Genetics: Probing the phenomics of noncoding RNA
- Garvan Institute of Medical Research, Australia
It has been known since the late 1970s that many DNA sequences are transcribed but not translated. Moreover, most protein-coding genes in mammals are fragmented, with only a small fraction of the primary RNA transcript being spliced together to form messenger RNA. For many years it was assumed that untranslated RNA molecules served no useful purpose but, starting in the mid-1990s, a small body of researchers, including the present author (Mattick, 1994), have been arguing that these RNAs transmit regulatory information, possibly associated with the emergence of multicellular organisms. This is supported by the observation that the proportion of noncoding genomic sequences broadly correlates with developmental complexity, reaching over 98% in mammals (Liu et al., 2013), although others have argued that the increase in genome size is due to the inefficiency of selection against non-functional elements as body size goes up and population size goes down (Lynch, 2007).
High-throughput sequencing analyses over the past decade have shown that the majority of mammalian genome is transcribed, often from both strands, and have revealed an extraordinarily complex landscape of overlapping and interlacing sense and antisense, alternatively spliced, protein-coding and non-protein-coding RNAs, the latter generally referred to as long noncoding RNAs (lncRNAs). Moreover, the repertoire of these lncRNAs is different in different cells (Carninci et al., 2005; Cheng et al., 2005; Birney et al., 2007; Mercer et al., 2012). While some transcripts may encode previously unrecognized small proteins, the function or otherwise of the vast majority of lncRNAs remains to be determined.
Because many lncRNAs appear to be expressed at low levels, and many have lower sequence conservation than messenger RNAs, one interpretation has been that these RNAs represent transcriptional noise from complex genomes cluttered with evolutionary debris. However, assessments of sequence conservation rely on assumptions about the non-functionality and representative distribution of reference sequences, which are not verified and cannot be directly tested (Pheasant and Mattick, 2007). Nonetheless, many lncRNAs show patches of relative sequence conservation (Derrien et al., 2012), and even more do so at the secondary structural level (Smith et al., 2013).
Expression analyses have shown that lncRNAs originate from all over the genome and are expressed at different times during differentiation and development (Dinger et al., 2008), often exhibiting highly cell-specific patterns (Mercer et al., 2008). The precision of lncRNA expression is consistent with evidence suggesting that many are associated with chromatin-modifying complexes, thereby acting as regulators of the epigenetic control of differentiation and development (Mercer and Mattick, 2013).
A number of lncRNAs have also been linked to complex diseases like cancer (Mattick, 2009) and other complex physiological processes (see, for example, Rapicavoli et al., 2013). However, these results seem at odds with the fact that few lncRNAs have been identified in traditional genetic screens. The reason for this is likely a combination of phenotypic, technical and expectational bias: mutations in protein-coding regions of the genome generally have phenotypes that are more severe, and are easier to identify, than those in non-coding regions. By contrast, in this context, it is worth noting that ∼95% of all variants associated with complex (as opposed to monogenic) diseases in humans map to non-coding, presumably regulatory, sequences (Freedman et al., 2011).
Still, the gold standard in this field is the targeted in vivo silencing or deletion of specific genes, and since few of these have been conducted to date, some researchers have remained sceptical about the biological significance of lncRNAs. Now, in eLife, John Rinn, Paolo Arlotta and co-workers at Harvard, MIT, the Broad Institute, Rutgers and Regeneron Pharmaceuticals—including Martin Sauvageau, Loyal Goff and Simona Lodata as joint first authors—report the results of the first large-scale attack on the question (Sauvageau et al., 2013). They selected 18 lncRNA genes in the mouse genome that had been stringently assessed for lack of protein-coding capacity and that did not overlap with known protein-coding genes or other known gene annotations—hence the name long intergenic noncoding RNAs (lincRNAs)—and generated knockout mouse mutants by replacing the lncRNA gene with a lacZ reporter cassette.
Sauvageau, Goff, Lodata et al. report discernable developmental problems in five of the 18 mutants, with three exhibiting embryonic or post-natal lethality, two of which exhibited growth defects in the survivors. The phenotypes of two of the mutants were analyzed in detail: one of the mutants that died showed defects in multiple organs (including the lung, heart and gastrointestinal tract), and one of the mutants that survived with growth defects also showed defects in the cerebral cortex. Other mutants that did not exhibit overt developmental defects showed brain-specific expression patterns and may be associated with cognitive defects that are not grossly apparent at the developmental level.
Another group (Grote et al., 2013) recently generated a different knockout allele for one of the 18 lincRNAs interrogated by Sauvageau et al., and also reported an embryonic lethal phenotype, albeit with some differences. Importantly, the approach used by Grote et al. also provided strong evidence that the mutant defects were not caused by an indirect effect on an overlapping genomic element, such as an enhancer for a nearby gene.
