Heliconius erato with one wing (right) altered by CRISPR gene. Dorsal view
Luca Livraghi.
Heliconius erato with one wing (right) altered by CRISPR gene. Ventral view
Luca Livraghi.
In reality, of course, it simply shows us that sometimes, evolution happens in unexpected ways.
The assumption, which is almost always valid and based on sound scientific evidence, is that DNA is transcribed into messenger RNA (mRNA) that mRNA is transcribed by ribosomes into proteins. Proteins then serve functions such as structural proteins or as enzymes catalysing chemical processes in the cell. In the special case of so-called homeobox genes (hox genes) these have a regulatory function in a developing embryo, stimulating groups of cells to organise into specific tissues such as limbs, eyes and other specialised tissues, by switching other genes off and on at certain times.
Scientists have discovered that a long non-coding RNA (lncRNA) transcribed from the 'cortex' locus is responsible for dark colors on butterfly and moth wings* How can they be sure it's non-coding? To determine whether a long non-coding RNA (lncRNA) is truly non-coding, scientists use several experimental and computational approaches to confirm that the RNA does not encode a protein. Here are some of the main methods they use to establish that an RNA transcribed from a particular locus, such as the 'cortex' locus in butterflies and moths, is non-coding:But what scientists working at George Washington University and the University of Cambridge have discovered is that the dark colors on some butterfly and moth wings is regulated not by a protein, but by a long strand of RNA which doesn't function as mRNA so is not transcribed into a functional protein but acts directly to regulate the placement of colored scales on a butterfly wing.
- Lack of an Open Reading Frame (ORF):
- The most direct way to determine if an RNA is non-coding is to analyze its sequence for the presence of an open reading frame (ORF), which is a continuous stretch of codons that could potentially encode a protein. If the RNA lacks a long ORF (typically considered to be less than 100 amino acids), it is a strong indication that it does not code for a protein.
- Codon Substitution Analysis:
- If the RNA contains an ORF, scientists can perform codon substitution analysis to check whether there is evidence of selection pressure to maintain a protein-coding sequence. Protein-coding genes tend to have specific patterns of synonymous (silent) versus nonsynonymous (amino acid-altering) mutations due to natural selection maintaining the amino acid sequence. A lack of such selective constraint suggests the RNA is not coding for a functional protein.
- Ribosome Profiling (Ribo-seq):
- Ribosome profiling is a technique that identifies which RNAs are actively being translated by ribosomes into proteins. In this method, researchers isolate ribosome-protected RNA fragments and sequence them. If the putative lncRNA does not show any ribosome occupancy, it suggests that the RNA is not being translated into a protein.
- Mass Spectrometry Proteomics:
- Mass spectrometry can be used to identify all the proteins present in a cell or tissue. If no peptides corresponding to the RNA in question are detected in proteomics datasets, it further supports the conclusion that the RNA does not encode a protein.
- Computational Tools and Databases:
- Bioinformatics tools and databases, such as the Coding Potential Calculator (CPC) and others, can predict whether an RNA has the potential to code for a protein based on features like ORF length, codon usage bias, sequence conservation, and secondary structure. RNAs that score low in coding potential are more likely to be non-coding.
- Subcellular Localization Studies:
- The localization of RNA within the cell can also provide clues about its function. Protein-coding RNAs (mRNAs) are typically found in the cytoplasm, where they are translated by ribosomes. In contrast, many lncRNAs are located primarily in the nucleus, which is consistent with a non-coding function. Techniques like fluorescence in situ hybridization (FISH) or subcellular fractionation followed by RNA sequencing can determine the RNA’s localization.
- Experimental Knockdown or Overexpression:
- Scientists can knock down or overexpress the lncRNA to observe its effect on cellular function. If altering the levels of the RNA leads to changes in gene expression, chromatin state, or other cellular processes without a corresponding change in protein levels, it suggests that the RNA is acting through a non-coding mechanism.
