Complex transcription interwoven between and within protein-coding genes produces many thousands of long non-coding RNAs (lncRNAs) that are greater than 200 nucleotides (nt) in length but that appear to lack protein-coding potential (Djebali et al., 2012). Nevertheless, even for the earliest discovered lncRNAs, such as mammalian H19, Xist or fruitfly roX, molecular effects and functional significance have proven difficult to establish (Gabory et al., 2010; Ilik et al., 2013; Sado and Brockdorff, 2013). Furthermore, no or only subtle mouse phenotypes were revealed by detailed loss-of-function studies of Malat1 or Evf-2. In contrast, mutation of Fendrr results in early lethality, and targeted replacement of BC1 results in seizures for some mice (Table 1). It is not possible to accurately predict from the level or extent of its expression, or its sequence composition, whether disruption of a lncRNA locus will result in an overt phenotype. This makes loss- or gain-of-function experiments crucial to understanding the roles of lncRNAs in vivo.

Many lncRNAs are known to act as primary host transcripts for classes of small non-coding RNAs (da Rocha et al., 2008; Royo and Cavaille, 2008). However, lncRNAs are also presumed to regulate the expression either of their neighbouring genes in cis, or of more distant genes in trans (Figure 1). The function of a lncRNA may be mediated by the gene's RNA product which can bind to proteins or to other nucleic acids thereby modulating their functions. This could act by competing with endogenous mRNAs for miRNA binding (Franco-Zorrilla et al., 2007; Poliseno et al., 2010; Jeck and Sharpless, 2014), providing binding sites for small RNAs that elicit transcriptional silencing (Wierzbicki et al., 2009), or through altering protein activity (Feng et al., 2006), binding or specificity (reviewed in Guttman and Rinn, 2012). Alternatively, the act of transcription per se through a lncRNA locus could be critical because of the changes this generates in chromatin structure, modification or protein binding: in this case the resultant RNA could be an incidental by-product (Petruk et al., 2006; Latos et al., 2012; Marquardt et al., 2014). In these latter cases, any technique intended to dissect mechanism must alter the act and extent of transcription rather than change RNA levels. This multiplicity of lncRNA functional mechanisms means that a toolkit of experimental strategies to dissect their modes of action will need to be added to those currently employed for investigating protein-coding genes. Protein-coding genes have been shown to contribute greatly to biological function, which is not yet the case for lncRNA loci, rendering their rigorous investigation particularly important.

Figure 1

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Analysis of lncRNA localisation both on a tissue and subcellular level by techniques such as fluorescent in situ hybridisation (FISH, Chakraborty et al., 2012) can give important insights into the cell types that are important for their function, and in which subcellular compartment they act. Understanding the mechanism of action of lncRNAs often relies on identification of interacting proteins or nucleic acids by RNA-protein (e.g., crosslinking immunoprecipitation, CLIP, Huppertz et al., 2014), RNA–RNA (e.g., crosslinking analysis of synthetic hybrids, CLASH, Helwak et al., 2013) or RNA-DNA (e.g., CHART, Simon et al., 2011; Vance and Ponting, 2014 and ChIRP, Chu et al., 2011) interaction assays. However, due to the nature of the RNA molecule, many assays are prone to non-specific binding, and it is critical to ensure that appropriate controls are performed (Brockdorff, 2013). Several of these techniques have therefore been designed to identify direct interactors by crosslinking, and subsequent use of denaturing conditions to remove non-specific interactions. These techniques are clearly important in determining the mechanism of action of lncRNAs, and are critical to guide experimental genetic knockout design. However, an understanding of the functional importance of lncRNAs in the context of the whole organism still relies on manipulating their expression by genetic modification, overexpression or knockdown strategies, and analysis of the resulting phenotypes.

