Thursday, October 01, 2015

How many RNA molecules per cell are needed for function?

One of the issues in the junk DNA wars is the importance of all those RNAs that are detected in sensitive assays. About 90% of the human genome is complementary to RNAs that are made at some time in some tissue or other. Does this pervasive transcription mean that most of the genome is functional or are most of these transcripts just background noise due to accidental transcription?

Opponents of junk DNA (e.g. John Mattick (Mattick and Dinger, 2013)) claim that many of these transcripts are regulatory RNAs that help create an intricate and complicated network of regulation in complex eukaryotes. They point out that many of the transcripts are only produced in certain cell types. According to them, this cell-specific transcription is an indication of function. In addition, many of these RNAs are not conserved in other species. These human-specific regulatory RNAs could be what makes humans different from other animals [see The Deflated Ego Problem].

Proponents of junk DNA (e.g. Pallazo and Gregory, 2014) claim that most of these transcripts are present in very low concentrations—usually less than one copy per cell. That suggests noise. They remind their opponents that noise is the result of non-specific binding of various transcription factors and some of these factors are only made in certain cells. Thus, you expect transcription noise to show specificity—it does not indicate function. Junk DNA proponents argue that the lack of conservation is more likely to be evidence of non-function.

How important is the concentration of RNA inside the cell? If it's a regulatory RNA then it has to bind to something and that usually means that there have to be lots of molecules.

Fei et al. (2015) looked at a model system in E. coli where a small regulatory RNA, SgrS, binds to several mRNAs and targets them for degradation.1 They looked at the binding interaction by estimating the association constant (Ka), or the dissociation constant (Kd). They also looked at the kinetics of the interaction by estimating the on-rate (kON) and the off-rate (kOFF).

Those of you who remember your introductory biochemistry class will recall that

                                                Ka = kON/kOFF

An equilibrium is established when the on-rate and the off-rate are equal and this equilibrium is determined by the strength of the biding. If you have strong binding then more of the RNA molecules are bound and fewer are free when the equilibrium is established.

When you have a situation where a regulatory RNA has to bind to a small number of mRNAs in a cell, the binding and kinetic parameters determine the concentration of the regulatory RNA required to get significant regulation. The values of the constants they determined are quite reasonable so it serves as a good standard to judge the concentrations needed for regulation. Fei et al. concluded that in E. coli cells you need about 1000 molecules of the regulatory RNA per cell in order to regulate the mRNAs.

This number has to be larger in eukaryotic cells because the cells are bigger and there are more mRNA molecules dispersed in the cytoplasm. The results strongly suggest that most of the transcripts detected are present at too low a concentration to be effective in regulation by binding to mRNA.

There are other ways that these RNAs could be involved in regulation. For example, they could bind directly to the DNA from which they are transcribed. Or, they could bind in the nucleus, That doesn't help very much because the nucleus is also quite large relative to the targets.

Maybe the "regulatory RNAs" don't bind to anything and it's just the fact that transcription occurs at a particluar locus that causes regulation. Maybe the regulatory RNAs bind chromatin to help form three-dimensional structures in which case the strength of binding might be very high, the target numbers are small (chromosome) and only a few RNAs are needed.

There are always imaginative ways of explaining regulation by small RNAs but there is a concentration problem if they are going to bind to other RNAs or proteins. Very few of the presumptive regulatory RNAs have a high enough concentration in cells to do anything significant. They are probably junk RNA or noise.

Keep this in mind the next time you hear anyone claiming that eukaryotic geneoms are full of genes for regulatory RNAs. How, exactly, do those regulatory RNAs work if there's only a few copies per cell? We know enough about binding constants and the kinetics of binding to rule out most adaptive just-so stories.


