Transcription-Associated Mutation Promotes RNA Complexity in Highly Expressed Genes—A Major New Source of Selectable Variation

Abstract

Alternatively spliced transcript isoforms are thought to play a critical role for functional diversity. However, the mechanism generating the enormous diversity of spliced transcript isoforms remains unknown, and its biological significance remains unclear. We analyzed transcriptomes in saker falcons, chickens, and mice to show that alternative splicing occurs more frequently, yielding more isoforms, in highly expressed genes. We focused on hemoglobin in the falcon, the most abundantly expressed genes in blood, finding that alternative splicing produces 10-fold more isoforms than expected from the number of splice junctions in the genome. These isoforms were produced mainly by alternative use of de novo splice sites generated by transcription-associated mutation (TAM), not by the RNA editing mechanism normally invoked. We found that high expression of globin genes increases mutation frequencies during transcription, especially on nontranscribed DNA strands. After DNA replication, transcribed strands inherit these somatic mutations, creating de novo splice sites, and generating multiple distinct isoforms in the cell clone. Bisulfate sequencing revealed that DNA methylation may counteract this process by suppressing TAM, suggesting DNA methylation can spatially regulate RNA complexity. RNA profiling showed that falcons living on the high Qinghai–Tibetan Plateau possess greater global gene expression levels and higher diversity of mean to high abundance isoforms (reads per kilobases per million mapped reads ≥18) than their low-altitude counterparts, and we speculate that this may enhance their oxygen transport capacity under low-oxygen environments. Thus, TAM-induced RNA diversity may be physiologically significant, providing an alternative strategy in lifestyle evolution.

Evolutionary Honing in and Mutational Replacement: How Long-Term Directed Mutational Responses to Specific Environmental Pressures Are Possible

“…They argued that this effect is strong in the hemoglobin genes because they are highly expressed and even stronger in a falcon population living at a high altitude, where there is increased oxygen demand. They also found that more than 80% of the hemoglobin mutations were to T, and that the most common mutation type was A→T, on the coding strand (Pan et al, 2018). These observations are of interest given that the HbS mutation is an A→T one and that it was the point mutation of highest de novo rate in the sub-Saharan African hemoglobin subunit beta (HBB) gene in the HbS de novo mutation study (Melamed et al, 2022).…”

“…While the general process responsible has been outlined above, how it may apply in detail to the HbS mutation remains to be uncovered, and below we provide some observations to help propel the quest for the missing detail. Focusing on hemoglobin genes in saker falcons, Pan et al (2018) observed a correlation between the expression levels of genes and their mutation rates in blood samples, along with characteristics of transcription-associated mutations (TAM). These single strand mutations become fixed as de novo mutations in the daughter cells after DNA replication during erythropoiesis, giving rise to numerous mutation variants in the soma and to an increased diversity of alternatively spliced mRNA variants due to de novo splice sites (Pan et al, 2018).…”

“…Focusing on hemoglobin genes in saker falcons, Pan et al (2018) observed a correlation between the expression levels of genes and their mutation rates in blood samples, along with characteristics of transcription-associated mutations (TAM). These single strand mutations become fixed as de novo mutations in the daughter cells after DNA replication during erythropoiesis, giving rise to numerous mutation variants in the soma and to an increased diversity of alternatively spliced mRNA variants due to de novo splice sites (Pan et al, 2018). They argued that this effect is strong in the hemoglobin genes because they are highly expressed and even stronger in a falcon population living at a high altitude, where there is increased oxygen demand.…”

“…Importantly, mutation rates in Pan et al's study were more complex than simply being based on transcription. They were lower in gene bodies in methylated regions, highly nonuniform across positions, and appeared particularly high (∼10 −4 ) at de novo splice sites based on the observation of many spliced variants among small cDNA samples (e.g., Pan et al, 2018Pan et al, , p. 1110. These observations appear to be more consistent with regulatory processes than with the errant, scattered genetic change implied by the random mutation concept.…”

Transcriptome-wide high-throughput mapping of protein–RNA occupancy profiles using POP-seq

“…Several studies suggest that single nucleotide variations (SNVs) play an important role in gene regulation via riboSNitches’ 101 i.e. by altering RNA secondary structure or TAM (Transcript associated mutation) 102 that further contribute to transcriptome complexity in higher eukaryotes. Therefore, it is imperative to investigate the genomic variations occurring in protein–RNA interaction sites identified by POP-seq protocols.…”