A Computational and Experimental Approach Reveals that the 5′-Proximal Region of the 5′-UTR has a Cis-Regulatory Signature Responsible for Heat Stress-Regulated mRNA Translation in Arabidopsis

Abstract

Translation of specific plant mRNAs is differentially regulated under certain abiotic stress conditions such as heat, oxygen deprivation and dehydration. The majority of transcripts exhibit varying degrees of translational repression, whereas a subset of transcripts escape such repression and remain actively translated. The underlying mechanisms that mediate this control, and in particular the identities of the regulatory RNA elements involved, remain poorly understood. Using a combined computational and experimental approach, we identified a novel cis-regulatory element in the 5'-untranslated region (5'-UTR) that affects differential translation in response to heat stress (HS) in Arabidopsis thaliana. First, we selected a set of genes with distinct translational responses to HS, based on our previously reported genome-wide data regarding changes in polysome loading induced by HS in A. thaliana cultured cells. We evaluated the 5'-UTRs of these messages for their ability to mediate expression, when fused to reporter mRNAs, in protoplasts under HS. The data from the reporter assay and the nucleotide sequences of the 5'-UTRs tested were used to define regulatory elements in the 5'-UTRs, with the help of a partial least square (PLS) regression model. The computational analysis using PLS and subsequent experimental characterization of a series of 5'-UTR mutants provided evidence that the 5'-proximal sequence of the 5'-UTR is a primary and position-dependent determinant of 5'-UTR-mediated differential translation in response to HS. Finally, we discuss the possible mechanism underlying HS regulation of differential mRNA translation.