Phylogenetic Analysis of the Plant U2 snRNP Auxiliary Factor Large Subunit A Gene Family in Response to Developmental Cues and Environmental Stimuli

In all organisms, splicing occurs through the formation of spliceosome complexes, and splicing auxiliary factors are essential during splicing. U2AF65 is a crucial splicing cofactor, and the two typical RNA-recognition motifs at its center recognize and bind the polypyrimidine sequence located between the intron branch site and the 3′-splice site. U2AF65A is a member of the U2AF65 gene family, with pivotal roles in diseases in mammals, specifically humans; however, few studies have investigated plant U2AF65A, and its specific functions are poorly understood. Therefore, in the present study, we systematically identified U2AF65A in plant species from algae to angiosperms. Based on 113 putative U2AF65A sequences from 33 plant species, phylogenetic analyses were performed, followed by basic bioinformatics, including the comparisons of gene structure, protein domains, promoter motifs, and gene expression levels. In addition, using rice as the model crop, we demonstrated that the OsU2AF65A protein is localized to the nucleus and cytoplasm, and it is involved in responses to various stresses, such as drought, high salinity, low temperature, and heavy metal exposure (e.g., cadmium). Using Arabidopsis thaliana and rice mutants, we demonstrated that U2AF65A is involved in the accumulation of plant biomass, growth of hypocotyl upon thermal stimulation, and reduction of tolerance of high temperature stress. These findings offer an overview of the U2AF65 gene family and its stress response functions, serving as the reference for further comprehensive functional studies of the essential specific splicing cofactor U2AF65A in the plant kingdom.

A cautionary tale on proper use of branch-site models to detect convergent positive selection

Comparative genomics has become a powerful tool to elucidate genotype-phenotype relationships, particularly through the study of convergently acquired phenotypes. By identifying genes under positive selection specifically on branches with the convergent phenotype we can potentially link genes to that phenotype. Such gene scans are often done using branch-site codon models. However, we have observed a recent troubling trend of misinterpretation of branch-site models in which phylogeny-wide positive selection is not distinguished from positive selection specific to convergent branches. Here, we use simulations and real data to demonstrate how failing to discern between these two cases leads to false inferences of positive selection associated with a convergent trait. We then present a "drop-out" test solution to distinguish the two cases and thereby truly capture positive selection events associated with convergent phenotypes, thus allowing for further insights into both genetic and phenotypic evolution.

Comparative Chloroplast Genomes of Zosteraceae Species Provide Adaptive Evolution Insights Into Seagrass

Seagrasses are marine flowering plants found in tropical and sub-tropical areas that live in coastal regions between the sea and land. All seagrass species evolved from terrestrial monocotyledons, providing the opportunity to study plant adaptation to sea environments. Here, we sequenced the chloroplast genomes (cpGenomes) of three Zostera species, then analyzed and compared their cpGenome structures and sequence variations. We also performed a phylogenetic analysis using published seagrass chloroplasts and calculated the selection pressure of 17 species within seagrasses and nine terrestrial monocotyledons, as well as estimated the number of shared genes of eight seagrasses. The cpGenomes of Zosteraceae species ranged in size from 143,877 bp (Zostera marina) to 152,726 bp (Phyllospadix iwatensis), which were conserved and displayed similar structures and gene orders. Additionally, we found 17 variable hotspot regions as candidate DNA barcodes for Zosteraceae species, which will be helpful for studying the phylogenetic relationships and interspecies differences between seagrass species. Interestingly, nine genes had positive selection sites, including two ATP subunit genes (atpA and atpF), two ribosome subunit genes (rps4 and rpl20), two DNA-dependent RNA polymerase genes (rpoC1 and rpoC2), as well as accD, clpP, and ycf2. These gene regions may have played key roles in the seagrass adaptation to diverse environments. The Branch model analysis showed that seagrasses had a higher rate of evolution than terrestrial monocotyledons, suggesting that seagrasses experienced greater environmental pressure. Moreover, a branch-site model identified positively selected sites (PSSs) in ccsA, suggesting their involvement in the adaptation to sea environments. These findings are valuable for further investigations on Zosteraceae cpGenomes and will serve as an excellent resource for future studies on seagrass adaptation to sea environments.

