A major obstacle to any explanation of the origins of eukaryotes is the emergence of the non-coding regions of genes known as introns which are largely absent from bacteria. Before we discuss how the origin of introns poses a significant challenge to evolution, it is first necessary to give a short introduction as to what introns are.

What Are Introns?

Introns (which, unlike exons, do not code for proteins) can be of considerable length in higher eukaryotes, even spanning many thousands of bases and sometimes comprising some 90 percent of the precursor mRNA.¹ In contrast, lower eukaryotes such as yeast possess fewer and shorter introns, which are typically fewer than 300 bases in length. Since introns are the non-coding segments of genes, they are removed from the mRNA before it is translated into a protein. The introns are excised from the pre-mRNA transcript, and the exons are spliced together by a riboprotein complex called the spliceosome.^2,3 This remarkable process is illustrated by the animation below:

What Functional Role Do Introns Play?

Introns, upon their discovery, were generally assumed to be non-functional junk DNA.^4,5,6,7 Francis Crick described these sequences as “‘nonsense’ stretches of DNA interspersed within the sense DNA.”⁸ This view of introns has, however, not aged well. Indeed, “introns in contemporary species fulfill a broad spectrum of functions, and are involved in virtually every step of mRNA processing.”⁹ Introns often show high levels of sequence conservation (particularly within classes of organisms), suggesting function.^10,11

We also now know that introns play an essential role in alternative splicing where the exons of pre-mRNA transcripts are spliced in different configurations producing multiple protein isoforms from the same gene.^12,13 According to one paper, the majority of alternatively spliced exons in humans and mice are flanked by introns, the sequences of which are highly conserved, implying an important role in alternative splicing.¹⁴

Another study documented that a six-nucleotide intronic sequence is “frequently located adjacent to tissue-specific alternative exons in the human genome.”¹⁵ Across taxa as widely divergent as dogs, rats, chickens, mice, and humans, this sequence exhibits conservation. These features, according to the paper, “mark it as a critical component of splicing switch mechanism(s) designed to activate a limited repertoire of splicing events in cell type-specific patterns.” In addition, direct evidence has been documented showing that introns contain other codes involved in the regulation of alternative splicing.^{16,17,18,19,20}

Genes for the majority of microRNAs (which are required for the expression of mRNAs during development) and small nucleolar RNAs (which play important roles in the processing of ribosomal RNAs) reside in introns.²¹ One paper reported that, 

Mirtrons are alternative precursors for microRNA biogenesis that were recently described in invertebrates. These short hairpin introns use splicing to bypass Drosha cleavage, which is otherwise essential for the generation of canonical animal microRNAs. Using computational and experimental strategies, we now establish that mammals have mirtrons as well.²²

Another study also reports that,

314 miRNAs (51%) were located in introns of protein-coding genes, 231 miRNAs (38%) were located in intergenic regions, while only 43 miRNAs (7%) were located in exons of noncoding RNAs or UTR of protein coding genes. Interestedly, 23 miRNAs (4%) were located in either an exon or an intron depending on alternative splicing of the host transcript.²³

It has also been established that the promoter sequences for those RNA genes occur in introns.²⁴Furthermore, as many as 52 percent of transcripts for chromatin-associated RNA, which play important roles in the structural organization of nuclear chromatin²⁵, are found in introns.²⁶

It is likely that the length of introns (and, consequently, the time taken to transcribe them) can contribute to timing mechanisms during development.²⁷ Since long introns take longer to transcribe, this can cause genes to respond more slowly to transcriptional activators or repressors compared to shorter genes, resulting in sequential gene activation, where genes with longer introns are expressed later even if they receive the same activating signal at the same time.

Explaining the Origins of Introns Is a Challenge to Evolution

How could introns arise in evolution without simultaneously having a mechanism in hand to facilitate their removal from the pre-mRNA transcript? Without the presence of such a mechanism, the introns would scramble the coding regions of the gene, and themselves be translated into a sequence of amino acids by the ribosome. Moreover, if the length of an intron is not a multiple of three, it could also potentially introduce a damaging frame shift into the coding region.

Self-Splicing Introns?

The most common explanation of the origins of eukaryotic introns is that they arose from self-splicing precursors, known as group II introns found in bacteria.^28,29,30,31 Self-splicing introns employ a mechanism very similar to eukaryotic pre-mRNA splicing, involving two sequential transesterification reactions. The first step is for a conserved “branchpoint” adenosine to initiate a nucleophilic reaction at the 5’ splice site, leading to cleavage of the RNA backbone and formation of a lariat structure. In the second stage, the 3’ splice site is attacked by the terminal 3’ hydroxyl group of the exon at the 5’ end. The result is the excision of the lariat intron and ligation of the flanking exons.