The work of Sauvageau, Goff, Lodata et al. is a mini tour-de-force that shows that there are lncRNAs with important developmental functions in vivo, and it joins a small number of studies from other pioneering groups that show the same thing (Lewejohann et al., 2004; Gutschner et al., 2013; Li et al., 2013), although not all of the targeted lncRNAs showed a phenotype. Similarly, other knockout experiments of widely expressed lncRNAs, as well as some of the most highly conserved elements in the mammalian genome, also did not yield discernable phenotypes (Ahituv et al., 2007; Nakagawa et al., 2011), which should sound a note of caution about the interpretation of negative results.
Indeed, since most lncRNAs are expressed in the brain (Mercer et al., 2008) and many are primate-specific (Derrien et al., 2012), it may be that much of the lncRNA-mediated genetic information in humans (and in mammals generally) is devoted to brain function, and therefore not easily detectable in developmental, as opposed to cognitive, screens. A good example is a noncoding RNA called BC1 that is widely expressed in the brain: knockout of BC1 causes no visible anatomical consequences, but it leads to a behavioural phenotype that would be lethal in the wild (Lewejohann et al., 2004).
Although evidence for the hypothesis that lncRNAs have a role in mammalian development, brain function and physiology is growing, there is also a clear need for more sophisticated and comprehensive phenotypic screens, especially with respect to cognitive function.
References
-
Principles for the post-GWAS functional characterization of cancer risk lociNature Genetics 43:513–518.https://doi.org/10.1038/ng.840
-
Role of a neuronal small non-messenger RNA: behavioural alterations in BC1 RNA-deleted miceBehavioural Brain Research 154:273–289.https://doi.org/10.1016/j.bbr.2004.02.015
-
Introns: evolution and functionCurrent Opinion in Genetics & Development 4:823–831.https://doi.org/10.1016/0959-437X(94)90066-3
-
The genetic signatures of noncoding RNAsPLOS Genetics 5:e1000459.https://doi.org/10.1371/journal.pgen.1000459
-
Specific expression of long noncoding RNAs in the mouse brainProceedings of the National Academy of Sciences of the United States of America 105:716–721.https://doi.org/10.1073/pnas.0706729105
-
Targeted RNA sequencing reveals the deep complexity of the human transcriptomeNature Biotechnology 30:99–104.https://doi.org/10.1038/nbt.2024
-
Structure and function of long noncoding RNAs in epigenetic regulationNature Structural and Molecular Biology 20:300–307.https://doi.org/10.1038/nsmb.2480
-
Paraspeckles are subpopulation-specific nuclear bodies that are not essential in miceJournal of Cell Biology 193:31–39.https://doi.org/10.1083/jcb.201011110
-
Raising the estimate of functional human sequencesGenome Research 17:1245–1253.https://doi.org/10.1101/gr.6406307
-
Widespread purifying selection on RNA structure in mammalsNucleic Acids Research 41:8220–8236.https://doi.org/10.1093/nar/gkt596
Article and author information
Author details
Publication history
Copyright
© 2013, Mattick
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 1,131
- views
-
- 136
- downloads
-
- 13
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Genetics: Probing the phenomics of noncoding RNA
eLife 2:e01968.
https://doi.org/10.7554/eLife.01968
Further reading
-
- Cell Biology
- Developmental Biology
In most murine species, spermatozoa exhibit a falciform apical hook at the head end. The function of the sperm hook is not yet clearly understood. In this study, we investigate the role of the sperm hook in the migration of spermatozoa through the female reproductive tract in Mus musculus (C57BL/6), using a deep tissue imaging custom-built two-photon microscope. Through live reproductive tract imaging, we found evidence indicating that the sperm hook aids in the attachment of spermatozoa to the epithelium and facilitates interactions between spermatozoa and the epithelium during migration in the uterus and oviduct. We also observed synchronized sperm beating, which resulted from the spontaneous unidirectional rearrangement of spermatozoa in the uterus. Based on live imaging of spermatozoa-epithelium interaction dynamics, we propose that the sperm hook plays a crucial role in successful migration through the female reproductive tract by providing anchor-like mechanical support and facilitating interactions between spermatozoa and the female reproductive tract in the house mouse.
-
- Developmental Biology
The morphogen FGF8 establishes graded positional cues imparting regional cellular responses via modulation of early target genes. The roles of FGF signaling and its effector genes remain poorly characterized in human experimental models mimicking early fetal telencephalic development. We used hiPSC-derived cerebral organoids as an in vitro platform to investigate the effect of FGF8 signaling on neural identity and differentiation. We found that FGF8 treatment increases cellular heterogeneity, leading to distinct telencephalic and mesencephalic-like domains that co-develop in multi-regional organoids. Within telencephalic regions, FGF8 affects the anteroposterior and dorsoventral identity of neural progenitors and the balance between GABAergic and glutamatergic neurons, thus impacting spontaneous neuronal network activity. Moreover, FGF8 efficiently modulates key regulators responsible for several human neurodevelopmental disorders. Overall, our results show that FGF8 signaling is directly involved in both regional patterning and cellular diversity in human cerebral organoids and in modulating genes associated with normal and pathological neural development.