Structural insights into how Prp5 proofreads the pre-mRNA branch site

During the splicing of introns from precursor messenger RNAs (pre-mRNAs), the U2 small nuclear ribonucleoprotein (snRNP) must undergo stable integration into the spliceosomal A complex—a poorly understood, multistep process that is facilitated by the DEAD-box helicase Prp5 (refs. 1–4). During this process, the U2 small nuclear RNA (snRNA) forms an RNA duplex with the pre-mRNA branch site (the U2–BS helix), which is proofread by Prp5 at this stage through an unclear mechanism5. Here, by deleting the branch-site adenosine (BS-A) or mutating the branch-site sequence of an actin pre-mRNA, we stall the assembly of spliceosomes in extracts from the yeast Saccharomyces cerevisiae directly before the A complex is formed. We then determine the three-dimensional structure of this newly identified assembly intermediate by cryo-electron microscopy. Our structure indicates that the U2–BS helix has formed in this pre-A complex, but is not yet clamped by the HEAT domain of the Hsh155 protein (Hsh155HEAT), which exhibits an open conformation. The structure further reveals a large-scale remodelling/repositioning of the U1 and U2 snRNPs during the formation of the A complex that is required to allow subsequent binding of the U4/U6.U5 tri-snRNP, but that this repositioning is blocked in the pre-A complex by the presence of Prp5. Our data suggest that binding of Hsh155HEAT to the bulged BS-A of the U2–BS helix triggers closure of Hsh155HEAT, which in turn destabilizes Prp5 binding. Thus, Prp5 proofreads the branch site indirectly, hindering spliceosome assembly if branch-site mutations prevent the remodelling of Hsh155HEAT. Our data provide structural insights into how a spliceosomal helicase enhances the fidelity of pre-mRNA splicing.

Structural basis of intron selection by U2 snRNP in the presence of covalent inhibitors

Intron selection during the formation of prespliceosomes is a critical event in pre-mRNA splicing. Chemical modulation of intron selection has emerged as a route for cancer therapy. Splicing modulators alter the splicing patterns in cells by binding to the U2 snRNP (small nuclear ribonucleoprotein)—a complex chaperoning the selection of branch and 3′ splice sites. Here we report crystal structures of the SF3B module of the U2 snRNP in complex with spliceostatin and sudemycin FR901464 analogs, and the cryo-electron microscopy structure of a cross-exon prespliceosome-like complex arrested with spliceostatin A. The structures reveal how modulators inactivate the branch site in a sequence-dependent manner and stall an E-to-A prespliceosome intermediate by covalent coupling to a nucleophilic zinc finger belonging to the SF3B subunit PHF5A. These findings support a mechanism of intron recognition by the U2 snRNP as a toehold-mediated strand invasion and advance an unanticipated drug targeting concept.

Insight into the Phylogenetic Relationships among Three Subfamilies within Heptageniidae (Insecta: Ephemeroptera) along with Low-Temperature Selection Pressure Analyses Using Mitogenomes

We determined 15 complete and two nearly complete mitogenomes of Heptageniidae belonging to three subfamilies (Heptageniinae, Rhithrogeninae, and Ecdyonurinae) and six genera (Afronurus, Epeorus, Leucrocuta, Maccaffertium, Stenacron, and Stenonema). Species of Rhithrogeninae and Ecdyonurinae had the same gene rearrangement of CR-I-M-Q-M-ND2, whereas a novel gene rearrangement of CR-I-M-Q-NCR-ND2 was found in Heptageniinae. Non-coding regions (NCRs) of 25–47 bp located between trnA and trnR were observed in all mayflies of Heptageniidae, which may be a synapomorphy for Heptageniidae. Both the BI and ML phylogenetic analyses supported the monophyly of Heptageniidae and its subfamilies (Heptageniinae, Rhithrogeninae, and Ecdyonurinae). The phylogenetic results combined with gene rearrangements and NCR locations confirmed the relationship of the subfamilies as (Heptageniinae + (Rhithrogeninae + Ecdyonurinae)). To assess the effects of low-temperature stress on Heptageniidae species from Ottawa, Canada, we found 27 positive selection sites in eight protein-coding genes (PCGs) using the branch-site model. The selection pressure analyses suggested that mitochondrial PCGs underwent positive selection to meet the energy requirements under low-temperature stress.

