Sequence features around cleavage sites are highly conserved among different species and a critical determinant for RNA cleavage position across eukaryotes.

RNA degradation is one of the critical steps for control of gene expression, and endonucleolytic cleavage-dependent RNA degradation is conserved among eukaryotes. Some cleavage sites are secondarily capped in the cytoplasm and identified using the Cap analysis of gene expression (CAGE) method. Although uncapped cleavage sites are widespread in eukaryotes, comparatively little information has been obtained about these sites using CAGE-based degradome analysis. Previously, we developed the truncated RNA-end sequencing (TREseq) method in plant species and used it to acquire comprehensive information about uncapped cleavage sites; we observed G-rich sequences near cleavage sites. However, it remains unclear whether this finding is general to other eukaryotes. In this study, we conducted TREseq analyses in fruit flies (Drosophila melanogaster) and budding yeast (Saccharomyces cerevisiae). The results revealed specific sequence features related to RNA cleavage in D. melanogaster and S. cerevisiae that were similar to sequence patterns in Arabidopsis thaliana. Although previous studies suggest that ribosome movements are important for determining cleavage position, feature selection using a random forest classifier showed that sequences around cleavage sites were major determinant for cleaved or uncleaved sites. Together, our results suggest that sequence features around cleavage sites are critical for determining cleavage position, and that sequence-specific endonucleolytic cleavage-dependent RNA degradation is highly conserved across eukaryotes.

Methods for detecting RNA degradation intermediates in plants.

RNA degradation is an important process for controlling gene expression and is mediated by decapping / deadenylation-dependent or endonucleolytic cleavage-dependent RNA degradation mechanisms. High-throughput sequencing of RNA degradation intermediates was initially developed in Arabidopsis thaliana and similar RNA degradome sequencing methods were conducted in other eukaryotes. However, interpreting results obtained by these sequencing methods is fragmented, and an overview is needed. Here we review the findings and limitations of these sequencing methods and discuss the missing experiments needed to understand RNA degradation intermediates accurately. This review provides direction for future research on RNA degradation and is a reference for RNA degradome studies in other species.

Feature selection for RNA cleavage efficiency at specific sites using the LASSO regression model in Arabidopsis thaliana.

BACKGROUND: RNA degradation is important for the regulation of gene expression. Despite the identification of proteins and sequences related to deadenylation-dependent RNA degradation in plants, endonucleolytic cleavage-dependent RNA degradation has not been studied in detail. Here, we developed truncated RNA end sequencing in Arabidopsis thaliana to identify cleavage sites and evaluate the efficiency of cleavage at each site. Although several features are related to RNA cleavage efficiency, the effect of each feature on cleavage efficiency has not been evaluated by considering multiple putative determinants in A. thaliana. RESULTS: Cleavage site information was acquired from a previous study, and cleavage efficiency at the site level (CSsite value), which indicates the number of reads at each cleavage site normalized to RNA abundance, was calculated. To identify features related to cleavage efficiency at the site level, multiple putative determinants (features) were used to perform feature selection using the Least Absolute Shrinkage and Selection Operator (LASSO) regression model. The results indicated that whole RNA features were important for the CSsite value, in addition to features around cleavage sites. Whole RNA features related to the translation process and nucleotide frequency around cleavage sites were major determinants of cleavage efficiency. The results were verified in a model constructed using only sequence features, which showed that the prediction accuracy was similar to that determined using all features including the translation process, suggesting that cleavage efficiency can be predicted using only sequence information. The LASSO regression model was validated in exogenous genes, which showed that the model constructed using only sequence information can predict cleavage efficiency in both endogenous and exogenous genes. CONCLUSIONS: Feature selection using the LASSO regression model in A. thaliana identified 155 features. Correlation coefficients revealed that whole RNA features are important for determining cleavage efficiency in addition to features around the cleavage sites. The LASSO regression model can predict cleavage efficiency in endogenous and exogenous genes using only sequence information. The model revealed the significance of the effect of multiple determinants on cleavage efficiency, suggesting that sequence features are important for RNA degradation mechanisms in A. thaliana.

Changes in mRNA Degradation Efficiencies under Varying Conditions Are Regulated by Multiple Determinants in Arabidopsis thaliana.

