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ENEMAL, an enhancer RNA transcribed from a distal MALAT1 enhancer, promotes NEAT1 long isoform expression.

ABSTRACT: Emerging evidence has shown that active enhancers are abundantly transcribed, generating long non-coding RNAs, called enhancer RNAs (eRNAs). While putative eRNAs are often observed from RNA sequencing, the roles of most eRNAs remain largely unknown. Previously, we identified putative enhancer regions at the MALAT1 locus that form chromatin-chromatin interactions under hypoxia, and one of these enhancers is located about 30 kb downstream of the NEAT1 gene and -20 kb upstream of the MALAT1 gene (MALAT1-20 kb enhancer). Here, we report that a novel eRNA, named eRNA of the NEAT1-MALAT1-Locus (eNEMAL), is transcribed from the MALAT1-20 kb enhancer and conserved in primates. We found that eNEMAL is upregulated in response to hypoxia in multiple breast cancer cell lines, but not in non-tumorigenic MCF10A cells. Overexpression and knockdown of eNEMAL revealed that alteration of eNEMAL level does not affect MALAT1 expression. Instead, we found that eNEMAL upregulates the long isoform of NEAT1 (NEAT1_2) without increasing the total NEAT1 transcript level in MCF7 breast cancer cells, suggesting that eNEMAL has a repressive effect on the 3'-end polyadenylation process required for generating the short isoform of NEAT1 (NEAT1_1). Altogether, we demonstrated that an eRNA transcribed from a MALAT1 enhancer regulates NEAT1 isoform expression, implicating the MALAT1-20 kb enhancer and its transcript eNEMAL in co-regulation of MALAT1 and NEAT1 in response to hypoxia in breast cancer cells.

SUBMITTER: Stone JK

PROVIDER: S-EPMC8139514 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Long noncoding RNAs (lncRNAs) are important regulators of chromatin; however, the mechanistic roles for many lncRNAs are poorly understood in part because their direct interactions with genomic loci and proteins are difficult to assess. We used CHART-seq to map the genomic binding sites for two highly expressed human lncRNAs, NEAT1 and MALAT1, which localize within the nucleus to paraspeckles and nuclear speckles, respectively. We show that NEAT1 and MALAT1 localize to hundreds of genomic sites in human cells, primarily over active genes. NEAT1 and MALAT1 exhibit colocalization to many of these loci, but display distinct gene body binding patterns at these sites, suggesting independent but complementary functions for these RNAs. Protein mass spectrometry analysis of CHART-enriched material (CHART-MS) identified numerous proteins enriched by both lncRNAs, supporting complementary binding and function, in addition to unique associated proteins. Transcriptional inhibition or stimulation affects the localization of NEAT1 to active chromatin sites, implying that DNA sequence itself does not target NEAT1 to chromatin and that localization responds to cues involved in the transcription process. Paired-end CHART-seq was performed for a single replicate of each capture oligonucleotide in untreated MCF-7 cells to establish binding sites of these RNAs, for a total of 6 samples. To investigate the effects of transcriptional inhibition and E2 stimulation on the localization of these RNAs, we performed paired-end CHART-seq with each capture oligonucleotide for two biological replicates of flavopiridol- and vehicle (DMSO)-treated MCF-7 cells and for two biological replicates of E2- and vehicle (ethanol)-treated MCF-7 cells. To establish the overlap of NEAT1 and MALAT1 binding sites with a known component of paraspeckles (NEAT1-containing subnuclear body), we performed paired-end ChIP-seq for the paraspeckle component PSF in MCF-7 cells, as well as a single-end biological replicate.

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ENEMAL, an enhancer RNA transcribed from a distal MALAT1 enhancer, promotes NEAT1 long isoform expression.

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