Probing the function of long noncoding RNAs in the nucleus

Abstract

The nucleus is a highly organized and dynamic environment where regulation and coordination of processes such as gene expression and DNA replication are paramount. In recent years, noncoding RNAs have emerged as key participants in the regulation of nuclear processes. There are a multitude of functional roles for long noncoding RNA (lncRNA), mediated through their ability to act as molecular scaffolds bridging interactions with proteins, chromatin, and other RNA molecules within the nuclear environment. In this review, we discuss the diversity of techniques that have been developed to probe the function of nuclear lncRNAs, along with the ways in which those techniques have revealed insights into their mechanisms of action. Foundational observations into lncRNA function have been gleaned from molecular cytology-based, single-cell approaches to illuminate both the localization and abundance of lncRNAs in addition to their potential binding partners. Biochemical, extraction-based approaches have revealed the molecular contacts between lncRNAs and other molecules within the nuclear environment and how those interactions may contribute to nuclear organization and regulation. Using examples of well-studied nuclear lncRNAs, we demonstrate that the emerging functions of individual lncRNAs have been most clearly deduced from combined cytology and biochemical approaches tailored to study specific lncRNAs. As more functional nuclear lncRNAs continue to emerge, the development of additional technologies to study their interactions and mechanisms of action promise to continually expand our understanding of nuclear organization, chromosome architecture, genome regulation, and disease states.

Keywords: Biochemical methods; Genome regulation; Microscopy; Molecular cytology; Noncoding RNA; Nuclear organization.

Fig 1

Key insights gained from molecular cytology on the nuclear function of NEAT1 and XIST lncRNAs. A) NEAT1 is transcribed and paraspeckle proteins are recruited to accumulated transcripts in the nucleus (Step 1). Paraspeckle components then organize (Step 2) into a core-shell structure, where NEAT-1 RNA transcripts are oriented with their 3’ and 5’ ends bundled along the shell while key paraspeckle proteins and the middle of NEAT-1 transcripts localize to the center (Step 3). B) XIST RNA localizes to, and “paints”, the inactive X chromosome in the nucleus, in a manner that is resistant to DNase or RNaseH treatment (Step 1). XIST coating of chromatin induces a suite of epigenetic effects ultimately resulting in heterochromatin formation (Step 2) and gene silencing within condensed folds of the chromosome, except for a few “escape genes” in transcriptionally permissive pockets (Step 3). Matrix-associating protein SAF-A is required for proper localization of Xist to the inactivated X chromosome (Hasegawa et al., 2010; Sunwoo et al., 2017), yet XIST’s localization dependency on SAF-A may vary in different cellular contexts (Kolpa et al., 2016).

Fig 2

A) Key insights gained from molecular cytology on the nuclear function of HSATII lncRNA. Larger HSATII genomic locations (Chr1q12) accumulate PRC1 polycomb marks (BMI-1 and UbH2A) in cancer cells leading to an abnormal distribution of these nuclear proteins in cancer cells (Step 1). Smaller HSATII loci do not recruit polycomb marks and instead are transcribed, where HSATII lncRNA accumulates in cis and recruits MeCP2 proteins in cancer cells (Steps 2 and 3), leading to sequestration of key nuclear regulatory proteins. B) Summary of findings from cytological studies of NEAT1, XIST, and HSATII.

Fig 3

Potential pipeline for investigating lncRNAs of interest. Initial steps include determining protein interactomes, RNA structure, DNA binding sites and RNA-RNA interactions for lncRNAs of interest. This is likely to be an iterative process (1). While difficult for lncRNAs that either form densely packed RNPs or primarily associate with DNA and/or RNA, lncRNA knockdown is a valuable tool for determining the function of lncRNAs of interest (2). Identified proteins that bind a lncRNA of interest are then cross referenced against an established database of identified RBPs in the same cell line to either confirm that the proteins are known to bind RNA (3) or, if not, the protein may be a novel RBP with the capability to bind a specific lncRNA of interest. The RBDs and RBRs within RBPs should be analyzed for known lncRNA interactions and regions of RBPs containing RBDs and RBRs should be established for newly identified proteins (4). Previous databases of all RBPs should be expanded by using lncRNAs to pulldown and identify additional RBPs (5), and by application to additional cell lines (6). After establishing RNA structure, the transcripts bound by RBPs and their DNA binding sites should be determined (7). Both of these techniques have the potential to be applied to proteins that specifically bind lncRNAs of interest (8,9). Individual steps are shaded in colored boxes to indicate their overall classification (lncRNA-centric, RNP-centric, RBP-centric, Next Steps). Refer to Table 1 for a summary of individual techniques designed to achieve each step within colored boxes.