Multiple mechanisms are involved in gene expression, with mRNA degradation being critical for the control of mRNA accumulation. In plants, although some trans-acting factors and motif sequences have been identified in deadenylation-dependent mRNA degradation, endonucleolytic cleavage-dependent mRNA degradation has not been studied in detail. Previously, we developed truncated RNA-end sequencing (TREseq) in Arabidopsis thaliana and detected G-rich sequence motifs around 5' degradation intermediates. However, it remained to be elucidated whether degradation efficiencies of 5' degradation intermediates in A. thaliana vary among growth conditions and developmental stages. To address this issue, we conducted TREseq of cultured cells under heat stress and at three developmental stages (seedlings, expanding leaves and expanded leaves) and compared 5' degradation intermediates data among the samples. Although some 5' degradation intermediates had almost identical degradation efficiencies, others differed among conditions. We focused on the genes and sites whose degradation efficiencies differed. Changes in degradation efficiencies at the gene and site levels revealed an effect on mRNA accumulation in all comparisons. These changes in degradation efficiencies involved multiple determinants, including mRNA length and translation efficiency. These results suggest that several determinants govern the efficiency of mRNA degradation in plants, helping the organism to adapt to varying conditions by controlling mRNA accumulation.

Different Plant Species Have Common Sequence Features Related to mRNA Degradation Intermediates.

mRNA degradation is an important cellular mechanism involved in the control of gene expression. Several genome-wide profiling methods have been developed for detecting mRNA degradation in plants and animals. However, because many of these techniques use poly (A) mRNA for library preparation, degradation intermediates are often only detected near the 3'-ends of transcripts. Previously, we developed the Truncated RNA End Sequencing (TREseq) method using Arabidopsis thaliana, and demonstrated that this method ameliorates 3'-end bias. In analyses using TREseq, we observed G-rich sequences near the 5'-ends of degradation intermediates. However, this finding remained to be confirmed in other plant species. Hence, in this study, we conducted TREseq analyses in Lactuca sativa (lettuce), Oryza sativa (rice) and Rosa hybrida (rose). These species including A. thaliana were selected to encompass a diverse range in the angiosperm phylogeny. The results revealed similar sequence features near the 5'-ends of degradation intermediates, and involvement of translation process in all four species. In addition, homologous genes have similar efficiencies of mRNA degradation in different plants, suggesting that similar mechanisms of mRNA degradation are conserved across plant species. These strong sequence features were not observed in previous degradome analyses among different species in plants.

Comprehensive analysis of mRNA internal cleavage sites in Arabidopsis thaliana.

The major obstacle of efficient transgene expression seems to be gene silencing, and one of the important factors in gene silencing is mRNA stability. Regulation of mRNA stability is an important aspect of the control of gene expression. mRNAs are degraded by both exonucleolytic digestion and endonucleolytic cleavage. However, with the exception of small RNA-guided cleavage, the mechanisms underlying endonucleolytic cleavage-dependent RNA degradation remain to be elucidated. High-throughput approaches for genome-wide profiling of RNA cleavage sites, collectively termed degradome sequencing, have been developed by several groups. These analyses have contributed to the identification of mRNA cleavage sites in plants, but due to selection of poly (A) mRNA in library preparation, these approaches cannot identify cleavage sites in a fully accurate manner. To address this issue, we developed a new experimental method, truncated RNA end sequencing (TREseq), which enabled us to accurately identify many cleavage sites. TREseq can also be used to estimate the efficiency of mRNA cleavage, revealing differences in base frequencies near cleavage sites that reflect differences in cleavage efficiency. These results will contribute to gain important knowledge about the stability of the transgene mRNA in the future.

Identification of 5'-untranslated regions that function as effective translational enhancers in monocotyledonous plant cells using a novel method of genome-wide analysis.

