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Spliceosomal introns, which have been found in most eukaryotic genes, are non-coding sequences excised from pre-mRNAs by a special complex called spliceosome during mRNA splicing. Introns occur in both protein- and RNA-coding genes and can be found in coding and untranslated gene regions. Because intron sequences vary greatly due to a high rate of polymorphism, the functions of intron had been for a long time associated only with alternative splicing, while intron evolution had been viewed not as an evolution of an individual genomic element, but rather considered within a framework of the evolution of the gene intron-exon structure. Here, we review the theories of intron origin, evolutionary events in the exon-intron structure, such as intron gain, loss, and sliding, intron functions known to date, and mechanisms by which changes in the intron features (length and phase) can affect the regulation of gene-mediated processes.

Cancer therapies that target epigenetic repressors can mediate their effects by activating retroelements within the human genome. Retroelement transcripts can form double-stranded RNA (dsRNA) that activates the MDA5 pattern recognition receptor 1 – 6 . This state of viral mimicry leads to loss of cancer cell fitness and stimulates innate and adaptive immune responses 7 , 8 . However, the clinical efficacy of epigenetic therapies has been limited. To find targets that would synergize with the viral mimicry response, we sought to identify the immunogenic retroelements that are activated by epigenetic therapies. Here we show that intronic and intergenic SINE elements, specifically inverted-repeat Alus, are the major source of drug-induced immunogenic dsRNA. These inverted-repeat Alus are frequently located downstream of ‘orphan’ CpG islands 9 . In mammals, the ADAR1 enzyme targets and destabilizes inverted-repeat Alu dsRNA 10 , which prevents activation of the MDA5 receptor 11 . We found that ADAR1 establishes a negative-feedback loop, restricting the viral mimicry response to epigenetic therapy. Depletion of ADAR1 in patient-derived cancer cells potentiates the efficacy of epigenetic therapy, restraining tumour growth and reducing cancer initiation. Therefore, epigenetic therapies trigger viral mimicry by inducing a subset of inverted-repeats Alus, leading to an ADAR1 dependency. Our findings suggest that combining epigenetic therapies with ADAR1 inhibitors represents a promising strategy for cancer treatment. Inverted-repeat Alu elements are the main source of drug-induced immunogenic double-stranded RNAs, which are destabilized by the RNA deaminase ADAR1, thereby limiting activation of the immune response.

Evolution of exon-intron structure of eukaryotic genes has been a matter of long-standing, intensive debate. The introns-early concept, later rebranded ‘introns first’ held that protein-coding genes were interrupted by numerous introns even at the earliest stages of life's evolution and that introns played a major role in the origin of proteins by facilitating recombination of sequences coding for small protein/peptide modules. The introns-late concept held that introns emerged only in eukaryotes and new introns have been accumulating continuously throughout eukaryotic evolution. Analysis of orthologous genes from completely sequenced eukaryotic genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists, suggesting that many ancestral introns have persisted since the last eukaryotic common ancestor (LECA). Reconstructions of intron gain and loss using the growing collection of genomes of diverse eukaryotes and increasingly advanced probabilistic models convincingly show that the LECA and the ancestors of each eukaryotic supergroup had intron-rich genes, with intron densities comparable to those in the most intron-rich modern genomes such as those of vertebrates. The subsequent evolution in most lineages of eukaryotes involved primarily loss of introns, with only a few episodes of substantial intron gain that might have accompanied major evolutionary innovations such as the origin of metazoa. The original invasion of self-splicing Group II introns, presumably originating from the mitochondrial endosymbiont, into the genome of the emerging eukaryote might have been a key factor of eukaryogenesis that in particular triggered the origin of endomembranes and the nucleus. Conversely, splicing errors gave rise to alternative splicing, a major contribution to the biological complexity of multicellular eukaryotes. There is no indication that any prokaryote has ever possessed a spliceosome or introns in protein-coding genes, other than relatively rare mobile self-splicing introns. Thus, the introns-first scenario is not supported by any evidence but exon-intron structure of protein-coding genes appears to have evolved concomitantly with the eukaryotic cell, and introns were a major factor of evolution throughout the history of eukaryotes. This article was reviewed by I. King Jordan, Manuel Irimia (nominated by Anthony Poole), Tobias Mourier (nominated by Anthony Poole), and Fyodor Kondrashov. For the complete reports, see the Reviewers’ Reports section.

