An initial pre-mRNA comprising of the entire gene is initially transcribed. Harnessing the sequences attained from the above genes, we designed two novel 5′ mRNA tags (TriTag-1 and TriTag-2) that targeted the translated GFP to chloroplast, peroxisome and/or cytosol using alternative splicing (Figure 1).
The translated proteins are targeted to the peroxisome if they retain the internal PTS2 site and to the cytosol if the site is removed. At least two spliceforms are produced from TTL from internal alternative acceptor junctions. This synthase catalyzes two steps in the allantoin biosynthesis pathway. The spliceforms produced from the 3′ transcription initiation site target the protein to the chloroplast when the targeting sequence is retained, and to the cytosol when it is not.Ī peroxisome targeting sequence, PTS2, containing the RLx 5HL nonapeptide, was taken from the transthyretin-like S-allantoin synthase gene ( TTL At5g58220). In nature, different isoforms are often produced from an individual gene, via the exclusion or inclusion of coding sequences from its mRNA by alternative splicing. Various mRNAs produced from PIMT2 are produced by alternative transcription initiation sites and alternative splicing events. PIMT2 is a ubiquitous repair protein, converting exposed isoaspartate residues to aspartate or asparagine residues in aging polypeptides. To construct TriTag-1 and TriTag-2, a chloroplast-targeting region (CTPa) was taken from protein-L-isoaspartate methyltransferase ( PIMT2, At5g50240). ĭesign for multiple-compartment localization by alternative splicing: TriTag-1 and TriTag-2 This combination of organelles is particularly interesting due to their close functional association in photorespiration, isoprenoid biosynthesis, β-oxidation and other metabolic processes. Here we describe a simple technique for targeting of transgenic proteins to multiple organelles, specifically the chloroplast, peroxisome, and cytosol. Although dual targeting to certain organelles may instead be achieved by adding a second localization peptide, this approach is limited to the possible combinations that can be made from available N- and C-terminal extensions. Coordinate expression may not be ensured due to context-dependent regulatory effects and/or homology-based silencing. These procedures are time-intensive and yield transformants with multiple spatially distinct copies of a protein expression cassette. Each copy must be introduced by successive retransformation, or alternatively, by backcrossing single transforms. One approach to target proteins to more than one location involves cloning multiple genetic copies, each containing a different localization peptide.
Issues around protein targeting have arisen in (1) studying protein function in a coordinated fashion, (2) improving holistic plant metabolic engineering efforts and (3) increasing yields attained by molecular farming and other protein factory applications. For example, re-engineering photorespiration and isoprenoid synthesis will involve both the chloroplasts and peroxisomes.Ī number of synthetic N-terminal and C-terminal extensions are readily available to target heterologous proteins to desired subcellular compartments, such as the chloroplast, peroxisome, mitochondrion, endoplasmic reticulum or the nucleus.
To enable complex metabolic engineering, plant engineers will require tools to direct single transgenes to multiple compartments. Plant cells harbor many distinct compartments that share some overlapping function, or are functionally associated in metabolic pathways and development. This work harnesses alternative splicing and signal embedding for engineering plants to express multi-functional proteins from single genetic constructs. Our novel signal sequences can reduce the number of cloning steps and the amount of genetic material required to target a heterologous protein to multiple locations in plant cells. TriTag-3 embeds a conserved peroxisomal targeting signal within a chloroplast transit peptide, directing GFP to the chloroplasts and peroxisomes. TriTag-1 shows a bias for targeting the chloroplast envelope while TriTag-2 preferentially targets the peroxisomes. TriTag-1 and TriTag-2 use alternative splicing to generate differentially localized GFP isoforms, localizing it to the chloroplasts, peroxisomes and cytosol. We designed novel hybrid signal sequences for multiple-compartment localization and characterize their function when fused to GFP in Nicotiana benthamiana leaf tissue. Plant bioengineers require simple genetic devices for predictable localization of heterologous proteins to multiple subcellular compartments.
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