Noncanonical Translation Initiation of theArabidopsisFlowering Time and Alternative Polyadenylation Regulator FCA  

Abstract

The RNA binding protein FCA regulates the floral transition and is required for silencing RNAs corresponding to specific noncoding sequences in the Arabidopsis thaliana genome. Through interaction with the canonical RNA 39 processing machinery, FCA affects alternative polyadenylation of many transcripts, including antisense RNAs at the locus encoding the floral repressor FLC. This potential for widespread alteration of gene regulation clearly needs to be tightly regulated, and we have previously shown that FCA expression is autoregulated through poly(A) site choice. Here, we show distinct layers of FCA regulation that involve sequences within the 59 region that regulate noncanonical translation initiation and alter the expression profile. FCA translation in vivo occurs exclusively at a noncanonical CUG codon upstream of the first in-frame AUG. We fully define the upstream flanking sequences essential for its selection, revealing features that distinguish this from other non-AUG start site mechanisms. Bioinformatic analysis identified 10 additional Arabidopsis genes that likely initiate translation at a CUG codon. Our findings reveal further unexpected complexity in the regulation of FCA expression with implications for its roles in regulating flowering time and gene expression and more generally show plant mRNA exceptions to AUG translation initiation.

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

“…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). Our data confirm that non-AUG initiation requires a strong context.…”

Super-resolution ribosome profiling reveals unannotated translation events inArabidopsis

“…Although RiboTaper only searches for "AUG" as the start codon, we wanted to see whether our data can validate predicted ORFs that use a non-AUG start, such as a "CUG" codon (43). Among the predicted genes, AT3G10985 is highly expressed in our root samples.…”

Mechanism of Cytoplasmic mRNA Translation

“…The latter encodes a RNA polymerase targeted to the plastid or mitochondrion based on the AUG selected (Riechmann et al, 1999;Wamboldt et al, 2009;Simpson et al, 2010). Mutational studies that evaluated the ramifications of initiation codon context, secondary structure, 5'UTR length and presence of uORFs on the rate of initiation on the protein coding ORFs of plants have provided insight into use of an CUG triplet as a functional initiation codon for the FCA transcript (Simpson et al, 2010).…”

“…The latter encodes a RNA polymerase targeted to the plastid or mitochondrion based on the AUG selected (Riechmann et al, 1999;Wamboldt et al, 2009;Simpson et al, 2010). Mutational studies that evaluated the ramifications of initiation codon context, secondary structure, 5'UTR length and presence of uORFs on the rate of initiation on the protein coding ORFs of plants have provided insight into use of an CUG triplet as a functional initiation codon for the FCA transcript (Simpson et al, 2010). Advances in nucleotide-level resolution of ribosome position (Liu et al, 2013;Juntawong et al, 2014) and in vivo secondary structure (Ding et al, 2014) are likely to yield additional examples of non-AUG initiation and information on the surrounding mRNA region that will provide new insight into flexibility in start site selection in plants.…”