Titus-McQuillan, J. | bioRxiv Science

bioRxiv2021

The evolution of splicing: transcriptome complexity and transcript distances implemented in TranD

Alternative splicing contributes to organismal complexity. Comparing transcripts between and within species is an important first step toward understanding questions about how evolution of transcript structure changes between species and contributes to sub-functionalization. These questions are confounded with issues of data quality and availability. The recent explosion of affordable long read sequencing of mRNA has considerably widened the ability to study transcriptional variation in non-model species. In this work, we develop a computational framework that uses nucleotide resolution distance metrics to compare transcript models for structural phenotypes: total transcript length, intron retention, donor/acceptor site variation, alternative exon cassettes, alternative 5/3 UTRs are each scored qualitatively and quantitatively in terms of number of nucleotides. For a single annotation file, all differences among transcripts within a gene are summarized and transcriptome-level complexity metrics: number of variable nucleotides, unique exons per gene, exons per transcript, and transcripts per gene are calculated. To compare two transcriptomes on the same co-ordinates, a weighted total distance between pairs of transcripts for the same gene is calculated. The weight function proposed has larger penalties for intron retention and exon skipping than alternative donor/acceptor sites. Minimum distances can be used to identify both transcript pairs and transcripts missing structural elements in either of the two annotations. This enables a broad range of functionality from comparing sister species to comparing different methods of building and summarizing transcriptomes. Importantly, the philosophy here is to output metrics, enabling others to explore the nucleotide-level distance metrics. Single transcriptome annotation summaries and pairwise comparisons are implemented in a new tool, TranD, distributed as a PyPi package and in the open-source web-based Galaxy (www.galaxyproject.org) platform.

genomics↗

bioRxiv2019

Resurrection of ancestral effector caspases identifies novel networks for evolution of substrate specificity

Apoptotic caspases evolved with metazoans more than 950 million years ago (MYA), and a series of gene duplications resulted in two subfamilies consisting of initiator and effector caspases. The effector caspase genes (caspases-3, -6, and -7) were subsequently fixed into the Chordata phylum more than 650 MYA when the gene for a common ancestor (CA) duplicated, and the three effector caspases have persisted throughout mammalian evolution. All caspases require an aspartate residue at the P1 position of substrates, so each caspase evolved discrete cellular roles through changes in substrate recognition at the P4 position combined with allosteric regulation. We examined the evolution of substrate specificity in caspase-6, which prefers valine at the P4 residue, compared to caspases-3 and -7, which prefer aspartate, by reconstructing the CA of effector caspases (AncCP-Ef1) and the CA of caspase-6 (AncCP-6An). We show that AncCP-Ef1 is a promiscuous enzyme with little distinction between Asp, Val, or Leu at P4. The specificity of caspase-6 was defined early in its evolution, where AncCP-6An demonstrates preference for Val over Asp at P4. Structures of AncCP-Ef1 and of AncCP-6An show a network of charged amino acids near the S4 pocket that, when combined with repositioning a flexible active site loop, resulted in a more hydrophobic binding pocket in AncCP-6An. The ancestral protein reconstructions show that the caspase-hemoglobinase fold has been conserved for over 650 million years and that only three substitutions in the scaffold are necessary to shift substrate selection toward Val over Asp.

biochemistry↗