Friday, January 17, 2014

On the function of lincRNAs

There's plenty of evidence that most of the DNA in mammalian genomes is junk [Five Things You Should Know if You Want to Participate in the Junk DNA Debate]. There's also plenty of evidence that as much as 10% of these genomes are functional in some way or another. This is a lot more DNA than the amount in coding regions but that shouldn't surprise anyone since we've known about functional noncoding DNA for half a century.

Lot's of genes specify functional RNA molecules. The best known ones are the genes for ribosomal RNAs, tRNAs, the spliceosomal RNAs, and a variety of other catalytic RNAs. A host of small regulatory RNAs have been characterized in bacteria over the past five decades (Waters and Storz, 2009) and in the past few decades a variety of different types of small RNAs have been identified in eukaryotes (see Sharp, 2009). These include miRNAs, siRNAs, piRNAs, and others (Malone and Hannon, 2009; Carthew and Sontheimer, 2009).

Theme

Genomes
& Junk DNA
Some of the most interesting RNAs are the long intergenic noncoding or lincRNAs (Ponting et al. 2009). The average size of these longer RNAs is about 1500 bp and there are about 1600 conserved lincRNA genes in the mammalian genome (Guttman et al., 2009). This makes up only about 0.08% of the genome but these RNAs are very curious.

Mammalian genomes are pervasively transcribed. Most of the transcripts are present at less than one copy per cell and they are not very stable. Their sequences are not conserved. Thus, they have all the characteristics of spurious transcripts and are undoubtedly junk RNA (Struhl, 2007). That's not what we're talking about here although lots of people are confused.

What we're talking about is true functional RNAs transcribed from noncoding DNA. They've been around for a long time and many are well characterized. We want to understand what the others are doing.

One way to decide if the genes for these RNAs are actually doing something is to disrupt them by knocking them out and looking for an effect. That's what Savageau et al. (2013) did with 18 genes for mouse link RNAs. They found that five of the mutant strains of mice had severe developmental defects that were often lethal (Fendrr, Peril, and Mdgt: mice with a deleted Mdgt lincRNA gene are shown in the photo). Two other strains, linc-Brn1b, and linc-Pint had less severe developmental defects.

What this shows it that five out of eighteen lincRNA genes are very important in mouse development. Some of the others may also be functional but the phenotypes may not as obvious. We now know functions for several dozen lincRNA genes. It is still an open question whether there are over a thousand functional lincRNA genes in mammalian genomes or whether most of them are spurious transcripts. Keep in mind that even if every single one is functional, it makes no significant impact on the amount of junk DNA in the genome.

What I like about this paper (Savageau et al., 2013) is that the authors are aware of the controversy. Here's what they say in the Discussion ...
In the post genomic era, thousands of long noncoding RNAs have been discovered as transcribed units in mammalian genomes. However, what fraction of these new transcripts have general functional significance in vivo is debated. While several studies have indicated a role for lincRNAs in diverse biological processes (Ponting et al., 2009; Rinn and Chang, 2012; Mercer and Mattick, 2013; Ulitsky and Bartel, 2013), it has been suggested that most transcripts could represent nonfunctional transcriptional by-products (Struhl, 2007; Kowalczyk et al., 2012). Early critical studies of knockout strains (e.g., Xist and Tsix) did find lncRNAs implicated in X inactivation to be required for life. Yet, of the relatively few lncRNA mouse models derived since, many have displayed subtle defects or no phenotype (Ripoche et al., 1997; Gordon et al., 2010; Anguera et al., 2011; Nakagawa et al., 2011; Zhang et al., 2012).
It's actually quite rare for people working on these RNAs to admit that most of them might not have a function.

John Mattick is a fierce opponent of junk DNA. He's never met an RNA that he didn't think was functional and he seemed in the past to be completely unaware of the evidence for junk DNA. Mattick wrote a review of the Savageau et al. et al. paper because he's quite excited about the fact that some more lincRNAs seem to be functional. Unfortunately, his review gets off on the wrong foot by stating that functional noncoding RNAs were only discovered in the 1990s and by perpetuating his false story about the correlation between genome size and complexity. Here's the beginning of his article ...
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).
The good news is that Mattick seems more willing that usual to admit to the controversy about the function of these small RNAs.
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).
Mattick still thinks that most of the lincRNA genes are doing something important. He falls back on the fact that a large number of these genes are expressed in mammalian brain tissue and it may be difficult to detect a phenotype in mice if the cognitive defects are subtle.