The earliest studied lncRNAs were those associated with imprinting, such as Airn and H19, or X chromosome regulation, such as Xist or roX1/2 (Table 1). In these cases, lncRNA expression was initially linked genetically to a known phenotype, and cell line models accurately reflected the in vivo models (Hao et al., 1993; Keniry et al., 2012; Latos et al., 2012; Helwak et al., 2013; Huppertz et al., 2014). These results highlight that the early models of lncRNAs involved in imprinting and X chromosomal dosage compensation could act as paradigms for the study of lncRNAs today (Kohtz, 2014). In the absence of a priori phenotypic associations, some lncRNAs have been chosen for study on the basis of their tissue restricted patterns of expression, sequence conservation, or cellular localisation. Others, such as MALAT1 (whose level of expression is associated with metastasis) have been selected on the basis of their suggested association with disease. Neat1 and Malat1 (also known as Neat2) are linked loci that produce highly expressed lncRNAs whose sequences are well conserved across diverse mammals and which have specific nuclear localisations (Chu et al., 2011). In cells, Neat1 was shown to be essential for nuclear paraspeckle assembly and maintenance (Clemson et al., 2009; Sasaki and Hirose, 2009; Sunwoo et al., 2009; Mao et al., 2011a, 2011b; Zhang et al., 2012) and Malat1/Neat2 binds to the Polycomb 2 (PC2) protein which is required for activating growth-control genes (Vance and Ponting, 2014). Nevertheless, in vivo disruption of either of these lncRNA loci results in viable and fertile mouse models (Table 1).

Confirmation or rejection of lncRNA functionality requires experimental evidence that clearly separates the role of the genomic locus from the role of its RNA products. Here we recommend experimental techniques that achieve this separation whilst minimising disruption of the DNA sequence. Furthermore, we propose some considerations that may assist in interpreting phenotypes arising from mutation of a lncRNA or lncRNA locus (Box 1).

In vivo, loss-of-function strategies

Different genetic loss-of-function strategies can be employed in vivo to study the function of lncRNAs (Figure 2). Prioritisation of strategy should depend on the lncRNA's known biology, including its localisation to one or more of the cytoplasm, nucleus or chromatin. In one study, the majority of human lncRNAs were enriched in the cytoplasm (van Heesch et al., 2014) and these may associate with ribosomes and, contrary to expectations, some may be translated (Guttman et al., 2013; Kim et al., 2014; Wilhelm et al., 2014). Nuclear lncRNAs, particularly those that are chromatin-associated, could act as cis-acting transcriptional regulators, whereas cytoplasmic or nucleoplasmic lncRNAs might be predicted to function in trans; by contrast, some nucleoplasmic lncRNAs may of course be non-functional products of transcription.

Figure 2

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Depletion of protein-coding transcripts is often achieved using RNAi-based techniques, which supply double-stranded RNA that is able to trigger post-transcriptional destabilisation of the mature mRNA and inhibit translation, predominantly in the cytoplasm. Although the presence of active RNAi factors in human cell nuclei has been proposed (Gagnon et al., 2014) the extent to which exclusively nuclear lncRNAs can be knocked down remains unclear. Whilst useful for studies of many trans-acting lncRNAs, RNAi-based knockdown acts post-transcriptionally, and therefore does not block the act of transcription, precluding analyses of lncRNAs which may produce their effects via this mechanism.

Another experimental approach is to genetically manipulate the lncRNA locus. When inserting transcriptional terminator sequences care must be taken to control for changes in spacing between DNA regulatory elements and to take account of regulatory elements that may be inadvertently inserted, such as promoters of resistance genes, since these may be able to drive expression of neighbouring genes or divert activities from nearby enhancers. Insertion of exogenous sequences can induce phenotypes (Steshina et al., 2006). Even single loxP sites can attract germline methylation that might potentially repress flanking regulatory elements (Rassoulzadegan et al., 2002). Extra controls are thus needed to identify possible gain-of-function effects arising from inserted sequences, such as reporters or selection cassettes. The advent of programmable nucleases (Kim and Kim, 2014) provides opportunities to investigate these possibilities. Transcriptional terminator sequences can vary in their efficacy depending on the genomic context into which they are inserted, which can cause termination to be highly inefficient. For example, a sequence that efficiently terminates transcription in multiple contexts in Airn, failed to do so when inserted close to a CpG island (Latos et al., 2012).

