**5. Conclusions and Unanswered Questions**

The *TRIOBP* locus therefore encodes a variety of distinct proteins (Figure 1) with TRIOBP-1 being a structured and ubiquitously expressed protein implicated in mental illness and TRIOBP-4 being a disordered protein with specialized expression pattern essential for hearing. The long isoforms TRIOBP-5 and 6 combine the structures and many of the functions of the shorter isoforms, but with distinct additional roles in the ear, and potentially elsewhere. In spite of this, all the isoforms are linked through their role in stabilizing actin (Table 2, Figure 5).

**Figure 5.** Illustrated representation of the expression and roles of TRIOBP-1 (green), TRIOBP-4 (blue), and TRIOBP-5/6 (red). Clockwise from top: Aggregation of TRIOBP-1 in the brain and mental illness; Role of TRIOBP-1 in F-actin stabilization throughout the body; Linking of actin to ion channel function in the heart by TRIOBP-1; role of TRIOBP-1 in the cell cycle; Importance of all major TRIOBP isoforms in metastasis; Distinct roles of TRIOBP-4 and TRIOBP-5 in the stereocilia of the inner ear and deafness. Protein interaction partners implicated in the various processes are indicated in gray.



<sup>1</sup> While TRIOBP-5 and 6 likely have at least partially differing roles, no attempt was made to differentiate here due to lack of data. <sup>2</sup> Differences in exact role within the stereocilia. <sup>3</sup> This mouse knockout and these human mutations would also affect TRIOBP-5/6.

The role of TRIOBP-1 in actin dynamics appears to be to bind directly to F-actin [3] and inhibit its depolymerization [12]. This appears to be a general function of TRIOBP-1 in many cell types and organs, with specific roles including the linking of adhesion receptors at the cell surface [13,30], neuronal outgrowth [28], cell migration [19], and signal transduction to mechanical force in the heart [15]. An additional, or possibly alternative, mechanism is that TRIOBP-1 can affect actin in certain circumstances through inhibition of TRIO [13]. TRIOBP-5 and/or 6, which share all the functional domains of TRIOBP-1, are also present in the brain and so can be presumed to participate in many functions there, but not in other TRIOBP-1 expressing tissues such as the heart or liver [4,5]. In contrast, TRIOBP-4 is an actin-bundling protein that likely uses its lack of rigid structure to wrap around actin fibers in the stereocilia, binding using its R1 repeat motif, and bundle them together during rootlet formation and early stereocilia development [45,50]. TRIOBP-5 (and possibly TRIOBP-6) then has a similar, but distinct role, in which it further "sculpts" and maintains the actin core of the stereocilia [18]. TRIOBP-1 is also present in the inner ear and stereocilia [4,5,20], but is seemingly not required for stereocilia formation or hearing [18].

An area where the *TRIOBP* isoforms show greater overlap is in the pathology of cancer. *TRIOBP-1* is expressed in many cancer cells and tissues, while *TRIOBP-4* and *5*/*6* are more specialized, in partial analogy to their normal expression patterns [42–46], although not necessarily in the same tissue types. Notably, all are implicated in metastasis. Specifically, suppression of TRIOBP-1 and 5/6 expression appears to be a means through which *miR-3178* suppresses metastasis [46], while TRIOBP-4 and 5/6 are implicated the in cell motility of pancreatic cancer cells [45].

While much has therefore been uncovered regarding these proteins, many questions remain. The exact mechanism through which TRIOBP-1 modulates actin is still only partially understood, and its roles in various organs and cell types need further analysis. While both its potential roles in mental illness and cancer are tantalizing, the relationship between it and specific mental illnesses and cancer subtypes needs to be established in larger patient samples. The role of TRIOBP-4 in the stereocilia is understood in more detail, and the role of frameshift and nonsense mutations of *TRIOBP-4* in hearing loss is well established. Nevertheless, the apparent role of more common variants of *TRIOBP-4* in hearing remains to be explored, as does its function in the retina. Perhaps the largest unexplored area of TRIOBP research, however, concerns the other splice variants. Little is known about shorter 3′ variants such as *TRIOBP-2*. For longer variants, putative roles have been found in the inner ear, but their role in the brain and other tissues is unclear, as is the relationship between TRIOBP-5 and TRIOBP-6.

The *TRIOBP* locus therefore provides a fascinating example of how multiple parts of a gene can cooperate in a single function, actin stabilization, through the generation of many different functional splice variants with distinct expression patterns and modes of action. The variety of different human diseases and conditions related to it highlights its importance, however, much work still needs to be done to clarify the exact relationships between these isoforms and with human health.

**Author Contributions:** Writing—original draft, N.J.B.; Writing—review & editing, B.Z. and B.S.; Visualization, B.Z., B.S., and N.J.B.; Funding acquisition: N.J.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Croatian Science Foundation (HRZZ: Hrvatska zaklada za znanost), grant numbers IP-2018–01–9424, DOK-2018–09–5395, and DOK-2020–01–8580.

**Acknowledgments:** We thank Shin-ichiro Kitajiri for sharing additional details regarding *TRIOBP* mutations as well as our colleagues who have worked with us on TRIOBP-1, notably Carsten Korth, Antony Yerabham, Maja Odorˇci´c, and Anja Hart.

**Conflicts of Interest:** The authors declare no conflict of interest.
