Characterization of two alternative splice variants associated with the Arabidopsis rhomboid protein gene At1g25290

Abstract

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.

Characterization of three alternative splice variants associated with theArabidopsisrhomboid protein geneAt1g74130

“…Confirmatory work related to the establishment of the At1g74130 mutant line was reported in Sedivy-Haley et al (2011). The construction of homozygous transgenic lines with altered Tic40 levels was reported in Ko et al (2005) and Sedivy-Haley et al (2012). The transgenic lines used here were 45-6 (very high Tic40 expression) and 59-3 (very low Tic40 expression) (the levels were disclosed in the supplementary material associated with the report by SedivyHaley et al 2012).…”

“…The number of working plastid rhomboids is further increased by alternative splicing, as reported for At1g25290 (Sedivy-Haley et al 2012). Alternative splicing of At1g25290 transcripts introduces into the population of At1g25290 rhomboid forms with and without a putative cyclin-binding RVL motif (Sedivy-Haley et al 2012). The use of alternative splicing to increase the number of rhomboid forms in play is not limited to Arabidopsis plastids.…”

“…Of the 13 expressed genes, there are four distinct plastid rhomboid genes predicted and at play in Arabidopsis plastids -two encoding active types (At1g25290 and At5g25752) and two encoding inactive forms (At1g74130 and At1g74140). The number of working plastid rhomboids is further increased by alternative splicing, as reported for At1g25290 (Sedivy-Haley et al 2012). Alternative splicing of At1g25290 transcripts introduces into the population of At1g25290 rhomboid forms with and without a putative cyclin-binding RVL motif (Sedivy-Haley et al 2012).…”

“…The results reported here advance our understanding of At1g74130's mode of operation and provide evidence that alternative splicing of At1g74130 bears functionality. Furthermore, the combined evidence from the two studies, At1g74130 (this study) and At1g25290 (Sedivy-Haley et al 2012), help uncover a potential mechanism for diversifying plastid rhomboid function.…”

“…Procedures and optimization details related to the analysis of transcript structure were the same as those described by Sedivy-Haley et al (2012). Briefly, all plant tissues destined for RNA extraction were collected between 1100 and 1300 during the light period to equalize the effects of circadian rhythms on transcript levels.…”