Conserved control signals in the transcriptome of higher plants.

Abstract

Understanding the mechanisms that regulate gene expression is an important goal in bioinformatic research. There are two major levels of gene regulation: transcriptional and post transcriptional control. Much attention has been directed to transcriptional control, but it is now clear that the untranslated regions (UTRs) of messenger RNA (mRNA) also play an important role in post-transcriptional control of gene expression. Two important control signals found in 5′-UTRs of both animal and plant mRNAs are stem-loop motifs and upstream open reading frames (uORFs). One strategy for identifying functional uORFs in plants is to use a comparative approach (Crowe et al. 2006; Hayden and Jorgensen 2007; Pavesi et al. 2007). There are extensive EST datasets for five important cereal crops (rice, wheat, barley, maize, and sorghum). Rice is the best characterised of these cereals with a sequenced genome (Yu et al. 2002) and a cDNA database containing 32,000 clones that are enriched for 5′ full-length sequences (Kikuchi et al. 2003). In this research, comparative R-nomics was used to identify conserved stem-loop motifs and uORFs in cereals using publicly available assembled EST data. To determine the prevalence of 5′-UTR stem-loop structures in plants a bioinformatics pipeline was developed to predict secondary structures. The pipeline used a program called RNAProfile to predict stem-loops that are conserved in both sequence and structure. The findings from this study concluded that conserved 5′-UTR stem-loops in long 5′-UTRs (200 to 1200 nt) are rare (~8%) in the cereal transcriptome, the genes themselves that contain conserved 5′-UTR stem-loop motifs are spread across different functions, and appear to have a biological role based on higher structure than sequence conservation in at least three out of four cereal species. Another control signal that is involved in post-transcriptional control is the uORF. A recent study in distantly related plants, such as rice and Arabidopsis, found that uORFs are rare in these transcriptomes (Hayden and Jorgensen 2007), but it is unclear how prevalent uORFs are in closely related plants. To address this question, the bioinformatics pipeline was modified to use a program called uORFSCAN to find conserved uORFs in five cereals that could potentially regulate translation. Major conclusions from this study are that the identified uORFs are highly conserved (50% median amino acid sequence similarity), are rare in cereal transcriptomes (<150 loci contain them), are generally short (less than 100 nt), position independent in their 5′-UTRs, and their start codon context and the usage of rare codons do not appear to be important for translation. Two candidate uORFs were selected for mutational analyses, and a quantitative in vitro transcription and translation system was used to determine if they function in translational control. The rice SAMDC small and S6K long uORFs were shown to be capable of down-regulating translation of a luciferase reporter gene. This study has provided evidence, for the first time, that the S6K uORF is involved in controlling translation. In conclusion, this study has identified new genes that may be controlled at the level of translation by stemloop motifs and conserved uORFs.