A few different lines of evidence are offered in support of the hypothesis that eukaryotic spliceosomal introns arose from group II introns. For example, a simpler way to achieve splicing presumably would be to bring the splice sites together in one step to directly cleave and rejoin them. The proposed scenario would explain the use of a lariat intermediate, since a lariat is generated by group II RNA intron sequences.^32,33 Moreover, it is argued that this hypothesis helps to clarify why RNA molecules play such an important part in the splicing process. Examples of self-splicing RNA introns still exist today, such as in the nuclear rRNA genes of the ciliate Tetrahymena.^34,35,36,37

It is unlikely that group II introns arose within protein-coding genes without pre-existing splicing mechanisms. For one thing, group II introns fold into a conserved six-domain secondary structure, which is required for their self-replicative capability.³⁸ The proper folding depends upon specific intra- and inter-domain base-pairing. Thus, key regions are quite sequence sensitive. The most highly conserved domain is domain V, which forms the catalytic center of the ribozyme.^39,40 Even single-nucleotide mutations can abolish or significantly reduce splicing activity. Domain I contains exon binding sites that have to form complementary base pairs with intron binding sites on the exons. There are also specific sequences that mediate long-range interactions, which are essential for proper folding. Given the need for sequence specificity, the right sequences would need to be generated by chance mutations — but one would expect the introduction of additional sequences within protein-coding genes to be detrimental to the organism, and thereby removed by purifying selection.

Because of this, it is generally assumed that group II introns must have first arisen in non-coding regions, which provided a safer “sandbox” for these elements to evolve the complex secondary structure and self-splicing capabilities, without immediate harmful effects. However, bacterial genomes are typically gene-dense with relatively little non-coding DNA. This raises the question of how much “safe space” would have been available for the origination of group II introns prior to their insertion into coding regions. For example, the E. coli genome has only approximately 10-12% non-coding DNA (~460,000-550,000 base pairs of DNA). An average group II intron is ~2,500 base pairs long. This only leaves room for a few dozen full length group II introns to exist in non-coding DNA without disrupting host fitness.

Even on this hypothesis, however, it seems implausible that, in a non-coding region, there would be selective pressure to preserve such highly constrained sequence features until the full intron, complete with its self-splicing capability, was fully realized. Furthermore, for splicing to be successful, group II introns must accurately recognize exon-intron boundaries and bind complementary sequences in the flanking exons. But the sandbox hypothesis would have us believe that introns evolved completely within non-coding sequences, without the necessary exonic contexts to evolve this coordinated interaction. Thus, even supposing that a fully operational group II intron were to arise in a non-coding region, its insertion into a coding gene would be highly disruptive unless precise splicing was already operational and accurately matched to the flanking exons.

There also is no plausible scenario for transitioning from group II introns to spliceosomal introns. The spliceosome machinery is far more complex and sophisticated than autocatalytic ribozymes, involving not just five RNAs but nearly one hundred core spliceosome proteins found in the simplest eukaryotes.

Eukaryotes and Prokaryotes Likely Do Not Share a Common Ancestor

As argued previously the disparity in mode of cell division between eukaryotes and prokaryotes evinces their separate origins.⁴¹ The abundance of introns in eukaryotic genomes, largely absent from prokaryotes, provides yet a further reason to suspect that eukaryotes and prokaryotes had independent origins. Current proposals speculate that introns in a proto-eukaryote may have arisen following infection by an alpha-proteobacterial endosymbiont (the hypothesized mechanism by which mitochondria and chloroplasts originated in eukaryotes).⁴² Group II intron retroelements originating from the endosymbiont could have profusely inserted themselves within the early host genome. This scenario is highly unlikely as spliceosomal introns of eukaryotes show negligible similarity with group II introns and would already require operative spliceosomes at this early evolutionary phase for their removal from mRNA. Thus, the origins of these diverse introns and the multitude of spliceosome components required for their removal are insufficiently described by this prominent evolutionary model. An alternative consideration to intron origins describes seven mechanisms that appear to give rise to rare, isolated introns but admission is presented that this falls woefully short of accounting for the vast majority of spliceosomal introns in eukaryotes.⁴³ Undoubtedly, this latter viewpoint more accurately communicates what we have learned regarding potential evolutionary explanations of introns.

Conclusion

In view of the problems outlined in the foregoing, the origin of spliceosomal introns represents a significant challenge to an evolutionary account of their origins. While the hypothesis that group II introns from bacterial endosymbionts gave rise to eukaryotic introns is the prevailing explanation, it faces a series of substantial obstacles. The complexity of intron splicing mechanisms suggest that their origin is better accounted for by a different paradigm than unguided evolutionary processes — one that allows for the rapid emergence of coordinated, information-rich systems. The only type of explanation that adequately accounts for this type of phenomenon is a goal-directed, or intelligent, cause.

Notes

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