RICHARD PEACHEY correspondence:  regarding the claim (shown at the bottom of this email) that DNA can be considered largely nonfunctional if removing it doesn’t seem to affect the phenotype.

Just recently I have encountered an article that discusses that very issue at length. (I have already added parts of it to my collection of quotes re evidence of functionality in “junk” DNA.)
Here, for your interest, are the key sections of that article (note especially the bits that I’ve turned green):

“Roles for MicroRNAs in Conferring Robustness to Biological Processes” (Margaret S. Ebert and Phillip A. Sharp, Cell, Vol. 149 [Apr 27, 2012], pp. 515-524)

[ABSTRACT:] “Increasing evidence suggests that, among their roles as posttranscriptional repressors of gene expression, microRNAs (miRNAs) help to confer robustness to biological processes by reinforcing transcriptional programs and attenuating aberrant transcripts, and they may in some network contexts help suppress random fluctuations in transcript copy number. These activities have important consequences for normal development and physiology, disease, and evolution.” (p. 515)

“MicroRNAs (miRNAs) are hairpin-derived RNAs ~20–24 nucleotides (nt) long, which posttranscriptionally repress the expression of target genes usually by binding to the 3′ UTR of messenger RNA (mRNA). As a class, miRNAs constitute about 1%–2% of genes in worms, flies, and mammals. . . . Their regulatory potential is vast: more than 60% of protein-coding genes are computationally predicted as targets based on conserved base-pairing between the 3′ UTR and the 5′ region of the miRNA, which is called the seed. . . . Although many miRNAs and their target binding sites are deeply conserved, which suggests important function, a typical miRNA-target interaction produces only subtle reduction (<2-fold) in protein level, and many miRNAs can be deleted without creating any obvious phenotype. Early observations of miRNA expression profiles revealed that miRNAs tend to be anticorrelated with target gene expression in contiguous developmental stages or tissues (Stark et al., 2005; Farh et al., 2005). Correspondingly, a view emerged that miRNA evolved primarily to play the role of a reinforcer, in that its activities cohere with transcriptional patterns to sharpen developmental transitions and entrench cellular identities. It is also possible that miRNAs buffer fluctuations in gene expression and more faithfully signal outcomes in the context of certain regulatory networks.
  “Robustness refers to a system’s ability to maintain its function in spite of internal or external perturbations. . . . The involvement of miRNAs in regulatory networks that provide developmental robustness is indicated by recent experiments in a variety of model organisms. It is also suggested by three general observations: (1) genes with tissue-specific expression have longer 3′ UTRs with more miRNA-binding sites . . .; (2) miRNA expression increases and diversifies over the course of embryonic development . . ., as 3′ UTRs are lengthened via alternative polyadenylation site choice . . .; and (3) the diversity of the miRNA repertoire in animal genomes has increased with increasing organismal complexity. . . . In this Review, we examine the current evidence for how miRNAs contribute to the robustness of biological processes.” (p. 515)

“One of the earliest functions attributed to miRNAs was sharpening developmental transitions by suppressing residual transcripts that were specific to the previous stage. Global gene expression analyses in fly, fish, and mouse have shown that miRNAs and their targets often have mutually exclusive RNA expression across tissues, especially in neighboring tissues derived from common progenitors. . . . This suggests that miRNAs can act to reinforce the transcriptional gene expression program by repressing leaky transcripts.
“More recently, sensitive gene expression profiling of cell types in the zebrafish embryo revealed not so much a stark mutual exclusion pattern but, rather, a tendency for anticorrelated but still overlapping expression of miRNAs and targets. . . . This suggests that miRNAs play a more prominent role than only reinforcing the patterns dictated by transcriptional regulation. In fact, a strongly transcribed, ubiquitously expressed actin transcript has its levels spatially sculpted by muscle-specific miRNAs in zebrafish. . . .” (pp. 515f.)

