checkpoint kinases in Saccharomyces cerevisiae.

CAF-1, Hir proteins, and Asf1 are histone H3/H4 binding proteins important for chromatin-mediated transcriptional silencing. We explored genetic and physical interactions between these proteins and S-phase/DNA damage checkpoint kinases in the budding yeast Saccharomyces cerevisiae. Although cells lacking checkpoint kinase Mec1 do not display defects in telomeric gene silencing, silencing was dramatically reduced in cells lacking both Mec1 and the Cac1 subunit of CAF-1. Silencing was restored in cac1Delta and cac1Delta mec1Delta cells upon deletion of Rad53, the kinase downstream of Mec1. Restoration of silencing to cac1Delta cells required both Hir1 and Asf1, suggesting that Mec1 counteracts functional sequestration of the Asf1/Hir1 complex by Rad53. Consistent with this idea, the degree of suppression of silencing defects by rad53 alleles correlated with effects on Asf1 binding. Furthermore, deletion of the Dun1 kinase, a downstream target of Rad53, also suppressed the silencing defects of cac1Delta cells and reduced the levels of Asf1 associated with Rad53 in vivo. Loss of Mec1 and Rad53 did not alter telomere lengths or Asf1 protein levels, nuclear localization, or chromosome association. We conclude that the Mec1 and Dun1 checkpoint kinases regulate the Asf1-Rad53 interaction and therefore affect the activity of the Asf1/Hir complex in vivo.

The budding yeast silencing protein Sir1 is a functional component of centromeric chromatin.

In fission yeast and multicellular organisms, centromere-proximal regions of chromosomes are heterochromatic, containing proteins that silence gene expression. In contrast, the relationship between heterochromatin proteins and kinetochore function in the budding yeast Saccharomyces cerevisiae remains largely unexplored. Here we report that the yeast heterochromatin protein Sir1 is a component of centromeric chromatin and contributes to mitotic chromosome stability. Sir1 recruitment to centromeres occurred through a novel mechanism independent of its interaction with the origin recognition complex (ORC). Sir1 function at centromeres was distinct from its role in forming heterochromatin, because the Sir2-4 proteins were not associated with centromeric regions. Sir1 bound to Cac1, a subunit of chromatin assembly factor I (CAF-I), and helped to retain Cac1 at centromeric loci. These studies reveal that although budding yeast and mammalian cells use fundamentally different mechanisms of forming heterochromatin, they both use silencing proteins to attract the histone deposition factor CAF-I to centromeric chromatin.

Chromatin assembly factor I and Hir proteins contribute to building functional kinetochores in S. cerevisiae.

Budding yeast centromeres are comprised of approximately 125-bp DNA sequences that direct formation of the kinetochore, a specialized chromatin structure that mediates spindle attachment to chromosomes. We report here a novel role for the histone deposition complex chromatin assembly factor I (CAF-I) in building centromeric chromatin. The contribution of CAF-I to kinetochore function overlaps that of the Hir proteins, which have also been implicated in nucleosome formation and heterochromatic gene silencing. cacDelta hirDelta double mutant cells lacking both CAF-I and Hir proteins are delayed in anaphase entry in a spindle assembly checkpoint-dependent manner. Further, cacDelta and hirDelta deletions together cause increased rates of chromosome missegregation, genetic synergies with mutations in kinetochore protein genes, and alterations in centromeric chromatin structure. Finally, CAF-I subunits and Hir1 are enriched at centromeres, indicating that these proteins make a direct contribution to centromeric chromatin structures.