Participation of Leaky Ribosome Scanning in Protein Dual Targeting by Alternative Translation Initiation in Higher Plants  

Abstract

Postendosymbiotic evolution has given rise to proteins that are multiply targeted within the cell. Various mechanisms have been identified to permit the expression of proteins encoding distinct N termini from a single gene. One mechanism involves alternative translation initiation (aTI). We previously showed evidence of aTI activity within the Arabidopsis thaliana organellar DNA polymerase gene POLg2. Translation initiates at four distinct sites within this gene, two non-AUG, to produce distinct plastid and mitochondrially targeted forms of the protein. To understand the regulation of aTI in higher plants, we used Polg2 as a model to investigate both cis-and trans-acting features of the process. Here, we show that aTI in Polg2 and other plant genes involves ribosome scanning dependent on sequence context at the multiple initiation sites to condition specific binding of at least one trans-acting factor essential for site recognition. Multiple active translation initiation sites appear to operate in several plant genes, often to expand protein targeting. In plants, where the mitochondrion and plastid must share a considerable portion of their proteomes and coordinate their functions, leaky ribosome scanning behavior provides adaptive advantage in the evolution of protein dual targeting and translational regulation.

Known and novel post-transcriptional regulatory sequences are conserved across plant families

“…This pattern, excepting the variable adenine of the canonical translation start codon, matches the strongest Kozak context for translation initiation in plants (Lukaszewicz et al 2000). In some prior cases, such sites are used to produce multiple protein isoforms for the same mRNA molecule, which can then be targeted to various organelles (Wamboldt et al 2009). Also, for seemingly different reasons, a key regulator of flowering time in plants, FCA, consistently uses CUG as an alternative translation initiation site (Simpson et al 2010).…”

Divergent Roles for the Two PolI-Like Organelle DNA Polymerases of Arabidopsis

“…In addition, the measured in vitro characteristics of these enzymes correspond almost perfectly to those measured ex vivo from the purified DNA-synthesis activity of mitochondria and plastids (Spencer and Whitfeld, 1969;Sala et al, 1980;McKown and Tewari, 1984;Heinhorst et al, 1990;Meissner et al, 1993). All these elements, plus the fact that they are present in both organelles (Elo et al, 2003;Christensen et al, 2005;Mori et al, 2005;Ono et al, 2007;Wamboldt et al, 2009), suggest that the PolI DNA polymerases are responsible for DNA replication in plant organelles.…”

“…Indeed, every plant genome that has been sequenced to date possesses two PolI-like genes which, in Arabidopsis and tobacco, encode proteins that localize to both organelles, thus suggesting an important conserved function (Elo et al, 2003;Christensen et al, 2005;Ono et al, 2007;Wamboldt et al, 2009). In addition, the recombinant proteins have a high processivity, which would allow them to replicate complete organelle genomes (Kimura et al, 2002;Mori et al, 2005;Ono et al, 2007).…”

Translational Regulation of Cytoplasmic mRNAs

“…Elements known to drive RNA localization (zipcodes) affect translation indirectly. Many translational control elements have been inferred on theoretical grounds, because they contain non-canonical start codons (Kobayashi et al, 2001;Wamboldt et al, 2009), a short coding region (uORF), or because they are conserved (Hayden and Jorgensen, 2007;Tran et al, 2008;Jorgensen and DorantesAcosta, 2012;Vaughn et al, 2012). In contrast, rather few plant translational control elements have been identified de novo by experimental structure-function analysis.…”