1. Small regulatory RNAs were discovered in bacteria in the 1970s.

Fei, J., Singh, D., Zhang, Q., Park, S., Balasubramanian, D., Golding, I., Vanderpool, C. K., and Ha, T. (2015) Determination of in vivo target search kinetics of regulatory noncoding RNA. Science, 347:1371-1374. [doi: 10.1126/science.1258849]

Mattick, J.S., and Dinger, M.E. (2013) The extent of functionality in the human genome. The HUGO Journal, 7:2. [doi: 10.1186/1877-6566-7-2]

Palazzo, A. F., and Gregory, T. R. (2014) The Case for Junk DNA. PLoS Genetics, 10(5), e1004351. [doi: 10.1371/journal.pgen.1004351]

40 comments :

  1. Prof. Moran, the link to the "The Deflated Ego Problem" post is broken

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    1. Hmm, a failed URL polyploidy? I think the correct links would be either

      http://sandwalk.blogspot.ca/2007/05/deflated-ego-problem.html

      or

      http://sandwalk.blogspot.com/2007/05/deflated-ego-problem.html

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  2. Great post. Do Fei et al. specifically make the point that this goes against ENCODE-type claims about millions of functional RNA's?

    If so, whom do they cite when they're shooting down the idea?

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  3. I don't think the mode of action studied in Fei et al. has much relevance to the ways that microRNAs and other small RNAs work.

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    1. Why not? Mature miRNA has to bind to its target transcript by complementary base pairing, just like the sRNA discussed in the paper.

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    2. MicroRNAs associate with their targets as parts of RNP complexes, not as free RNAs. So RNA-RNA pairing, free from associated protein partners, would not seem to be particularly relevant to the matter of miRNA regulation.

      Also, Larry, your statement that "(t)he values of the constants they determined are quite reasonable" is a bit puzzling. Fe et al. determine Kd's in the micro molar range. This seems to me to be rather high for a regulatory interaction - the implied affinity is pretty feeble. What is the range of affinities that are typically associated with regulatory RNP-nucleic acid interactions in eukaryotes? Does it really tend to the micromolar range?

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  4. Here is the link to the ENCORE paper. At the bottom of the paper is the gene group by gene group analysis in detail.
    Nature. 2012 Sep 6; 489(7414): 57–74.
    doi: 10.1038/nature11247
    PMCID: PMC3439153
    NIHMSID: NIHMS381381
    An Integrated Encyclopedia of DNA Elements in the Human Genome
    The ENCODE Project Consortium

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  5. From Palazzo...It seems the hypothesis below could be tested. How many mutations does it take to make a ncRNA non functional. Does the data match with the predictions below?

    Using statistical methods, it has been estimated that humans sustain 2.1–10 deleterious mutations per generation [66]–[68]. These data would suggest that at most 10% of the human genome exhibits detectable organism-level function and conversely that at least 90% of the genome consists of junk DNA. These figures agree with measurements of genome conservation (∼9%, see above) and are incompatible with the view that 80% of the genome is functional in the sense implied by ENCODE.
    Using statistical methods, it has been estimated that humans sustain 2.1–10 deleterious mutations per generation [66]–[68]. These data would suggest that at most 10% of the human genome exhibits detectable organism-level function and conversely that at least 90% of the genome consists of junk DNA. These figures agree with measurements of genome conservation (∼9%, see above) and are incompatible with the view that 80% of the genome is functional in the sense implied by ENCODE.

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  6. @Larry

    Are you certain about the results of the studies you based your opinion on? You didn't do one study or experiment on the theme? What is it that makes you so certain that you, and the other people are right? Evidence please!

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    1. WTH? Evidence, like the data presented in the paper discussed?
      You're welcome.

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    2. There's a new trend that's emerged on the internet of idiotic people seeing the common request for "evidence" for baseless claims and parroting it in the most stupid of contexts because they think using that line atuomatically proves their point.
      All it shows is that they don't even comprehend what evidence means.

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    3. JoJo
      Have you identified any or part of the functional content of the human genome? How much do you think is undiscovered? Larry asked a question that stimulates interesting conversation. When you ask for evidence you are saying that he has not provided any. Larry sighted 3 papers in his article that were analysis based on evidence. You may question the evidence but IMHO it is not reasonable to ask for something he has already provided.

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    4. Have you identified any or part of the functional content of the human genome? How much do you think is undiscovered?