High expression of a transgene is often necessary to produce useful substances in plants. The efficiency of mRNA translation is an important determinant of the level of transgene expression. In dicotyledonous plants, the 5'UTR of certain mRNAs act as translational enhancers, dramatically improving transgene expression levels. On the other hand, translation enhancers derived from dicotyledonous plants are not so much effective in monocotyledonous plants, which are important as industrial crops and as hosts for production of useful substances. In this study, we evaluated the polysome association on a large scale with high resolution for each 5'UTR variant from multiple transcription start site in normal and heat-stressed Oryza sativa suspension cultures. Translational enhancer candidates were selected from the resultant large-scale data set, and their enhancer activities were evaluated by transient expression assay. In this manner, we obtained several translational enhancers with significantly higher activities than previously reported enhancers. Their activities were confirmed in a different monocotyledonous plant, Secale cereale, and using a different reporter gene. In addition, enhancer activities of tested 5'UTRs were different between monocotyledonous and dicotyledonous plants, suggesting that the enhancer activities were not compatible between them. Overall, we demonstrate these useful 5'UTRs as enhancer sequence for transgene expression in monocotyledonous plants.

Arabidopsis thaliana cold-regulated 47 gene 5'-untranslated region enables stable high-level expression of transgenes.

Transgene expression is regulated through several steps, this study focuses on the mRNA translation step. The expression level of transgenes can be increased by 5'-untranslated region (5'UTR) sequences in certain genes which act as translational enhancers. On the other hand, translation in most mRNA species is repressed by growth, development, and stress events. There is a possibility that transgene mRNA is also repressed in these conditions, despite the use of a translational enhancer. Therefore, a consistently efficient translational enhancer is needed to develop a reliable transgene expression system. Herein we searched for mRNAs translated stably under different growth, development and environmental conditions using data sets of polysome fraction assays and microarray analysis. Correct 5'UTR sequences of candidate genes were determined by cap analysis of gene expression and we tested translational ability of the candidate 5'UTRs by reporter assays. We found the 5'UTR of cold-regulated 47 gene to be an effective translational enhancer, contributing to stable high-level expression under various conditions.

Changes in Polysome Association of mRNA Throughout Growth and Development in Arabidopsis thaliana.

Translational control is a key regulatory step in the expression of genes as proteins. In plant cells, the translational efficiency of mRNAs differs for different mRNA species, and the efficiency dynamically changes in various conditions. To gain a global view of translational control throughout growth and development, we performed genome-wide analysis of polysome association of mRNA during growth and leaf development in Arabidopsis thaliana by subjecting the mRNAs in polysomes to DNA microarray. This analysis revealed that the degree of polysome association of mRNA was different depending on the mRNA species, and the polysome association changed greatly throughout growth and development for each. In the growth stage, transcripts showed varying changes in polysome association from strongly depressed to unchanged, with the majority of transcripts showing dissociation from ribosomes. On the other hand, during leaf development, the polysome association of transcripts showed a normal distribution from repressed to activated mRNAs when comparing expanding and expanded leaves. In addition, functional category analysis of the microarray data suggested that translational control has a physiological significance in the plant growth and development process, especially in the categories of signaling and protein synthesis. In addition to this, we compared changes in polysome association of mRNAs between various conditions and characterized translational controls in each. This result suggested that mRNA translation might be controlled by complicated mechanisms for response to each condition. Our results highlight the importance of dynamic changes in mRNA translation in plant development and growth.

Efficient transgene expression by alleviation of translational repression in plant cells.

Global translational repression under abiotic stress influences translation of both endogenous and transgene mRNAs. Even in plant cell culture, hypoxia and nutrient deficient stress arise during the growth process. In this study, we first demonstrated the existence of global translational repression in Arabidopsis T87 cultured cells over a time course following inoculation. Next, we performed genome-wide analysis, which revealed that the translational states of endogenous mRNAs differed significantly between growth and stationary phase cells. This analysis showed that translation from most mRNAs was repressed upon stationary phase. Otherwise, a part of mRNA including alcohol dehydrogenase (ADH) gene was recalcitrant to the repression. Furthermore, by polysome analysis and followed quantitative reverse transcription PCR analysis of transformants having 5'untranslated regions (UTRs) of ADH or translationally repressed At3g47610 mRNA fused to reporter gene, we demonstrated that polysomal associations of reporter mRNAs were in accordance with those the mRNAs from which their 5'UTR derived, suggesting that the 5'UTR is an important determinant of the translational state of mRNAs in stationary phase cells. Finally, we demonstrated the effectiveness of 5'UTR of ADH mRNA in transformants derived from the BY-2 tobacco cell line. These results suggested that 5'UTR of ADH mRNA would be a useful element for efficient transgene expression upon stationary phase.