Other approaches include deletion of the full-length lncRNA locus or its promoter sequence, mutation of putative functional domains or targeted interruption between the promoter and the RNA sequence through an engineered inversion (Figure 2; Table 1). Whilst useful, such strategies may not always be successful. Promoter inversion, for instance, may not always abrogate transcription, because of the bidirectionality of promoters (Wu and Sharp, 2013), and promoter deletion may also disrupt the expression level of protein-coding transcripts with which lncRNAs share a bidirectional promoter. In all of these cases, it is important to minimise the removal or reorganisation of regulatory factor binding sites or other regulatory elements within the DNA, and to control for the addition of novel binding sites. For example, it should be borne in mind that many lncRNAs initiate within enhancers (Marques et al., 2013) and in these cases disruption of the lncRNA promoter could also cause unintended changes in gene expression. In the case of transcription terminators, to ensure effects are due to changes in RNA rather than DNA, inversions of the terminator sequence or a variety of different terminators can be used. In the experimental design it is also important to consider alternatively spliced transcripts and additional transcriptional start sites to ensure full abrogation of lncRNA expression.

Antisense oligonucleotides might provide an alternative technique for analysis of lncRNA function. They are thought to act by forming a DNA/RNA hybrid with the nascent RNA transcript, and triggering RNase H-dependent degradation of the RNA in the nucleus (Figure 2). This reduces the level of the RNA before the mature transcript is produced, but the nature and extent of off-target effects are not fully understood and may be substantial (Sahu et al., 2007). Also, it is not possible to generate stable transgenic lines, which restricts analysis to cell lines or to systems where the oligonucleotides can be supplied by injection. Other approaches to disrupting lncRNA function use morpholino antisense oligos targeting e.g. splice sites (Ulitsky et al., 2011), or locked nucleic acid antisense oligonucleotides (Sarma et al., 2010).

Recent developments in rational design of DNA binding factors using transcription activator-like effector (TALE) proteins or the clustered regularly interspersed palindromic repeats (CRISPR) system have enabled recruitment of transcriptional activation (Cheng et al., 2013) or repression domains (Cong et al., 2012; Gilbert et al., 2013) to defined sites within the genome to modulate transcription, or to directly interfere with the passage of the RNA polymerase. These techniques could be used to modulate the rate of transcriptional initiation or elongation of the lncRNA (Figure 2), but care must be taken to control for direct effects of these factors on the transcription of neighbouring genes.

Separating RNA- from DNA-sequence dependent effects

Deletion of a lncRNA genomic locus does not cleanly separate a role of the lncRNA per se from a role of other functional elements contained within the underlying DNA. Such elements might be irrelevant to the lncRNA's function, yet critical to the normal function of a neighbouring protein-coding gene. Eighteen mouse knockout lines were recently described in which genomic regions containing intergenic lncRNA loci (21.6 kb mean size, 4.8 kb–49.7 kb range) were deleted and replaced by a lacZ reporter cassette (Sauvageau et al., 2013). For 13 of these lines no overt phenotypes were reported. In contrast, strong phenotypes from 5 knockout lines were observed: Peril^−/− or Fendrr^−/− mice have reduced viability; Mdgt^−/− and linc-Pint^−/− mice show growth defects; and linc-Brn1b^−/− mice exhibit abnormal cortical anatomy. The authors conclude that these developmental disorders generated by DNA deletions demonstrate the critical roles that lncRNAs play in vivo (Sauvageau et al., 2013).