The effect of an individual miRNA on a target’s protein level tends to be subtle, usually less than 2-fold. . . . Most loss-of-function mutations are recessive; thus, organisms are commonly able to compensate for a 2-fold loss of gene expression. Such differences may even be within the range of random variation in mRNA or protein level between different cells in a genetically identical population or in a given cell at different times. So how do miRNAs and target sites experience selective pressure, and how do miRNAs accomplish any significant regulation? For starters, there are miRNA-target interactions that involve multiple sites for a given target and confer much stronger repression, such as the interaction between the micro-RNA let-7 and the oncogene HMGA2. . . . More often, different miRNAs work together to cotarget a given mRNA, so their combined repressive effect greatly exceeds the individual contributions. On average, there are more than four highly conserved seed match sites per UTR considering all miRNAs and many more sites when more weakly conserved sequences are considered. . . .
  “. . . a small change in the level of protein can sometimes have a large physiological effect, such as when a positive feedback loop amplifies the change. . . .
  “Another mechanism by which a miRNA can increase its impact is by targeting a set of genes that are in a shared pathway or protein complex.” (p. 517)

In spite of the large numbers of target genes predicted to be affected by miRNA loss of function, gene knockout experiments for individual miRNAs have yielded many disappointing results. In worms, most individual miRNA mutants show no gross phenotype . . .; the same is true for several of the mouse knockouts generated to date, including miR-21, miR-210, miR-214, miR-206, and miR-143. . . . A partial explanation for these results resides in the functional redundancy of many miRNAs that share their seed sequence with others. For example, the let-7 family members miR-48, miR-84, and miR-241 operate redundantly to control the L2-to-L3 larval transition in C. elegans. . . . Additionally, many miRNAs of different seed families work together to cotarget a given gene or set of genes, providing overlapping functions. To generate an observable impairment in the animal, it might be necessary to delete all members of a seed family and also nonseed family members that have a high degree of cotargeting.
  “It is also possible that a mutant phenotype would only arise upon acute miRNA deletion if, during development, miRNA loss can be compensated at the level of gene expression or by one cell type populating a niche to assist an impaired or underpopulated cell type within an organ or system such as the immune system. . . . Even once an organ has developed, miRNAs may be required for maintenance: Dicer loss in the mouse thymic epithelium or the highly structured retina leads to progressive degeneration of tissue architecture. . . . However, there are several contrary examples in which deletion of Dicer and loss of all miRNAs in mature tissue do not appear to generate a phenotype. Deletion of Dicer in the mouse olfactory system had no apparent phenotype over periods of several months . . ., whereas the same deletion in developing olfactory tissue led to severe neurogenesis defects.
  “Finally, a miRNA phenotype may appear only upon the application of certain internal or external stresses. The most well-characterized example of this mechanism is in the Drosophila eye, in which miR-7 plays a role in the determination of sensory organs. . . . Loss of miR-7 had little observable impact on the development of the sensory organs under normal, uniform conditions, and expression of the proneural transcription factor Atonal was also detected at wild-type level. . . . But when an environmental perturbation was added during larval development (i.e., fluctuating the temperature between 31°C and 18°C roughly every 90 min), the miR-7 mutant eyes showed abnormally low Atonal expression and abnormally high, irregular expression of the antineural transcription factor Yan. Sensory organ precursor (SOP) defects also appeared: some groups of antennal SOPs failed to develop or developed with abnormal patterning; their cells showed low Atonal levels. The ability of miR-7 to confer developmental robustness against temperature perturbations likely depends on its placement in a network of feedback and feedforward loops with Atonal and Yan. . . .
  “In mice, deletion of the heart muscle-specific miRNA miR-208 has little phenotype under normal conditions but results in a failure to induce cardiac remodeling upon stress. . . . When the mice were treated to induce pressure overload or hypothyroidism, miR-208 activity was required in the cardiomyocytes to upregulate βMHC by targeting the thyroid receptor signaling pathway. The embryonic stem cell-specific miR-290-295 cluster is not required for cell viability until DNA damage stress, upon which it promotes cell survival. . . . In worms sensitized by mutations in a variety of regulatory pathways, 25 of 31 deleted miRNAs revealed a mutant phenotype . . .; these same deletions in a wild-type background did not produce a phenotype. These examples show the utility of assessing animal systems not only under standard laboratory conditions, but also with treatments that mimic the natural hardships and flaws that they might experience in the wild.” (pp. 517f.)