- Lack of Conservation at the Amino Acid Level:
- If the RNA sequence is conserved across species at the nucleotide level but not at the amino acid level, this indicates it is not constrained by the requirements of protein coding. Conservation at the nucleotide level often suggests a functional role in RNA form rather than a protein product.
- Mutational Analysis:
- Introducing mutations that would normally disrupt a protein-coding sequence (such as introducing stop codons) can help determine if the RNA is non-coding. If such mutations do not affect the RNA’s function, this suggests it does not act by producing a protein.
By using a combination of these methods, scientists can be confident in classifying the RNA transcribed from the 'cortex' locus as non-coding. Each method contributes a piece of evidence supporting the conclusion that the RNA does not code for a protein and instead likely has a regulatory or structural function.
These long non-coding RNAs (lncRNA) have been known about for some time, but this is the first to be shown to have such a wide-ranging function, spread across several genera of lepidoptera (moths and butterflies) that diverged some 80 million years ago.
Sadly, the team's paper in Proceedings of the National Academy of Sciences is behind an expensive paywall and neither the abstract nor the news release from George Washington University have details of how they ascertained that the lncRNA was indeed non-coding, but the AI panel to the right spells out how this is determined in general. I assume the authors thought readers would be aware of it.
Genomic dark matter solves butterfly evolutionary riddle
New study reveals how an unexpected genetic mechanism influences the evolution of butterfly wing coloration.
A team of international researchers has uncovered a surprising genetic mechanism that influences the vibrant and complex patterns on butterfly wings. In a study published in the Proceedings of the National Academy of Sciences, the team, led by Luca Livraghi at the George Washington University and the University of Cambridge, discovered that an RNA molecule, rather than a protein as previously thought, plays a pivotal role in determining the distribution of black pigment on butterfly wings.
Precisely how butterflies are able to generate the vibrant patterns and colors on their wings has fascinated biologists for centuries. The genetic code contained within the cells of developing butterfly wings dictates the specific arrangement of the color on the wing’s scales—the microscopic tiles that form wing patterns—similar to the arrangement of colored pixels to form a digital image. Cracking this code is fundamental to understanding how our own genes build our anatomy. In the lab, researchers can manipulate that code in butterflies with gene-editing tools and observe the effect on visible traits, such as coloration on a wing.
Scientists have long known that protein-coding genes are crucial to these processes. These types of genes create proteins that can dictate when and where a specific scale should generate a particular pigment. When it comes to black pigments, researchers thought this process would be no different, and initially implicated a protein-coding gene. The new research, however, paints a different picture.
The team discovered a gene that produces an RNA molecule—not a protein—controls where dark pigments are made during butterfly metamorphosis. Using the genome-editing technique CRISPR, the researchers demonstrated that when you remove the gene that produces the RNA molecule, butterflies completely lose their black pigmented scales, showing a clear link between RNA activity and dark pigment development.
The researchers further explored how the RNA molecule functions during wing development. By examining its activity, they observed a perfect correlation between where the RNA is expressed and where black scales form.What we found was astonishing. This RNA molecule directly influences where the black pigment appears on the wings, shaping the butterfly’s color patterns in a way we hadn’t anticipated.
Dr Luca Livraghi, first author
Department of Biological Sciences
The George Washington University, Washington, DC, USA And The Department of Zoology
University of Cambridge, Cambridge UK.We were amazed that this gene is turned on where the black scales will eventually develop on the wing, with exquisite precision. It is truly an evolutionary paintbrush in this sense, and a creative one, judging by its effects in several species.
Associate Professor Arnaud Martin, co-corresponding author
Department of Biological Sciences
The George Washington University, Washington, DC, USA.
The researchers examined the newly discovered RNA in several other butterflies whose evolutionary history diverged around 80 million years ago. They found that in each of these species, the RNA had evolved to control new placements in the patterns of dark pigments.
Gulf fritillary butterfly, Agraulis incarnata, wing scales displaying clonal CRISPR mutations for the ivory lncRNA.Luca Livraghi.The consistent result obtained from CRISPR mutants in several species really demonstrate that this RNA gene is not a recent invention, but a key ancestral mechanism to control wing pattern diversity.
