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	FIGURE 1. Drosophila melanogaster DBT differs from DBT of ancestral insect Thermobia domestica and both mouse (M. musculus) homologs, CKIε and CKIδ. (A) A detail of the kinase domain with highlighted conserved Lysine K224 and Drosophila-specific differences: N220, L233, S236, V238, and F244. (B) A schematic depiction of proteins with highlighted kinase domain, N- and C-terminal tails, and positions of detailed alignments shown in panels (A,C). (C) Detail of the C-terminal tail, where grey boxes indicate residues conserved among CKIε, CKIδ, and T. domestica DBT.
	FIGURE 2. Phylogeny of bilaterian Casein kinase I (CKI) reveals seven clearly separated CKI-coding genes in fruit flies (Drosophila genus). (A) A tree illustrating relatedness among CKI proteins (values above branches indicate bootstrap support), in which well-separated clusters are color-coded. Insect DBTs (cerulean) branch together with deuterostomian sequences (cobalt blue) including CKIε and CKIδ from fish, amphibia, reptiles, and mammals. Besides well-established isoforms CKIα and CKIγ (the latter encoded by gilgamesh in Drosophila), four Drosophila genus-specific clusters were detected. Two of them branch at the base of CKIα and are therefore labeled as CKIα-like I (peanut) and CKIα-like II (cinnamon brown). Two additional clusters are separated from established CKI isoforms and are labeled according to the D. melanogaster nomenclature as CG9962 (orange) and CG2577 (apricot). Tau-tubulin kinases (a.k.a. asator in D. melanogaster) serve as an outgroup. Positions of the fruit fly D. melanogaster and the mouse Mus musculus proteins are highlighted by arrows in sage green and lavender, respectively. In established CKI terminology, the term isoform (α, γ, δ, ε) is used to refer to kinases encoded by distinct genes, although some of these genes might also encode different splice variants (for clarity, we use the term “splicing isoforms”). Multiple arrows in the mouse refer to gene multiplications, not to splicing isoforms. The phylogenetic analysis strongly supports the existence of the new groups of CKI genes in Drosophila and confirms already established groups. However, the relationship among CKI groups is sometimes poorly supported and the tree should not be interpreted as a focused analysis of CKI history. The tree was inferred using RAxML maximum likelihood of 239 protein sequences (final GAMMA-based score of the best tree -72993.763796) using Geneious 11 software (Biometters). Bootstrap support was calculated from 100 replications. See Supplementary Tables S1, S2 for accession numbers of analyzed sequences. (B) Schematic illustration of CKI proteins with highlighted kinase domain (brown), N- and C-terminal tails are shown as empty rectangles. (C) Details of protein alignment with highlighted residues that are important for the function of CKIδ (Shinohara et al., 2017; Philpott et al., 2020) and DBT (Dahlberg et al., 2009).
	FIGURE 3. DBT and CKIδ/ε genes and proteins mapped on insect and deuterostomian phylogeny. The phylogenetic tree corresponds to a consensus of recent phylogenomic studies (Misof et al., 2014; Johnson et al., 2018; Kawahara et al., 2019; McKenna et al., 2019; Wipfler et al., 2019). Representative species are shown at the terminal nodes. The first column indicates the presence of DBT-coding genes (note a Petromyzon-specific gene duplication). Greek letters refer to the presence of CKIδ and CKIε; note two CKIδ paralogs in Danio and Xenopus and two CKIε paralogs in Xenopus (for details on CKIδ and CKIε phylogeny, see Figure 4). The second column indicates how many splicing isoforms affecting the C-terminal tail protein sequence were identified. The C-terminal tail of the longest isoform is depicted for each gene in each species (see Supplementary Figures S2–S7 for all isoforms and additional species). The color bars indicate in silico predicted phosphorylation patterns of threonine (T, green), tyrosine (Y, blue), and serine residues (S, pink). The bar’s height refers to the predicted score between 0.5–1.0. K224 region indicates whether NKRQK or TKRQK motif was found in the region corresponding to the 220–224 position within the catalytic domain of D. melanogaster DBT. Major changes in the circadian clock setup are depicted: presence of CRY mammalian (mCRY) type, loss of TIMELESS-drosophila type (dTIM), loss of PERIOD (PER) (Kotwica-Rolinska et al., 2022b), and transition of BMAL (activation domain is present) to CYC (activation domain lost) (Supplementary Figure S8). DBT-interacting protein paralogs BRIDE of DBT (BDBT) was found only in Diptera (for details and BDBT phylogeny see Figure 5).