While this may be the correct interpretation, the strong phenotypes observed in these lines may derive from the engineered deletion of cis-regulatory DNA elements lying within these large DNA deletions that are critical for the normal functions of proximal protein-coding genes. For instance Fendrr is 1.4 kb from Foxf1, and Mdgt starts only 84 bp from the 5′ exon of Hoxd1 and terminates close to Hoxd3 (Figure 3). Consistent with this notion, data from the ENCODE project indicate that the genomic region deleted in Mdgt^−/− lines contains binding sites for several transcription factors and chromatin regulatory proteins (Figure 3). Whilst the authors detected no global change of neighbouring protein-coding gene expression as assessed by limited RNAseq of tissues, it is still possible that altered cell type or developmental stage specific expression of these genes escaped detection. LncRNAs are often transcribed in a highly restricted cell population and a global, high-throughput analysis of even the full embryo may not have been informative. Ultimately, the best evidence for RNA-dependent lncRNA function derives from loss-of-function, followed by complementation approaches, as for example described in Grote et al. (2013).

Figure 3

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This issue is also relevant for other lncRNAs transcribed from within Hox gene clusters. In the case of Hotair (Rinn et al., 2007), a several kb large deletion of the entire Hotair genomic DNA in vivo induces a subtle morphological phenotype in the spine, which was interpreted as a gain-of-function of Hoxd genes in trans (Li et al., 2013). However, Hotair is embedded in the HoxC gene cluster and topological modifications or re-arrangements in such a dense series of transcription units are likely to modify the expression of neighbouring genes. Further insights have been acquired by removing the entire HoxC locus, including both the lncRNA locus and flanking genes (Suemori and Noguchi, 2000; Schorderet and Duboule, 2011). Even when multiple alleles are available, as for Hotair, lncRNA function remains difficult to evaluate.

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Thank you for sending your work entitled “Considerations when investigating lncRNA function in vivo” for consideration at eLife. Your article has been favorably peer reviewed by Detlef Weigel and two outside reviewers.

There has been considerable recent excitement about the role of long noncoding RNAs (lncRNAs) in gene regulation. A major difficulty in studying lncRNA function genetically is that they can overlap with other elements of the genome that have proven (in the case of antisense RNA) or potential separate functions (such as insulators, enhancers, etc). Therefore, simple knockout experiments, using either insertion of large foreign sequences or sequence deletions may produce misleading results.

The article provides several examples from the literature that clearly illustrate how studies of the same lncRNA locus can come to different conclusions. The article is timely given the apparent lack of defined “gold standards” for this kind of work. A variety of approaches for the functional analysis of lncRNA loci is discussed, highlighting the pitfalls that can be encountered when trying to distinguish the contributions of genomic DNA from the act of transcription or the RNA product itself to the observed phenotypes. The difficulties associated with distinguishing cis versus trans effects of lncRNAs are discussed and rigorous and standardized approaches to evaluating lncRNA function in vivo are proposed. The article will thus serve as an important guide to those performing genetic analyses of lncRNA loci.

The discussion is overall very balanced and the reviewers had only a few suggestions:

1) Include either at the beginning or at the end a series of bullet points, with an introductory sentence that lncRNA functional evidence should include at least x out of y of these types of evidence.

2) Mention the need for appropriate controls, when genetic manipulations, e.g., in ES or similar cells, are followed by selection of clones derived from single cells. ES and other cultured cells (e.g., derived from tumors) are genetically not perfectly stable, and one needs to ensure that phenotypes are not due to second-site mutations. Multiple clones or transgenic rescue experiments should be the norm (obviously this applies not only to lncRNA analyses).

3) It would be best to distinguish true “loss of function”, in which transcription is completely abolished, from “reduction of function”, where a truncated transcript is produced or RNAi is used. To this end, Figure 2 should be amended.

4) A brief paragraph that mentions flanking cell biological (e.g., FISH) and biochemical (e.g., RNP analysis) approaches for evaluating lncRNA function would be worthwhile.

1) Include either at the beginning or at the end a series of bullet points, with an introductory sentence that lncRNA functional evidence should include at least x out of y of these types of evidence.

The intention of Box 1 “Considerations when interpreting phenotypes resulting from lncRNA mutation” was to provide such suggestions. However, we believe that each lncRNA has to be considered on its own merits, depending on its mechanism of action and it is therefore not possible to define a set of general guidelines that would be applicable to all cases. We have therefore deliberately given a set of “considerations” rather than “guidelines”.