miRNAs are surely not the only regulatory factors that contribute to system robustness. Whole-genome bioinformatic analysis of worm and fly reveal transcription factors enriched in feedforward loops as well. . . . Compared to transcriptional regulators, however, miRNAs do have some distinguishing features that may make them well suited in this role. As posttranscriptional regulators acting in the cytoplasmic compartment, miRNAs can intervene late in the pipeline of gene expression to counteract variation from the upstream processes of transcription, splicing, and nuclear export. They are able to regulate transcripts in special compartments, such as maternally deposited transcripts in the early embryo . . . and locally translated transcripts of dendrites far from the cell body of neurons. They can also be present at high concentrations (10,000s of molecules per cell) by virtue of being very stable (e.g., the heart muscle-specific miR-208 has an in vivo half-life of > 1 week). . . . This is consistent with theoretical constraints indicating the need for many more molecules of a regulator to achieve a small reduction in the noise of a target gene. . . . miRNA expression profiling from progressive stages of T-lymphocyte development found that the total number of miRNAs expressed per cell changed in parallel with changes in total cellular RNA content, suggesting that global miRNA levels are tuned to the translational capacity of the cell. . . .” (pp. 519f.)

[CONCLUDING REMARKS:] “Multicellular organisms must manage the tasks of development and physiology in unpredictable, changing environments and with imperfect genetic and biochemical components. Random noise in gene expression must be dampened or, as in the case of some cell fate decisions, harnessed in a system control network to designate one fate or another among neighboring cells. Robustness goes beyond the job of keeping one state the same in the face of perturbations. In development, it can mean not sending a signal until the right time and then sending it strongly and irreversibly. Although miRNAs act to confer accuracy and uniformity to developmental transitions, the loss of a miRNA may result not in catastrophic defects but, rather, in imprecise, variable phenotypes. If other feedback or back-up mechanisms are in place, then the loss of robustness may only be detected by applying additional perturbations. The addition of miRNAs to metazoan genomes over time and the diversity of miRNA repertoires among different tissues of developing animals suggest that miRNAs are involved in reinforcing developmental decisions to make organismal complexity reliable and heritable from one generation to the next.” (p. 521)

———- Forwarded message ———-
From: Richard Peachey <>
Date: Fri, Sep 7, 2012 at 3:50 PM
Subject: Further on Junk DNA

Hey, note part 5 (“Junk DNA”) of this Wikipedia article: <>

After giving some obviously outdated info, the article brings up ENCODE but then goes on to claim:
Still, a significant amount of the sequence of the genomes of eukaryotic organisms currently appears to fall under no existing classification other than “junk”.

[Seems the evolutionists aren’t going to give up easily! I guess this is just another round in what Robert W. Carter in 2009 called “the slow, painful death of junk DNA.” <>]

That Wikipedia article continues:
For example, one experiment removed 0.1% of the mouse genome with no detectable effect on the phenotype. This result suggests that the removed DNA was largely nonfunctional.

It’s not always that simple, however, to detect phenotypic changes. Some can be subtle: even if a stretch of DNA is not essential for life, it may still be adaptive in some way. (Non-subtle illustration: Your left arm is helpful though not absolutely essential for survival/reproduction!) Also, survival in the wild can be quite different from survival in the cozy laboratory. Consider the following statement:

  “Another way to seek evidence of function en masse is to get rid of long non-coding RNAs and watch how animals cope. But such an experiment may produce only subtle changes in an organism as a whole, and could still miss the importance of a transcript. ‘I think the cell will use these transcripts at very different times and in very different cell types and conditions.’ [Geneticist Thomas] Gingeras says. ‘You may need to see them in a very specific context to see the function.’
“That is what Jürgen Brosius of the University of Münster, Germany, and his colleagues found when they removed a 150-nucleotide RNA from mouse neurons, where it is normally transported down the cellular fingers that communicate with other cells. The engineered animals looked and acted more or less the same as the control animals — but Brosius says that on close inspection they weren’t as inquisitive and had unusual exploratory behaviours. Such activity might be lethal in the wild, Mattick [i.e., John Mattick, the director of the Centre for Molecular Biology and Biotechnology at the University of Queensland in Brisbane] says, ‘but it was affecting their behaviour in ways that were far too subtle to be assessed in a cage.’ ” (Anna Petherick, “The Production Line.” Nature 454:1044f., Aug. 28, 2008)

Evolutionists have at times accused creationists and Intelligent Design proponents of holding to a “God of the gaps,” but it looks to me like they’re desperately clutching a “Junk DNA of the gaps” in this losing battle!