      I don't have to. Larry cherry-picked (es) the papers that support his OWN view. That is why I asked him whether he was the one to verify the claims himself. It's easy to link studies and take no responsibility for it. If one wants to be considered as scientists, he/she needs to prove their claims. Creationists are performing hundreds of experiments and they base their interpretation of their experiments on the data that anyone can review and criticize. What has Larry done in this case? We all know even if we don't like it. No further comments are necessary Larry. We all know you will remove my post and continue to pretend nothing happened...

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    5. Creationists are performing hundreds of experiments and they base their interpretation of their experiments on the data that anyone can review and criticize

      Show us those experiments please

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    6. JoJo
      Did you read the papers? One of the papers was supporting a high level of functionality in the genome. My experience with Larry is that if you make an argument with facts he will evaluate it fairly. "I don't have to" if you want to argue credibly, yes you do. Please cite a creationist experiment that identifies functionality to the genome that is new.

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    7. "We all know you will remove my post and continue to pretend nothing happened..."

      No, Jojo, I don't think you are getting off that easily. I would bet Larry will leave the big rotten egg you laid here in this thread and let it stink up the place.

      Yes you do need to provide evidence to back up your claims, Jojo. That is how science, and every other rational human endeavor works. You claim Larry has "cherry picked" those three references because they support his view. Do you realize that Mattick disagrees with junk DNA and Larry references Mattick's paper as being opposed to his own view?

      But get involved in the scientific discussion, Jojo. Present three creationist papers out of those hundreds that present your view. Feel free to cherry pick. I eagerly await your contribution.

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    8. Give him a break Chris. The difference between hundreds and zero may not be all that apparent to a creationist

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  7. JoJo
    Here are two papers from a creationists website that talk about nc regions of the genome.

    Shapiro JA, von Sternberg R (2005) Why repetitive DNA is essential to genome function. Biological Reviews 80: 227-250. Review. PMID: 15921050

    von Sternberg R, Shapiro JA (2005) How repeated retroelements format genome function. Cytogenetic and Genome Research 110: 108-116. PMID: 16093662

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  8. I do see evidence that the perception of Junk DNA in the Scientific community is changing:

    "with roughly 45 percent of the human genome made up of such genetic flotsam and jetsam.” Collins 2006

    I would say, in terms of junk DNA, we don’t use that term any more ‘cause I think it was pretty much a case of hubris to imagine that we could dispense with any part of the genome as if we knew enough to say it wasn’t functional … most of the genome that we used to think was there for spacer turns out to be doing stuff and most of that stuff is about regulation and that’s where the epigenome gets involved, and is teaching us a lot (Collins 2015)

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    1. Is that the only evidence of changing opinion that you see?

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    2. Here is a paper on lncRNA and association with heart failure
      and heart development Decoding the Noncoding Transcripts in Human Heart Failure
      Xinshu (Grace) Xiao, PhD; Marlin Touma, MD; Yibin Wang, PhD

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    3. What is your point? Nobody disputes the idea that some lincRNAs have a biological function?

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  9. Here is a paper lncRNA's and their possible association with cancer.

    doi: 10.1038/onc.2014.456. Epub 2015 Jan 26. Junk DNA and the long non-coding RNA twist in cancer genetics.

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  10. This paper is one of 41 papers on LncRNA and cancer in pubmed.gov in September. This is a biggie because the hedgehog path is the path that creates stem cells when it is activated. Stem cells are the root cause of metastasis as they can grow in multiple tissue types.

    LncRNA-Hh Strengthen Cancer Stem Cells Generation in Twist-Positive Breast Cancer via Activation of Hedgehog Signaling Pathway. 2015 Sep 29. doi: 10.1002/stem.2219. [Epub ahead of print]

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    1. What percentage of the genome is devoted to genes for functional RNAs, in your opinion? I think that only about 300 have been identified over the past two decades. I suspect there aren't more than 5,000. That number (5,000) would take up about 0.2% of the genome.

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  11. I have not spent any time trying to figure this out and have no reason to doubt your estimates. The information that I found yesterday indicating that these non coding areas are important to cancer research says to me that we need to encourage their discovery.