There is massive variation in intron numbers across eukaryotic genomes, yet the major drivers of intron content during evolution remain elusive. Rapid intron loss and gain in some lineages contrast with long-term evolutionary stasis in others. Episodic intron gain could be explained by recently discovered specialized transposons called Introners, but so far Introners are only known from a handful of species. Here, we performed a systematic search across 3,325 eukaryotic genomes and identified 27,563 Introner-derived introns in 175 genomes (5.2%). Species with Introners span remarkable phylogenetic diversity, from animals to basal protists, representing lineages whose last common ancestor dates to over 1.7 billion years ago. Aquatic organisms were 6.5 times more likely to contain Introners than terrestrial organisms. Introners exhibit mechanistic diversity but most are consistent with DNA transposition, indicating that Introners have evolved convergently hundreds of times from nonautonomous transposable elements. Transposable elements and aquatic taxa are associated with high rates of horizontal gene transfer, suggesting that this combination of factors may explain the punctuated and biased diversity of species containing Introners. More generally, our data suggest that Introners may explain the episodic nature of intron gain across the eukaryotic tree of life. These results illuminate the major source of ongoing intron creation in eukaryotic genomes.

Removal of introns from messenger RNA precursors (pre-mRNA splicing) is an essential step for the expression of most eukaryotic genes. Alternative splicing enables the regulated generation of multiple mRNA and protein products from a single gene. Cancer cells have general as well as cancer type-specific and subtype-specific alterations in the splicing process that can have prognostic value and contribute to every hallmark of cancer progression, including cancer immune responses. These splicing alterations are often linked to the occurrence of cancer driver mutations in genes encoding either core components or regulators of the splicing machinery. Of therapeutic relevance, the transcriptomic landscape of cancer cells makes them particularly vulnerable to pharmacological inhibition of splicing. Small-molecule splicing modulators are currently in clinical trials and, in addition to splice site-switching antisense oligonucleotides, offer the promise of novel and personalized approaches to cancer treatment.Alternative splicing enables the regulated generation of multiple mRNA and protein products from a single gene. This Review outlines the splicing process and its alterations in cancer before highlighting related opportunities for the development of innovative therapeutic approaches.

Previous evolutionary reconstructions have concluded that early eukaryotic ancestors including both the last common ancestor of eukaryotes and of all fungi had intron-rich genomes. By contrast, some extant eukaryotes have few introns, underscoring the complex histories of intron–exon structures, and raising the question as to why these few introns are retained. Here, we have used recently available fungal genomes to address a variety of questions related to intron evolution. Evolutionary reconstruction of intron presence and absence using 263 diverse fungal species supports the idea that massive intron reduction through intron loss has occurred in multiple clades. The intron densities estimated in various fungal ancestors differ from zero to 7.6 introns per 1 kb of protein-coding sequence. Massive intron loss has occurred not only in microsporidian parasites and saccharomycetous yeasts, but also in diverse smuts and allies. To investigate the roles of the remaining introns in highly-reduced species, we have searched for their special characteristics in eight intron-poor fungi. Notably, the introns of ribosome-associated genes RPL7 and NOG2 have conserved positions; both intron-containing genes encoding snoRNAs. Furthermore, both the proteins and snoRNAs are involved in ribosome biogenesis, suggesting that the expression of the protein-coding genes and noncoding snoRNAs may be functionally coordinated. Indeed, these introns are also conserved in three-quarters of fungi species. Our study shows that fungal introns have a complex evolutionary history and underappreciated roles in gene expression.