Professor Riccardo Papa, co-author
Department of Biology
University of Puerto Rico at Río Piedras, San Juan, Puerto Rico.
We and others have now looked at this genetic trait in many different butterfly species, and remarkably we are finding that this same RNA is used again and again, from longwing butterflies, to monarchs and painted lady butterflies. It’s clearly a crucial gene for the evolution of wing patterns. I wonder what other, similar phenomena biologists might have been missing because they weren’t paying attention to the dark matter of the genome.
Dr Joseph J. Hanly, co-author
Department of Biological Sciences
The George Washington University, Washington, DC, USA.
The findings not only challenge long-standing assumptions about genetic regulation but also open up new avenues for studying how visible traits evolve in animals.
The study, “A long non-coding RNA at the cortex locus controls adaptive coloration in butterflies,” was published on August 30, 2024 in the Proceedings of the National Academy of Sciences. The research was supported by the National Science Foundation and the Biotechnology and Biological Sciences Research Council.
SignificanceThis evidence of common descent will probably be ignored by creationist frauds who, if they don't ignore it altogether, will be misrepresented as falsifying all we ever thought we knew about evolutionary biology, with the unspoken assumption that therefore the locally-popular god did it.
Deciphering the genetic underpinnings of adaptive variation is fundamental for a comprehensive understanding of evolutionary processes. Long noncoding RNAs (lncRNAs) represent an emerging category of genetic modulators within the genome, yet they have been overlooked as a source of phenotypic diversity. In this study, we unveil the pivotal role of a lncRNA in orchestrating color transitions between dark and light patterns during butterfly wing development. Remarkably, this lncRNA gene is nested within the cortex locus, a genetic region known to control multiple cases of adaptive variation in butterflies and moths, including iconic examples of natural selection. These findings highlight the significant influence of lncRNAs in developmental regulation and underscore their potential as key genetic players in the evolutionary process itself.
Abstract
Evolutionary variation in the wing pigmentation of butterflies and moths offers striking examples of adaptation by crypsis and mimicry. The cortex locus has been independently mapped as the locus controlling color polymorphisms in 15 lepidopteran species, suggesting that it acts as a genomic hotspot for the diversification of wing patterns, but functional validation through protein-coding knockouts has proven difficult to obtain. Our study unveils the role of a long noncoding RNA (lncRNA) which we name ivory, transcribed from the cortex locus, in modulating color patterning in butterflies. Strikingly, ivory expression prefigures most melanic patterns during pupal development, suggesting an early developmental role in specifying scale identity. To test this, we generated CRISPR mosaic knock-outs in five nymphalid butterfly species and show that ivory mutagenesis yields transformations of dark pigmented scales into white or light-colored scales. Genotyping of Vanessa cardui germline mutants associates these phenotypes to small on-target deletions at the conserved first exon of ivory. In contrast, cortex germline mutant butterflies with confirmed null alleles lack any wing phenotype and exclude a color patterning role for this adjacent gene. Overall, these results show that a lncRNA gene acts as a master switch of color pattern specification and played key roles in the adaptive diversification of wing patterns in butterflies.
Livraghi, Luca; Hanly, Joseph J.; Evans, Elizabeth; Wright, Charlotte J.; Loh, Ling S.; Mazo-Vargas, Anyi; Kamrava, Kiana; Carter, Alexander; van der Heijden, Eva S. M.; Reed, Robert D.; Papa, Riccardo; Jiggins, Chris D.; Martin, Arnaud
A long noncoding RNA at the cortex locus controls adaptive coloration in butterflies Proceedings of the National Academy of Sciences 121(36), e2403326121. DOI: 10.1073/pnas.2403326121.
© 2024 PNAS.
Reprinted under the terms of s60 of the Copyright, Designs and Patents Act 1988.
It is, of course nothing of the sort and the authors show no hint of a doubt that the explanation lies in how these butterflies have evolved and diversified over time according to environmental pressures so is evidence of common ancestry as far back as 80 million years ago - well before creationists believe there was an Earth to have evolving life on it.
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