	FIGURE 4. Phylogeny of DBT, CKIδ, and CKIε indicates several gene duplications in vertebrates. In the lancelet Branchiostoma, only one CKI gene was found to precede the δ- and ε-isoforms. The major duplication that gave rise to the δ and ε isoforms dates back to the ancestor of Gnatostomata (vertebrates with jaws), while two CKI genes in the sea lamprey Petromyzon marinus resulted from Petromyzon-specific gene duplication. CKIδ was duplicated in the zebrafish Danio rerio, resulting in the so-called CKIδ-A and CKIδ-B. In the African clawed frog Xenopus laevis, a large genome duplication resulted in two CKIδ (L and S) and CKIε (L and S) genes. DBT sequences from protostomian representatives (the firebrat Thermobia domestica, the honey bee Apis mellifera, the marmorated sting bud Halyomorpha halys, the fruit fly Drosophila melanogaster, and the horseshoe crab Limulus polyphemus) were used as outgroups. The tree was inferred using RAxML maximum likelihood of 31 protein sequences using Geneious 11 software (Biomatters). Bootstrap support was calculated using 100 replications.
	FIGURE 5. Phylogeny of FK506-binding proteins revealed a clear separation of dipteran BRIDE of DBT (BDBT) from all remaining clusters. Major insect orders are highlighted. Arrows indicate the position of sequences from representative species. FK506-binding proteins from Mecoptera and Siphonaptera do branch far away from Diptera. The tree was inferred using RAxML maximum likelihood of 280 protein sequences (final GAMMA-based score of the best tree −104683.7791) using Geneious 11 software (Biomatters). Bootstrap support was calculated using 100 replications.
	FIGURE 6. Alternative splicing of doubletime (dbt) gene in the linden bug Pyrrhocoris apterus. (A) A gene model illustrating the length of all dbt exons and introns. Two alternative transcription starts and splicing at the 5’end (exon 1 or 2, respectively) do not influence the predicted protein coding sequence. Alternative skipping of exons 9 and/or 10 results in four isoforms (dbt-iso1, 2, 3, 4). The expression level of each dbt isoform mRNA in the brain was detected from the Oxford nanopore transcriptome and depicted as % contribution to all dbt molecules (100%—all isoforms). (B) Protein alignment of the C-terminal tail illustrating the isoform-specific and variable regions (the more conserved residue the darker the color). Color rectangles refer to the theoretical prediction of phosphorylation pattern (>0.5; NetPhos-3.1) for serine (S), threonine (T), and tyrosine (Y).
	FIGURE 7. C-terminal tail of DBT in 15 representative species of Diptera. Protein alignment points to variable and conserved regions in the protein sequence (the more conserved the darker the color; a hyphen corresponds to a gap in the alignment). Color rectangles refer to the theoretical prediction of phosphorylation pattern (>0.5, NetPhos-3.1) for serin (S), threonine (T), and tyrosine (Y). The C-terminal tail contains a motif conserved in Diptera (light purple), which can be further extended in Cyclorhapha (Deep Purple).
	FIGURE 8. Functional analysis of new dbt mutants created in D. melanogaster. (A) A schematic depiction of DBT protein with highlighted mutations; here-created mutants are in bold, the asterisk in the scheme refers to a premature protein termination due to a frameshift. (B) Detail of the C-terminal tail in the wild type (wt) and three mutant lines. The dipteran and cyclorrhaphan motifs are highlighted by light and deep purple, respectively. (C–I) Circadian clock phenotype [each dot in panels (C–I) represents the free-running period tau, τ, of individual male flies] was recorded for 10 days in DD at the specified temperature. Blue dots connected with a blue line indicate the percentage of rhythmic individuals at a particular temperature. A scale indicating the percentage rhythmicity is on the right y-axis, the blue numbers in the chart represented ntotal/nrhythmic. (J) The temperature compensation depicted as Q10 was calculated from data presented in (C–I). Magenta bars represent means ± SEM.

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