• Premise of the study: Noncoding chloroplast DNA (NC-cpDNA) sequences are the staple data source of low-level phylogeographic and phylogenetic studies of angiosperms. We followed up on previous papers (tortoise and hare II and III) that sought to identify the most consistently variable regions of NC-cpDNA. We used an exhaustive literature review and newly available whole plastome data to assess applicability of previous conclusions at low taxonomic levels.• Methods: We aligned complete plastomes of 25 species pairs from across angiosperms, comparing the number of genetic differences found in 107 NC-cpDNA regions and matK. We surveyed Web of Science for the plant phylogeographic literature between 2007 and 2013 to assess how NC-cpDNA has been used at the intraspecific level.• Key results: Several regions are consistently the most variable across angiosperm lineages: ndhF-rpl32, rpl32-trnL(UAG), ndhC-trnV(UAC), 5′rps16-trnQ(UUG), psbE-petL, trnT(GGU)-psbD, petA-psbJ, and rpl16 intron. However, there is no universally best region. The average number of regions applied to low-level studies is ∼2.5, which may be too little to access the full discriminating power of this genome.• Conclusions: Plastome sequences have been used successfully at lower and lower taxonomic levels. Our findings corroborate earlier works, suggesting that there are regions that are most likely to be the most variable. However, while NC-cpDNA sequences are commonly used in plant phylogeographic studies, few of the most variable regions are applied in that context. Furthermore, it appears that in most studies too few NC-cpDNAs are used to access the discriminating power of the cpDNA genome.

While splicing changes caused by somatic mutations in SF3B1 are known, identifying full-length isoform changes may better elucidate the functional consequences of these mutations. We report nanopore sequencing of full-length cDNA from CLL samples with and without SF3B1 mutation, as well as normal B cell samples, giving a total of 149 million pass reads. We present FLAIR (Full-Length Alternative Isoform analysis of RNA), a computational workflow to identify high-confidence transcripts, perform differential splicing event analysis, and differential isoform analysis. Using nanopore reads, we demonstrate differential 3’ splice site changes associated with SF3B1 mutation, agreeing with previous studies. We also observe a strong downregulation of intron retention events associated with SF3B1 mutation. Full-length transcript analysis links multiple alternative splicing events together and allows for better estimates of the abundance of productive versus unproductive isoforms. Our work demonstrates the potential utility of nanopore sequencing for cancer and splicing research. Long-read sequencing is useful in determining exon-connectivity of full-length mRNA isoforms. Here, by long-read nanopore sequencing, the authors report that intron retention is downregulated in SF3B1 mutant chronic lymphocytic leukemia cells than normal B cells.

Intron retention (IR) by alternative splicing is a conserved regulatory mechanism that can affect gene expression and protein function during adult development and age‐onset diseases. However, it remains unclear whether IR undergoes spatial or temporal changes during different stages of aging or neurodegeneration like Alzheimer's disease (AD). By profiling the transcriptome of Drosophila head cells at different ages, we observed a significant increase in IR events for many genes during aging. Differential IR affects distinct biological functions at different ages and occurs at several AD‐associated genes in older adults. The increased nucleosome occupancy at the differentially retained introns in young animals suggests that it may regulate the level of IR during aging. Notably, an increase in the number of IR events was also observed in healthy older mouse and human brain tissues, as well as in the cerebellum and frontal cortex from independent AD cohorts. Genes with differential IR shared many common features, including shorter intron length, no perturbation in their mRNA level, and enrichment for biological functions that are associated with mRNA processing and proteostasis. The differentially retained introns identified in AD frontal cortex have higher GC content, with many of their mRNA transcripts showing an altered level of protein expression compared to control samples. Taken together, our results suggest that an increased IR is an conserved signature that is associated with aging. By affecting pathways involved in mRNA and protein homeostasis, changes of IR pattern during aging may regulate the transition from healthy to pathological state in late‐onset sporadic AD.