The androgen receptor (AR) plays a central role in establishing an oncogenic cascade that drives prostate cancer progression. Some prostate cancers escape androgen dependence and are often associated with an aggressive phenotype. The oestrogen receptor alpha (ERα) is expressed in prostate cancers, independent of AR status. However, the role of ERα remains elusive. Using a combination of chromatin immunoprecipitation (ChIP) and RNA-sequencing data, we identified an ERα-specific non-coding transcriptome signature. Among putatively ERα-regulated intergenic long non-coding RNAs (lncRNAs), we identified nuclear enriched abundant transcript 1 (NEAT1) as the most significantly overexpressed lncRNA in prostate cancer. Analysis of two large clinical cohorts also revealed that NEAT1 expression is associated with prostate cancer progression. Prostate cancer cells expressing high levels of NEAT1 were recalcitrant to androgen or AR antagonists. Finally, we provide evidence that NEAT1 drives oncogenic growth by altering the epigenetic landscape of target gene promoters to favour transcription.

Transcriptomic analyses have identified tens of thousands of intergenic, intronic, and cis-antisense long noncoding RNAs (lncRNAs) that are expressed from mammalian genomes. Despite progress in functional characterization, little is known about the post-transcriptional regulation of lncRNAs and their half-lives. Although many are easily detectable by a variety of techniques, it has been assumed that lncRNAs are generally unstable, but this has not been examined genome-wide. Utilizing a custom noncoding RNA array, we determined the half-lives of ∼800 lncRNAs and ∼12,000 mRNAs in the mouse Neuro-2a cell line. We find only a minority of lncRNAs are unstable. LncRNA half-lives vary over a wide range, comparable to, although on average less than, that of mRNAs, suggestive of complex metabolism and widespread functionality. Combining half-lives with comprehensive lncRNA annotations identified hundreds of unstable (half-life < 2 h) intergenic, cis-antisense, and intronic lncRNAs, as well as lncRNAs showing extreme stability (half-life > 16 h). Analysis of lncRNA features revealed that intergenic and cis-antisense RNAs are more stable than those derived from introns, as are spliced lncRNAs compared to unspliced (single exon) transcripts. Subcellular localization of lncRNAs indicated widespread trafficking to different cellular locations, with nuclear-localized lncRNAs more likely to be unstable. Surprisingly, one of the least stable lncRNAs is the well-characterized paraspeckle RNA Neat1, suggesting Neat1 instability contributes to the dynamic nature of this subnuclear domain. We have created an online interactive resource (http://stability.matticklab.com) that allows easy navigation of lncRNA and mRNA stability profiles and provides a comprehensive annotation of ∼7200 mouse lncRNAs.

Paraspeckles are subnuclear structures formed around nuclear paraspeckle assembly transcript 1 (NEAT1)/MENε/β long noncoding RNA (lncRNA). Here we show that paraspeckles become dramatically enlarged after proteasome inhibition. This enlargement is mainly caused by NEAT1 transcriptional up-regulation rather than accumulation of undegraded paraspeckle proteins. Of interest, however, using immuno–electron microscopy, we find that key paraspeckle proteins become effectively depleted from the nucleoplasm by 50% when paraspeckle assembly is enhanced, suggesting a sequestration mechanism. We also perform microarrays from NEAT1-knockdown cells and find that NEAT1 represses transcription of several genes, including the RNA-specific adenosine deaminase B2 (ADARB2) gene. In contrast, the NEAT1-binding paraspeckle protein splicing factor proline/glutamine-rich (SFPQ) is required for ADARB2 transcription. This leads us to hypothesize that ADARB2 expression is controlled by NEAT1-dependent sequestration of SFPQ. Accordingly, we find that ADARB2 expression is strongly reduced upon enhanced SFPQ sequestration by proteasome inhibition, with concomitant reduction in SFPQ binding to the ADARB2 promoter. Finally, NEAT1^−/− fibroblasts are more sensitive to proteasome inhibition, which triggers cell death, suggesting that paraspeckles/NEAT1 attenuates the cell death pathway. These data further confirm that paraspeckles are stress-responsive nuclear bodies and provide a model in which induced NEAT1 controls target gene transcription by protein sequestration into paraspeckles.