Whatever.

The point is that scientists are beginning to find out what some of these lincRNAs are doing but that it has nothing to do with the junk DNA debate.


Carthew, R.W. and Sontheimer, E.J. (2009) Origins and mechanisms of miRNAs and siRNAs. Cell 136, 642-655. [doi: 10.1016/j.cell.2009.01.035]

Guttman, M., Amit, I., Garber, M., French, C., Lin, M.F., Feldser, D., Huarte, M., Zuk, O., Carey, B.W., Cassady, J.P. and Cabili, M.N. (2009) Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature 458, 223-227. [doi: 10.1038/nature0767]

Malone, C.D. and Hannon, G.J. (2009) Small RNAs as guardians of the genome. Cell 136, 656-668. [doi: 10.1016/j.cell.2009.01.045]

Mattick, J.S. (2013) Probing the phenomics of noncoding RNA. eLife 2.
[doi: 10.7554/eLife.01968]

Ponting, C.P., Oliver, P.L. and Reik, W. (2009) Evolution and functions of long noncoding RNAs. Cell 136, 629-641. [doi: 10.1016/j.cell.2009.02.006]

Sauvageau, M., Goff, L.A., Lodato, S., Bonev, B., Groff, A.F., Gerhardinger, C., Sanchez-Gomez, D.B., Hacisuleyman, E., Li, E. and Spence, M. (2013) Multiple knockout mouse models reveal lincRNAs are required for life and brain development. eLife 2. [doi: 10.7554/eLife.01749]

Sharp, P.A. (2009) The centrality of RNA. Cell 136, 577-580. [doi: 10.1016/j.cell.2009.02.007]

Struhl, K. (2007) Transcriptional noise and the fidelity of initiation by RNA polymerase II. Nature structural & molecular biology 14, 103-105. [doi: 10.1038/nsmb0207-103]

Waters, L.S. and Storz, G. (2009) Regulatory RNAs in bacteria. Cell 136, 615-628. [doi: 10.1016/j.cell.2009.01.043]

15 comments :

  1. Sorry, I got a bit confused there. Is 1600 the total number of lincRNAs in the mammalian genome, or is it the total number of conserved lincRNAs only? If it is the latter, then do we have an estimate for the overall number of lincRNAs in the genome?

    The study that you cited (Guttman 2009) seems to suggest that their technique found 1600 lincRNAs in total, 95% of which are highly conserved. Does that mean that most lincRNAs are functional?

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    1. lincRNA genes, that is. The number of transcripts is 17993

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    2. Thanks, Georgi. How many of those ~18,000 transcripts are actually conserved across mammalian genomes? Is there any official figures out there? Or is it a question that's still being debated?

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    3. Well, how do you define conservation for these things? They are often quite divergent

      This paper came out this week, it might be of interest to you:

      http://www.ncbi.nlm.nih.gov/pubmed/24429298

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  2. Larry, here you present lincRNAs just as long non-coding RNAs, but I read they're more specificly long *intergenic* non-coding RNAs.

    Is it perhaps thar most long RNAs happen to be lincRNAs and you are generalising?

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    1. Sorry, I forgot to include "intergenic" in my definition. I fixed it.

      However, quite a few putative lincRNA genes are located in introns and on the reverse strands of known genes so "intergenic" isn't really a very precise restriction.

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  3. I would like to understand about the junk DNA

    "Genetic Load
    Every newborn human baby has about 100 mutations not found in either parent. If most of our genome contained functional sequence information, then this would be an intolerable genetic load. Only a small percentage of our genome can contain important sequence information suggesting strongly that most of our genome is junk."

    The above suggests me that having these so called junk DNA's is actually very important for our survival. If it's so important (vital) why should we call it junk? Can you call junk something that is necessary?

    One of the strongest argument in favor of random evolution was the presence of the junk DNA. I assume then that our far ancestors had much less of these junk DNA.Doesn't the above argument about the intolarable genetic load apply to them also? Than why for our ancestors the genetic load was not only tolerated ,but they actually evolved.

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    1. You have that completely backwards.

      If all of our DNA was functional, the genome would be around 100Mb.

