Changing transcriptional initiation sites and alternative 5′‐ and 3′‐splice site selection of the first intron deploys Arabidopsis PROTEIN ISOASPARTYL METHYLTRANSFERASE2 variants to different subcellular compartments

Abstract

SummaryArabidopsis thaliana (L.) Heynh. possesses two PROTEIN-L-ISOASPARTATE METHYLTRANSFERASE (PIMT) genes encoding enzymes (EC 2.1.1.77) capable of converting uncoded L-isoaspartyl residues, arising spontaneously at L-asparaginyl and L-aspartyl sites in proteins, to L-aspartate. PIMT2 produces at least eight transcripts by using four transcriptional initiation sites (TIS; resulting in three different initiating methionines) and both 5¢-and 3¢-alternative splice site selection of the first intron. The transcripts produce mature proteins capable of converting L-isoaspartate to L-aspartate in small peptide substrates. PIMT:GFP fusion proteins generated a detectable signal in the nucleus. However, whether the protein was also detectable in the cytoplasm, endo-membrane system, chloroplasts, and/or mitochondria, depended on the transcript from which it was produced. On-blot-methylation of proteins, prior to the completion of germination, indicated that cruciferin subunits contain isoaspartate. The implications of using transcriptional mechanisms to expand a single gene's repertoire to protein variants capable of entry into the cell's various compartments are discussed in light of PIMT's presumed role in repairing the proteome.

PROTEIN l-ISOASPARTYL METHYLTRANSFERASE2 Is Differentially Expressed in Chickpea and Enhances Seed Vigor and Longevity by Reducing Abnormal Isoaspartyl Accumulation Predominantly in Seed Nuclear Proteins

“…Previously, AtPIMT1 was also shown to be cytosolic and AtPIMT2 as primarily nucleus localized, although various splice variants and transcriptional start site variants were shown to have adopted different subcellular localizations, including mitochondria and plastid (Xu et al, 2004;Dinkins et al, 2008). PIMT activity and protein have been reported to localize to the nucleus in other organisms as well (O'Connor, 1987;O'Connor and Germain, 1987).…”

“…Previous reports based on the transiently expressed GFP fusion proteins revealed differential subcellular localization of AtPIMT1 and AtPIMT2s (Xu et al, 2004;Dinkins et al, 2008). Interestingly, AtPIMT2 transcriptional start sites were shown to target different subcellular compartments.…”

“…Interestingly, two differentially regulated PIMT coding genes (AtPIMT1 and AtPIMT2) have been reported in Arabidopsis (Xu et al, 2004;Dinkins et al, 2008), in contrast to bacterial or animal systems, where such enzymes are encoded by a single gene.…”

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

“…These different messenger RNAs can encode for different isoforms of the same protein, or in the case of a frameshift, a different protein altogether. Although alternative splicing has been described in the literature for many years, the mechanisms of action and conserved donor and receptor splice sites are only being elucidated more recently 6 . As these sites are being better described, they open up opportunities for engineering.…”

“…For example, re-engineering photorespiration 7,8 and isoprenoid synthesis 9,10 involves both the chloroplasts and peroxisomes. In our case we took advantage of splice sites as observed in a natural Arabidopsis thaliana system described previously 6 . We rationally redesigned , and a peroxisome targeting sequence from Arabidopsis thaliana transthyretin-like S-allantoin synthase 16,17 were determined.…”