The role of G-Quadruplex DNA in Paraspeckle formation in cancer

Highlights

• NEAT1_2 lncRNA and DNA G-quadruplexes are altered in multiple cancers .

• We identify and characterise potential G-quadruplexes in the NEAT1 gene promoter.

• Stabilization of G-quadruplexes alters NEAT1_2 expression and paraspeckle formation.

Abstract

Paraspeckles are RNA–protein structures within the nucleus of mammalian cells, capable of orchestrating various biochemical processes. An overexpression of the architectural component of paraspeckles, a long non-coding RNA called NEAT1 (Nuclear Enriched Abundant Transcript 1), has been linked to a variety of cancers and is often associated with poor patient prognosis. Thus, there is an accumulating interest in the role of paraspeckles in carcinogenesis, however there is a limited understanding of how NEAT1 expression is regulated. Here, we demonstrate that both nuclear G-quadruplex (G4) and paraspeckle formation are significantly increased in a human breast cancer cell line compared to non-tumorigenic breast cells. Moreover, we identified and characterized G4-forming sequences within the NEAT1 promoter and demonstrate stabilization of G4 DNA with a G4-stabilizing small molecule results in a significant alteration in both paraspeckle formation and NEAT1 expression. This G4-mediated alteration of NEAT1 at both the transcriptional and post-transcriptional levels was evident in U2OS osteosarcoma cells, MCF-7 breast adenocarcinoma and MDA-MB-231 triple negative breast cancer cells.

Introduction

Paraspeckles orchestrate a variety of biochemical processes within the cell nucleus, including the regulation of both rhythmic and developmental pathways [1]. Previously described as ‘nuclear hubs’, these paraspeckles are repositories of regulatory proteins and RNAs, whose retention and release has been shown to co-ordinate cellular processes such as gene expression, immune response, and the maturation of microRNAs [[1], [2], [3], [4]]. The paraspeckle core is formed by the interaction between Drosophila Behavior Human Splicing (DBHS) proteins, Fused in Sarcoma (FUS) protein, and the long non-coding RNA (lncRNA) Nuclear Enriched Abundant Transcript 1 (NEAT1) (specifically, the NEAT1_2 22.3 kb isoform) [5]. NEAT1_2 is the main architectural component of paraspeckles and its expression is both an absolute requirement for and exclusive to paraspeckle formation [6,7]. Due to the dynamic nature of paraspeckles, their precise biological function is not well understood. Paraspeckles have however been shown to mediate gene expression, and have been highlighted within recent literature as important regulatory elements [[8], [9], [10]]. Functional studies have shown that NEAT1_2 expression is required for the normal development of mammary tissues, with NEAT1_2 knockout mice displaying compromised lactation, reduced mammary epithelial cell proliferation, and defective areola development [11]. These results indicate that NEAT1_2, and therefore paraspeckles, have an important role in breast tissue development.

Of particular interest is the role of paraspeckles within a pathological framework. NEAT1_2 overexpression has been observed in a majority of solid tumor types, including breast cancer and is considered an important driver in processes such as aberrant cell proliferation [[12], [13], [14], [15], [16], [17], [18], [19], [20], [21]]. Increased NEAT1_2 expression has previously been reported to promote cell survival and decreased apoptosis [22,23]. Recent literature has demonstrated NEAT1_2 expression is induced by DNA damage and stress conditions in order to promote cell survival, and as a consequence, NEAT1 has been suggested as a potential biomarker and therapeutic target for a range of pathologies [8,24]. With regard to breast cancer, high expression of NEAT1_2 has been correlated with shorter survival in patients, attributed to both accelerated tumor cell proliferation and reduced cell death [25]. Conversely, a reduction of NEAT1 expression inhibited growth and induced apoptosis [16]. Thus, it has been suggested that NEAT1_2 may be used as a diagnostic and prognostic biomarker for breast cancer [[26], [27], [28], [29]]. A number of transcription factors bind and regulate the NEAT1 promoter following different stress triggers and RNA-binding proteins mediate additional post-transcriptional regulation of NEAT1 to enable paraspeckle formation [30]. However, the precise mechanism of NEAT1 regulation is only now being explored, and a more complete understanding of NEAT1 regulation at the transcriptional level, and subsequent paraspeckle formation, is required.

Similarly, non-canonical secondary structures known as G-quadruplexes (G4s) are widely known to have critical roles in the development and progression of cancer. G4s are self-assembled non-canonical structures that form within certain guanine-rich sequences in DNA and RNA [31,32]. Two or more planar G-tetrads, consisting of four guanine bases held together through Hoogsteen hydrogen bonds, pi-stack to form the G4 structure [33]. The formation of G4s leads to alterations in the chromatin landscape and thus has major implications on genome stability and regulation of gene expression [34]. Unresolved G4s can stall molecular machinery to inhibit transcription or enhance transcription by providing a binding site for proteins such as transcription factors, dependent on gene context [35]. This extends to regulation of telomere shortening, DNA replication processes, and expression of many oncogenes and proto-oncogenes [36]. G4-formation in itself is significantly associated with oncogenes and tumor suppressors, furthering links to genomic instability and carcinogenesis [32,[37], [38], [39], [40], [41]]. Furthermore, G4s have been identified as an attractive target for gene regulation, with numerous small molecules being reported to target G4s in oncogene promoters, with some compounds entering clinical trials [[42], [43], [44], [45]].

Whilst transcriptome-wide studies have revealed the formation of G4-RNA within NEAT1 [[46], [47], [48]] that are bound by DBHS proteins, there is little knowledge of G4-DNA in NEAT1-associated regulatory elements. Using G4 prediction tools [49,50], we screened the NEAT1 gene, including its CpG island promoter region, to identify potential G4 DNA formation sites in order to determine the relationship between G4-DNA and paraspeckle formation.

Herein, we describe the relationship between G4-DNA formation and NEAT1 expression, at both a transcriptional (gene expression) and post-transcriptional (paraspeckle) level. We demonstrate increased paraspeckle and G4-DNA abundance within the breast adenocarcinoma cell line, MCF-7, when compared to non-tumorigenic MCF-10A cells and observe that by stabilizing G4s within the cellular environment with G4-specific ligands, paraspeckle formation can be regulated. G4-mediated regulation of NEAT1 expression was also examined in U2OS osteosarcoma cells and MDA-MB-231 triple negative breast cancer cells to investigate cell-type specific behaviors. Furthermore, we characterized G4-forming sequences within the NEAT1 promoter region which are highly stable in physiologically relevant conditions.