*4.1. The Structure of the Long Splice Variants*

While TRIOBP-1 and 4 share no common amino acid sequence with each other, they do with the longer TRIOBP splice variants. These contain the entire coding sequence of TRIOBP-1 and 4, except for the optionally translated extreme N-terminus of TRIOBP-1 (Figure 1). In human, the longest isoform, TRIOBP-6, is derived from a 24 exon long transcript, of which all but exons 1 and 24 are coding. This leads to a 2365 amino acid peptide, which forms the basis of numbering for all *TRIOBP* putative pathological mutations (Table 1). The majority of biological experiments, however, have instead focused on TRIOBP-5 (also called TRIOBP-3 in some earlier articles), which is a 2193 amino acid protein in humans. TRIOBP-5 is also the longest established isoform in mice. It derives from a transcript lacking exons 1 and 5, and whose open reading frame only begins on exon 6. This is because

the Kozak sequence used for *TRIOBP-6* is encoded across exons 1 and 2, and is therefore incomplete in *TRIOBP-5* transcripts. As a result, TRIOBP-5 begins its reading frame at the same point as TRIOBP-4, but is otherwise identical in the amino acid sequence to TRIOBP-6.

TRIOBP-6 has some isoform-specific amino acid sequence at its N-terminus, plus both it and TRIOBP-5 share some of the coding sequence, which lies between the coding exons of *TRIOBP-1* and *5* (Figure 1a,b). These additional sequences are predicted to be predominantly unstructured, with the exception of a possible short stretch of α-helix in the isoform specific N-terminus of TRIOBP-6, and another near the center of the long isoforms (Figure 1c). Neither of these regions show significant sequence similarity to known protein structures. The long variants are therefore predicted to be intrinsically disordered for most of their length, but with the PH domain and coiled-coil domains of TRIOBP-1 at their C-terminal ends. The coiled-coil regions of TRIOBP-5 have been shown to lead to oligomerization, in a similar manner to TRIOBP-1 [12,18]. These proteins also possess multiple actin binding domains, sharing both the R1 repeat of TRIOBP-4 and coiled-coil domains of TRIOBP-1.