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    1. Is that a veiled shot at junk DNA? Do you understand that non-coding DNA is not the same thing as junk DNA?

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    2. Hi Dazz: Yes, I know that Junk DNA an expression used to describe DNA that is non functional while NC and LNC RNA is functional. This statement stands on its own. I really have no stake in the Junk DNA battles and really don't see them as particularly meaningful in the evolutionist /creationist debates.

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    3. Bill Cole, as for your last sentence:
      I suppose you mean "... in the scientist/creationist debates", as "evolutionist' is a (creationist) term that is not used by scientists.

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  12. Cold spring Harbor Labs demonstrated that a set of low concentration RNA's appear to have significant importance since it is deeply conserved across species and cell types and they concentration amounts must be fine tuned, and if fine tuned, it is likely not junk.

    http://www.cshl.edu/1265-variation-in-expression-of-thousands-of-genes-is-kept-under-tight-constraint-in-mice-and-humans-in-all-cells-and-tissue-types.html

    One can't just write this sort of research off.

    "Cells need these genes to behave within a relatively narrow range in order to work properly.

    The 6600 genes identified by the team have evolved in mice and people to show expression that varies over about a hundred-fold range, whether, for example, in breast tissue or lung tissue; whether in an epithelial cell or a nerve cell. This is compared to the remaining two-thirds of expressed genes, which are capable of varying their expression over a 100,000-fold range.

    Gingeras and collaborators additionally found that when six very different animal species are compared, ranging from chickens to humans, there are still a set of 2500 genes they share in common whose expression is sharply constrained in the same narrow range. This shows that this regulatory mechanism is evolutionarily very old and thus important, the scientists say."

    How can the RNAs be used rather than just plain ole' binding? In regulatory computation. RNA's are an ideal substrate for nano-molecular computing as suspected in the CeRNA hypothesis:


    http://www.cell.com/cell/abstract/S0092-8674(11)00812-9?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867411008129%3Fshowall%3Dtrue&cc=y=

    "Here, we present a unifying hypothesis about how messenger RNAs, transcribed pseudogenes, and long noncoding RNAs “talk” to each other using microRNA response elements (MREs) as letters of a new language. We propose that this “competing endogenous RNA” (ceRNA) activity forms a large-scale regulatory network across the transcriptome, greatly expanding the functional genetic information in the human genome and playing important roles in pathological conditions, such as cancer."

    If we are computing differentiation of 100 trillion cells in a human in all stages of development, how are the computations done to determine how cells are marked and differentiated and managed? There needs to be a repository to track all this.

    The cytosine methylations, the histone modifcations which wrap around the "junk DNA", who knows what else --- is needed to manage all these transcriptomes and means of getting cells to know and fulfill their specialized roles.

    We have RNAs whose expression levels are modulated by factors of 100,000 but others that remain in a tight constraint (hence concentration). This suggests fine tuning of how things work in the cell, hence are not junk.

    Too early to tell. But I wouldn't rely on the junk science of evolutionary theory to decide the matter, better to actually examine the question mechanically like the ENCODE and mouse ENCODE consortium are actually doing.

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    1. "This suggests fine tuning of how things work in the cell,"

      Why would that be ?

      "But I wouldn't rely on the junk science of evolutionary theory"

      Oh, that is why ... and your blogname already hinted at that, but hey - one never knows.

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    2. I wouldn't rely on the junk science of evolutionary theory

      Stop quoting evolutionary biologists and their papers then, fucktard

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    3. liars,

      The paper you cite was an approach to identify 'housekeeping' genes that control basic cellular functions, in this case genes that are likely common mammalian housekeeping genes. Their approach is explicitly evolutionary in its rationale. They in fact did rely on the well supported science of evolutionary theory to guide their study.

      They compared transcription levels of genes between human and mouse from a number of tissue types. These were all known orthologous genes between mouse and human. This paper was not about studying junk DNA. And analysis of expression levels of these constrained genes showed that most of the variation was found within a cell line, while variation among cell lines accounted for very little. In other words, the expression of these genes did not vary much between cell types.

