Rhomboid proteases are involved in various cellular activities, from development to cancer, and cellular processes and substrates associated with rhomboid proteases or rhomboid-like proteins have been identified for a range of organisms. Plant rhomboid proteases or rhomboid-like proteins are the least understood of the group. Moreover, the general phenomenon of alternative splicing and rhomboid protein genes has yet to be studied robustly. This study thus focused on the alternative splicing events associated with the Arabidopsis rhomboid protein gene At1g25290. The patterns obtained through RT-PCR and DNA sequencing provided evidence of alternative splicing in the At1g25290 transcript population, especially in the region spanning exons 5 and 6. The levels of the two splice variants involving exons 5 and 6 appear to be sufficiently abundant to possess functional significance and appear to adjust relative to each other in different contexts. Adjustments were observed in tissues of different developmental stages, in an Arabidopsis plant bearing a mutation in another rhomboid protein, and in response to transgenic manipulations affecting the levels of Tic40, a plastid translocon component. The resulting change to the protein sequence may, in turn, affect how At1g25290 proteins interact with their substrates. Collectively, the evidence suggests that alternative splicing of At1g25290 bears functional significance in Arabidopsis.
Background: Rhomboid serine proteases are present across many 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 Arabidopsis showed that the number of rhomboid proteins working 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 Arabidopsis plastid rhomboids, there has yet to be an assessment as to whether this mechanism extends to other rhomboids and to other species. Methods: We thus conducted a comparative analysis of select databases to determine if the alternative splicing mechanism observed for the two Arabidopsis plastid rhomboids was utilized in other species to expand the repertoire of rhomboid proteins. To help verify the in silico observations, 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. Results: A comparison of database entries of six widely used eukaryotic experimental models (human, mouse, Arabidopsis, Drosophila, nematode, and yeast) revealed robust usage of alternative splicing to diversify rhomboid protein structure across the various motifs or regions, especially in human, mouse and Arabidopsis. Subsequent validation studies uncover evidence that the splice variants selected for testing displayed functionality in the different activity assays. Conclusions: The combined results support the hypothesis that alternative splicing is likely used to diversify and expand rhomboid protein functionality, and this potentially occurred across the various motifs or regions of the protein.
Background: Four distinct rhomboid genes appear to function in Arabidopsis plastids, two “active” types from the secretases and presenilin-like associated rhomboid-like (PARL) categories (At1g25290 and At5g25752) and two “inactive” rhomboid forms (At1g74130 and At1g74140). The number of working rhomboids is further increased by alternative splicing, two reported for At1g25290 and three for At1g74130. Since At1g25290 and At1g74130 exist as alternative splice variants, it would be necessary to assess the splicing patterns of the other two plastid rhomboid genes, At5g25752 and At1g74140, before studying the Arabidopsis plastid rhomboid system as a whole. Methods: This study thus specifically focused on an analysis of the At1g74140 transcript population using various RT-PCR strategies. Results: The exon mapping results indicate splicing patterns different from the close relative At1g74130, despite similarity between the exonic sequences. The splicing patterns indicate a high level of sequence “discontinuity” in the At1g74140 transcript population with a significant portion of the discontinuity being generated by two regions of the gene. Conclusion: The overall discontinuous splicing pattern of At1g74140 may be reflective of its mode of involvement in activities like controlling gene expression.
Rhomboid serine proteases are present in many species with Background: sequenced genomes, 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 inshowed that the number of rhomboid proteins working Arabidopsis simultaneously can be further diversified by alternative splicing. This phenomenon was confirmed for the plastid rhomboid proteins Arabidopsis At1g25290 and At1g74130. Although alternative splicing was determined to be a significant mechanism for diversifying these two plastid Arabidopsis rhomboids, there has yet to be an assessment as to whether this mechanism extends to other rhomboids and to other species.We thus conducted a multi-year analysis of databases to determine Methods: if the alternative splicing mechanism observed for the two plastid Arabidopsis rhomboids was utilized in other species to expand the repertoire of rhomboid proteins. To help verify the findings, select splice variants from different in silico 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.The multi-year assessment for six model experimental Results:in silico species (human, mouse, , , nematode, and yeast) Arabidopsis Drosophila 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 evidence that the splice Arabidopsis 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 protein.
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