Rhomboid serine proteases are present across many Background: species and are often encoded in each species by more than one predicted gene. Based on protein sequence comparisons, rhomboids can be differentiated into groups -secretases, presenilin-like associated rhomboid-like (PARL) proteases, iRhoms, and "inactive" rhomboid proteins. Although these rhomboid groups are distinct, the different types can operate simultaneously. Studies in showed that the number of Arabidopsis rhomboid proteins

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... rking simultaneously can be further diversified by alternative splicing. This phenomenon was confirmed for the Arabidopsis plastid rhomboid proteins At1g25290 and At1g74130. Although alternative splicing was determined to be a significant mechanism for diversifying these two plastid rhomboids, there has yet to be an Arabidopsis assessment as to whether this mechanism extends to other rhomboids and to other species. We thus conducted a comparative analysis of select databases Methods: to determine if the alternative splicing mechanism observed for the two plastid rhomboids was utilized in other species to expand the Arabidopsis repertoire of rhomboid proteins. To help verify the observations, in silico select splice variants from different groups were tested for activity using transgenic-and additive-based assays. These assays aimed to uncover evidence that the selected splice variants display capacities to influence processes like antimicrobial sensitivity. A comparison of database entries of six widely used eukaryotic Results: experimental models (human, mouse, , , nematode, Arabidopsis Drosophila and yeast) revealed robust usage of alternative splicing to diversify rhomboid protein structure across the various motifs or regions, especially in human, mouse and . Subsequent validation studies uncover Arabidopsis evidence that the splice variants selected for testing displayed functionality in the different activity assays. The combined results support the hypothesis that alternative Conclusions: splicing is likely used to diversify and expand rhomboid protein functionality, and this potentially occurred across the various motifs or regions of the Reviewer Status splicing is likely used to diversify and expand rhomboid protein functionality, and this potentially occurred across the various motifs or regions of the protein.