      The reason we have a 3Gb genome is that selection in our lineage has been too weak to get rid of the rest for hundreds of millions of years.

      The rest of the DNA is not there to protect the important parts of the genome from mutations, it's there because it can be.

      Also, it is very simplistic to look at this only in such terms. The mutation rate itself is not constant and depends on the population genetic environment. And once you have transposons in your genome, you develop mechanisms to suppress them, which makes them more tolerable, and so on...

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    2. LOL! Unknown seems to think that there is some external force causing every human born to have 100 mutations, regardless of the size of the genome, so junk DNA serves as a kind of decoy to attract these mutations to themselves and protect the functional DNA.

      No, Unknown. Mutations occur at a more or less constant rate. So if the genome was smaller, then the number of mutations occurring would be proportionally smaller as well. The difference would be, if the genome was reduced to the point that only functional DNA was present, then the tolerance for any mutations would be reduced such that they would be more likely deleterious or lethal, so it would be very unlikely to find someone walking around with 100 de novo mutations. However, the odds for any individual actually suffering a deleterious mutation would be unchanged, since that would depend on the odds for any particular gene suffering a mutation and this is independent of the size of the genome.

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  4. Thanks Georgi.

    I wonder what you and Larry think of this recently published study on the matter: The evolution of lncRNA repertoires and expression patterns in tetrapods. The study is behind a paywall (at least for me), but the press release has some pretty good details about what they did and what they discovered. I was particularly interested in the following:

    In the second phase of the research, a comparison among the different species allowed the scientists to pinpoint the emergence of these genes in the evolutionary history. While 11,000 long non-coding RNAs are shared by all primates, 2,500 go back to an ancestor common to man and mouse, about 90 million years ago. Only a hundred genes of this kind stem from an ancestor common to all eleven species considered, including birds and amphibians. "One of our main findings is that the activity of these non-coding genes is controlled by the same transcription factors that regulate protein-coding gene activity. Even more strikingly, we found that the 2,500 oldest long noncoding RNA genes are regulated by factors that are important for embryonic development. This suggests that, among the 2500 long non-coding RNAs conserved during the evolution of placental mammals, a large percentage may function specifically in embryonic development."

    So it seems that they traced the lncRNAs that they looked at across different lineages, and while some of them are shared by primates only, others (a smaller number) are present in other mammals, and a smaller number still are present in birds and amphibians.

    But I'm a bit skeptical about their conclusions regarding the functional roles of these transcripts. As Larry said many times, the mere existence of TF binding sites near a transcript doesn't mean that it is performing an essential function in the cell.

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    1. The press releases is horrible, as press releases usually are.

      The paper has some interesting results and some subtle things that make my head hurt. But that's how it usually is.

      P.S. Pay attention to the testes results, in this one and in other papers

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    3. Regarding function:

      We have known of instances of critically important lincRNAs for a very long time, long before people did genome-wide scans for them and found thousands of candidates. Xist was discovered in 1991 or something, and it had grown to a few dozens of examples before the late 00s when it exploded.

      There is no question that there are functional lincRNAs. There is also little doubt that there are a lot of them. But "a lot" means a few hundreds to the low thousands. The most one can find is ~10,000 candidates, and that is hardly enough to overturn our understanding of how the genome is structured, how it evolved, and how it works. And I highly doubt all of these candidates will turn out to be important - the high evolutionary turnover suggest otherwise.

      BTW, Xist is very informative for its evolutionary history - it used to be a protein coding gene that got pseudogenized and then turned into a lincRNA. There is probably a lot of this kind of thing going on - you begin with lincRNAs arising from intergenic space by the acquisition of the capacity to be transcribed, then they either disappear (this is probably what happens most often) or get fixed as lincRNAs. And on some occasions they might even evolve into new protein coding genes. The inverse also happens (as illustrated by Xist) - protein coding genes become inactivated, then turn into lincRNAs, which then can evolve in other directions, and so on.

      That you see so many of these in testes is very telling. We never published that paper, but in our analysis of ENCODE and HBM data, we saw 3-4 times as many lincRNAs expressed in testes than in any other tissue or cell line. This is what everyone else has observed too. Transcriptional regulation in testis is known to be messed up in all kinds of ways (though very poorly understood) and chromatin there is very permissible to transcription. So that makes a lot of intuitive sense

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