The Conservation of Chloroplast Genome Structure and Improved Resolution of Infrafamilial Relationships of Crassulaceae

Crassulaceae are the largest family in the angiosperm order Saxifragales. Species of this family are characterized by succulent leaves and a unique photosynthetic pathway known as Crassulacean acid metabolism (CAM). Although the inter- and intrageneric relationships have been extensively studied over the last few decades, the infrafamilial relationships of Crassulaceae remain partially obscured. Here, we report nine newly sequenced chloroplast genomes, which comprise several key lineages of Crassulaceae. Our comparative analyses and positive selection analyses of Crassulaceae species indicate that the overall gene organization and function of the chloroplast genome are highly conserved across the family. No positively selected gene was statistically supported in Crassulaceae lineage using likelihood ratio test (LRT) based on branch-site models. Among the three subfamilies of Crassulaceae, our phylogenetic analyses of chloroplast protein-coding genes support Crassuloideae as sister to Kalanchoideae plus Sempervivoideae. Furthermore, within Sempervivoideae, our analyses unambiguously resolved five clades that are successively sister lineages, i.e., Telephium clade, Sempervivum clade, Aeonium clade, Leucosedum clade, and Acre clade. Overall, this study enhances our understanding of the infrafamilial relationships and the conservation of chloroplast genomes within Crassulaceae.

Evolutionary ecology of the visual opsin gene sequence and its expression in turbot (Scophthalmus maximus)

Background As flatfish, turbot undergo metamorphosis as part of their life cycle. In the larval stage, turbot live at the ocean surface, but after metamorphosis they move to deeper water and turn to benthic life. Thus, the light environment differs greatly between life stages. The visual system plays a great role in organic evolution, but reports of the relationship between the visual system and benthic life are rare. In this study, we reported the molecular and evolutionary analysis of opsin genes in turbot, and the heterochronic shifts in opsin expression during development. Results Our gene synteny analysis showed that subtype RH2C was not on the same gene cluster as the other four green-sensitive opsin genes (RH2) in turbot. It was translocated to chromosome 8 from chromosome 6. Based on branch-site test and spectral tuning sites analyses, E122Q and M207L substitutions in RH2C, which were found to be under positive selection, are closely related to the blue shift of optimum light sensitivities. And real-time PCR results indicated the dominant opsin gene shifted from red-sensitive (LWS) to RH2B1 during turbot development, which may lead to spectral sensitivity shifts to shorter wavelengths. Conclusions This is the first report that RH2C may be an important subtype of green opsin gene that was retained by turbot and possibly other flatfish species during evolution. Moreover, E122Q and M207L substitutions in RH2C may contribute to the survival of turbot in the bluish colored ocean. And heterochronic shifts in opsin expression may be an important strategy for turbot to adapt to benthic life.

Positive Selection in Gene Regulatory Factors Suggests Adaptive Pleiotropic Changes During Human Evolution

Gene regulatory factors (GRFs), such as transcription factors, co-factors and histone-modifying enzymes, play many important roles in modifying gene expression in biological processes. They have also been proposed to underlie speciation and adaptation. To investigate potential contributions of GRFs to primate evolution, we analyzed GRF genes in 27 publicly available primate genomes. Genes coding for zinc finger (ZNF) proteins, especially ZNFs with a Krüppel-associated box (KRAB) domain were the most abundant TFs in all genomes. Gene numbers per TF family differed between all species. To detect signs of positive selection in GRF genes we investigated more than 3,000 human GRFs with their more than 70,000 orthologs in 26 non-human primates. We implemented two independent tests for positive selection, the branch-site-model of the PAML suite and aBSREL of the HyPhy suite, focusing on the human and great ape branch. Our workflow included rigorous procedures to reduce the number of false positives: excluding distantly similar orthologs, manual corrections of alignments, and considering only genes and sites detected by both tests for positive selection. Furthermore, we verified the candidate sites for selection by investigating their variation within human and non-human great ape population data. In order to approximately assign a date to positively selected sites in the human lineage, we analyzed archaic human genomes. Our work revealed with high confidence five GRFs that have been positively selected on the human lineage and one GRF that has been positively selected on the great ape lineage. These GRFs are scattered on different chromosomes and have been previously linked to diverse functions. For some of them a role in speciation and/or adaptation can be proposed based on the expression pattern or association with human diseases, but it seems that they all contributed independently to human evolution. Four of the positively selected GRFs are KRAB-ZNF proteins, that induce changes in target genes co-expression and/or through arms race with transposable elements. Since each positively selected GRF contains several sites with evidence for positive selection, we suggest that these GRFs participated pleiotropically to phenotypic adaptations in humans.