**1. Introduction**

Actin filaments are one of the key elements of the cytoskeleton, and are vital for processes including cellular motility, neuronal differentiation, and cell–cell junctions. The core of these are composed of filamentous F-actin. These are formed by the polymerization of globular units of G-actin, and fibers can in turn depolymerize back to G-actin again [1]. The correct regulation of this key molecular process therefore impacts upon a wide array of cellular functions, and incorrect regulation is associated with various diseases [2]. Among the regulators of actin discovered in the last few decades are the proteins encoded by the *TRIO* and *F-actin Binding Protein* (*TRIOBP*) locus [3]. The *TRIOBP* gene is subject to complicated alternative splicing (Figure 1a). Multiple long splice variants exist [4,5], of which the longest is *TRIOBP-6*, although the slightly shorter *TRIOBP-5* is more often studied. The majority of published work into TRIOBP proteins, however, has instead focused on the products of two shorter transcripts. Of these, *TRIOBP-1* is transcribed from the 3′ end of the *TRIOBP* gene and encodes a largely structured protein [3] with a ubiquitous expression pattern [4,5]. In contrast, *TRIOBP-4* is transcribed from the 5′ end of the gene and encodes a structurally disordered protein, expressed predominantly in the inner ear and retina [4]. The TRIOBP-1 and TRIOBP-4 proteins share no common amino acid sequence, however, both share most or all of their primary structure with the longer variants (Figure 1b).

**Figure 1.** (**a**) Scale schematic of the alternative splicing of *TRIOBP* in humans. Exons (vertical bars) on the four most studied isoforms are shown, with introns represented by horizontal lines. Blue exons are entirely or mainly coding, black exons are entirely or mainly non-coding. Exon numbering is according to Park et al. [6]. (**b**) Scale schematic of the human TRIOBP-1, 4, 5, and 6 proteins with structural regions highlighted: R1, R2: First and second repeat domains, PH: Pleckstrin homology domain, Central: Central coiled coil domain, CT: C-terminal coiled coil domain. The number of amino acids (AA) in humans is also indicated. (**c**) The level of conservation of each section of the TRIOBP-6 amongst mammalian orthologues and predictions of three forms of secondary structure: disordered/unstructured protein, α-helix, and β-sheet. These are displayed as heat maps to scale with the schematic in part (**b**). Conservation determined using AL2CO [7], based on amino acid sequences of TRIOBP-6 (or similar splice variants) from 57 different mammalian genera. These were identified using BLAST (reference sequence human: TRIOBP-6, NP\_001034230.1), aligned with CLUSTAL Omega 1.2.4 [8] and the alignment was then manually curated. Secondary structure predictions were made using PSIPDRED 4.0 and DISOPRED3 [9–11] with protein analyzed in three overlapping sections. All results were averaged over an 11 amino acid sliding window for clarity. The N-terminal 61 amino acids of TRIOBP-1 from exon 11a that are not present in TRIOBP-6 were not evaluated here, but were previously predicted to be disordered with comparatively poor conservation [12].