      Go and read the Conclusions section of the paper you cite; it does a good job of explaining the evolutionary context of the study.

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    4. Chris B,

      Thanks for your response. However, the paper by Gingeras extended the analysis to ncRNA "genes". From the paper:

      "we extended the mouse gene and transcript candidate set, and enhanced the current set of orthologuos genes between these genomes to include long non-coding RNAs (lncRNAs) and pseudogenes."

      So the lncRNAs and pseudogenes are fine tuned. We have evidence that coding genes in humans that are pseudogenes in mice have different roles. The pseudogenes in mice are used in regulation if the CeRNA hypothesis is correct.

      Furthermore, as pointed out before, the supposed junk DNA in mice has a dual role: that of making optical lenses to assist in nocturnal vision.

      See:
      http://www.cell.com/abstract/S0092-8674(09)00137-8

      "The inverted rod nuclei act as collecting lenses, and computer simulations indicate that columns of such nuclei channel light efficiently toward the light-sensing rod outer segments"

      Junk DNA wraps around histones, the histone code provides a means of information storage of differentiated cell data as well as possibly a means of brain memory!

      This who focus on coding is wrong headed.

      DNA has multiple uses that just the ACGT base pairs. The cytosine methylation markings, the histone modifications are information bearing. So its not just about the RNA transcriptome. In nocturnal mammals it is used as part of a optical lensing system. Who knows what other ways the junk DNA is used?

      If the human brain leverages histone modifications for learning and cognition, then in that case the junk DNAs importance in the brain isn't just associated with the RNA transcriptome, but is part of a neurological substrate of helping us learn an remember. It's not about modifying the ACGT DNA bases, it's about providing a substrate for the epigenetic real time changes that help our brains actually work!

      The quasi repetitive architecture of DNA makes a lot of sense if indeed it is used as an addressing scheme to locate the methylation markings and information bearing histones. If the histones and methylation marks are critical to human cognition, then the junk DNA debate takes on a whole new perspective. We'd be brain dead without all that junk DNA!

      See:
      https://en.wikipedia.org/wiki/Epigenetics_in_learning_and_memory

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    5. "So the lncRNAs and pseudogenes are fine tuned."

      That's what you LIKE to believe, but how on Earth would that follow from what these authors wrote ? Why do you think you can deduce fine-tuning from that ?

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    6. liar,
      I have been through this Gish gallop in other threads with you. So let's cut through the obfuscation and to the chase:

      1. lnc RNAs, even if there are thousands of genuinely functional ones, will account for <1% of the human genome.

      2. Evolutionary theory makes no a priori prediction about junk DNA. Lots of nonfunctional DNA, or very little nonfunctional DNA, poses no problem whatsoever for evolutionary theory.

      3. How do you define "fine-tuned", and by what method do you detect it? How is it distinguished from "evolutionarily well-adapted"?

      4. With regard to nocturnal mice, the role of junk DNA in vision is consistent with junk DNA being exapted for that purpose. Is there evidence that non-nocturnal mice lack junk DNA? What about blind rodents?

      5. "DNA has multiple uses that just the ACGT base pairs. The cytosine methylation markings, the histone modifications are information bearing. So its not just about the RNA transcriptome." True, and science doesn't make that mistake. The Wikipedia page you cite summarizes a lot of science showing that.

      6. "If the human brain leverages histone modifications for learning and cognition, then in that case the junk DNAs importance in the brain isn't just associated with the RNA transcriptome, but is part of a neurological substrate of helping us learn an remember."
      That is a hypothesis that would need to be tested. Where would you start?

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  13. http://www.lncrnablog.com/category/news/commentary/
    A site that covers lncrna

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    1. From Palazzo & Gregory 2014: "Besides protein-coding sequences (including associated untranslated regions), which make up 1.5%–2.5% of the human genome [24], data from ENCODE suggest that conserved long noncoding RNAs (lncRNAs) are generated from about 9,000 loci that add up to less than an additional 0.4% [46], [47]. Thus, even if a vast new untapped world of functional noncoding RNA is discovered, this will probably be transcribed from a small fraction of the human genome."

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