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 0 -untranslated region (5 0 -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 0 -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 0 -UTRs tested were used to define regulatory elements in the 5 0 -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 0 -UTR mutants provided evidence that the 5 0 -proximal sequence of the 5 0 -UTR is a primary and position-dependent determinant of 5 0 -UTR-mediated differential translation in response to HS. Finally, we discuss the possible mechanism underlying HS regulation of differential mRNA translation.

Germination Potential of Dormant and Nondormant Arabidopsis Seeds Is Driven by Distinct Recruitment of Messenger RNAs to Polysomes

“…This can be achieved by carrying out a series of mutations in 59UTR (Dvir et al, 2013;Kim et al, 2014) in order to identify important regions for differential mRNA translation during seed germination or dormancy maintenance. Such an approach has already proved to be efficient for demonstrating the role of 59UTR in the regulation of mRNA translation during heat stress in Arabidopsis (Khurana et al, 2013;Matsuura et al, 2013).…”

Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) Enables Time-Resolved Analysis of Protein Synthesis in Native Plant Tissue

“…We opted to study heat stress because it has been well characterized in Arabidopsis at the level of translation (Matsuura et al, 2010(Matsuura et al, , 2013Mittler et al, 2012), and because thermotolerance has been shown to be induced by heating seedlings to 38°C for 90 min (Larkindale and Vierling, 2008), a timescale that is easily accessible to BONCAT analysis (Fig. 2B).…”

Translational Regulation of Cytoplasmic mRNAs

“…To give one example, upon heat stress, heat shock protein mRNAs continue to be actively translated, whereas other mRNAs are sequestered into stress granules and translationally suppressed (Nover et al, 1989;Matsuura et al, 2010a;Sormani et al, 2011a). Some sequence elements responsible for continued translation under heat shock reside in the 5' UTR (Matsuura et al, 2013). For example, in Arabidopsis HSP81-3, a 47 nucleotide (nt) pyrimidine rich element stimulates cap-independent translation after heat shock (Matsuura et al, 2008).…”

“…To date, such elements qualify as cap-independent, i.e., they may still involve eIF4E and scanning (Vanderhaeghen et al, 2006;Mardanova et al, 2008). For example, the preferential translation of a heat shock protein mRNA at high temperature is mediated by a feature of its 5' UTR that renders its translation cap-independent (Stuger et al, 1999;Dinkova et al, 2005;Matsuura et al, 2008;Matsuura et al, 2013).…”