2) Mention the need for appropriate controls, when genetic manipulations, e.g., in ES or similar cells, are followed by selection of clones derived from single cells. ES and other cultured cells (e.g., derived from tumors) are genetically not perfectly stable, and one needs to ensure that phenotypes are not due to second-site mutations. Multiple clones or transgenic rescue experiments should be the norm (obviously this applies not only to lncRNA analyses).

We agree with the reviewers that this is an important point, since genetic background will have a large effect, especially when the phenotypes are subtle, and that this can vary substantially between different clones or lines. We have added this concept to the text.

3) It would be best to distinguish true “loss of function”, in which transcription is completely abolished, from “reduction of function”, where a truncated transcript is produced or RNAi is used. To this end, Figure 2 should be amended.

We have amended Figure 2 and the text to make this clear.

4) A brief paragraph that mentions flanking cell biological (e.g., FISH) and biochemical (e.g., RNP analysis) approaches for evaluating lncRNA function would be worthwhile.

We have added a paragraph describing some of the complimentary techniques that can be used to evaluate lncRNA mechanism, and how these may be used to guide mutant design.

Author details

1. Andrew R Bassett

   MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom

   Present address

   Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom

   Contribution

   ARB, Conception and design, Drafting or revising the article

   For correspondence

   andrew.bassett@path.ox.ac.uk

   Competing interests

   No competing interests declared.

2. Asifa Akhtar

   Department of Chromatin Regulation, Max-Planck-Institut für Immunbiologie und Epigenetik, Freiburg im Breisgau, Germany

   Contribution

   AA, Drafting or revising the article

   Competing interests

   AA: Reviewing Editor, eLife.

3. Denise P Barlow

   CeMM, Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria

   Contribution

   DPB, Drafting or revising the article

   Competing interests

   No competing interests declared.

4. Adrian P Bird

   Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom

   Contribution

   APB, Drafting or revising the article

   Competing interests

   No competing interests declared.

5. Neil Brockdorff

   Department of Biochemistry, University of Oxford, Oxford, United Kingdom

   Contribution

   NB, Drafting or revising the article

   Competing interests

   No competing interests declared.

6. Denis Duboule

   School of Life Sciences, Ecole Polytechnique Fédérale Lausanne, Lausanne, Switzerland

   Contribution

   DD, Drafting or revising the article

   Competing interests

   No competing interests declared.

7. Anne Ephrussi

   Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany

   Contribution

   AE, Drafting or revising the article

   Competing interests

   No competing interests declared.

   "This ORCID iD identifies the author of this article:" 0000-0002-5061-4620

8. Anne C Ferguson-Smith

   Department of Genetics, University of Cambridge, Cambridge, United Kingdom

   Contribution

   ACF-S, Drafting or revising the article

   Competing interests

   ACF-S: Reviewing Editor, eLife.

9. Thomas R Gingeras

   Functional Genomics Group, Cold Spring Harbor Laboratory, Cold Spring Harbor, United States

   Contribution

   TRG, Drafting or revising the article

   Competing interests

   TRG: Reviewing Editor, eLife.

10. Wilfried Haerty

    MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom

    Contribution

    WH, Drafting or revising the article

    Competing interests

    No competing interests declared.

11. Douglas R Higgs

    MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford, United Kingdom

    Contribution

    DRH, Drafting or revising the article

    Competing interests

    No competing interests declared.

12. Eric A Miska

    Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, United Kingdom

    Contribution

    EAM, Drafting or revising the article

    Competing interests

    No competing interests declared.

13. Chris P Ponting

    MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom

    Contribution

    CPP, Conception and design, Drafting or revising the article

    For correspondence

    chris.ponting@dpag.ox.ac.uk

    Competing interests

    AA: Reviewing Editor, eLife.

    "This ORCID iD identifies the author of this article:" 0000-0003-0202-7816

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