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.

Isolation of dominant Arabidopsis seiv mutants defective in VND7-induced xylem vessel cell differentiation.

The plant-specific NAC transcription factor VASCULAR-RELATED NAC-DOMAIN 7 (VND7) functions in xylem vessel cell differentiation in Arabidopsis thaliana. To identify novel factors regulating xylem vessel cell differentiation, we previously performed ethyl methanesulfonate mutagenesis of a transgenic 35S::VND7-VP16-GR line in which VND7 activity can be induced post-translationally by glucocorticoid treatment. We successfully isolated mutants that fail to form ectopic xylem vessel cells named seiv (suppressor of ectopic vessel cell differentiation induced by VND7) mutants. Here, we isolated eight novel dominant seiv mutants: seiv2 to seiv9. In these seiv mutants, ectopic xylem vessel cell differentiation was inhibited in aboveground but not underground tissues. Specifically, the shoot apices of the mutants, containing shoot apical meristems and leaf primordia, completely lacked ectopic xylem vessel cells. In these mutants, the VND7-induced upregulation of downstream genes was reduced, especially in shoots compared to roots. However, endogenous xylem vessel cell formation was not affected in the seiv mutants. Therefore, the seiv mutations identified in this study have repressive effects on cell differentiation in shoot meristematic regions, resulting in inhibited ectopic xylem vessel cell differentiation.

Stable and transient transformation, and a promoter assay in the selective lignin-degrading fungus, Ceriporiopsis subvermispora.

A genetic transformation system was developed for the selective white rot basidiomycete Ceriporiopsis subvermispora using a modified protocol with polyethylene glycol and CaCl2 treatment of the protoplasts and plasmids harboring recombinant hygromycin phosphotransferase (hph) driven by a homologous promoter. During repeated transfer on fresh potato dextrose agar plates containing 100 µg/ml hygromycin B, most transformants lost drug resistance, while the remaining isolates showed stable resistance over five transfers. No drug-resistant colonies appeared in control experiments without DNA or using a promoter-less derivative of the plasmid, indicating that a transient expression of the recombinant hph was driven by the promoter sequence in these unstable drug-resistant transformants. Southern blot analysis of the stable transformants revealed random integration of the plasmid DNA fragment in the chromosome at different copy numbers. This transformation system yielding mostly transient transformants was successfully used for promoter assay experiments, and only a 141-bp fragment was found to be essential for the basic promoter function of glyceraldehyde dehydrogenase gene (gpd) in this fungus. Subsequent mutational analyses suggested that a TATAA sequence is important for the basic promoter function of gpd gene. The promoter assay system will enable the functional analysis of gene expression control sequences quickly and easily, mostly in the absence of undesirable effects from differences in copy number and chromosomal position of an integrated reporter gene among stable transformants.

Evidence that thiol-based redox state is critical for xylem vessel cell differentiation.

Nitric oxide (NO), which plays essential roles in a variety of cell signaling processes, is the precursor of a family of NO-derived molecules, including toxic reactive nitrogen species. The NO-based regulation of cellular activity is mediated by the reversible modification of cysteine thiol groups in redox-sensitive proteins. One such modification is protein S-nitrosylation, i.e., the addition of an NO moiety to a cysteine thiol, and this S-nitrosylation is regulated by enzymes such as S-nitrosoglutathione reductase (GSNOR). Recently, we reported a novel loss-of-function allele of gsnor1, named suppressor of ectopic vessel cell differentiation induced by VND7-1 (seiv1), based on the VND7-inducible system, in which almost all cell types are transdifferentiated into xylem vessel cells upon activation of the NAC transcription factor VND7. We also found that VND7 can be S-nitrosylated and that the target cysteine residues for S-nitrosylation are critical for VND7 transactivation activity. Here, we further discuss roles for GSNOR1 in xylem vessel cell differentiation, and provide additional data on the effects of cellular NO level on VND7 activity.

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.

Protein S-Nitrosylation Regulates Xylem Vessel Cell Differentiation in Arabidopsis.

Post-translational modifications of proteins have important roles in the regulation of protein activity. One such modification, S-nitrosylation, involves the covalent binding of nitric oxide (NO)-related species to a cysteine residue. Recent work showed that protein S-nitrosylation has crucial functions in plant development and environmental responses. In the present study, we investigated the importance of protein S-nitrosylation for xylem vessel cell differentiation using a forward genetics approach. We performed ethyl methanesulfonate mutagenesis of a transgenic Arabidopsis 35S::VND7-VP16-GR line in which the activity of VASCULAR-RELATED NAC-DOMAIN7 (VND7), a key transcription factor involved in xylem vessel cell differentiation, can be induced post-translationally by glucocorticoid treatment, with the goal of obtaining suppressor mutants that failed to differentiate ectopic xylem vessel cells; we named these mutants suppressor of ectopic vessel cell differentiation induced by VND7 (seiv) mutants. We found the seiv1 mutant to be a recessive mutant in which ectopic xylem cell differentiation was inhibited, especially in aboveground organs. In seiv1 mutants, a single nucleic acid substitution (G to A) leading to an amino acid substitution (E36K) was present in the gene encoding S-NITROSOGLUTATHIONE REDUCTASE 1 (GSNOR1), which regulates the turnover of the natural NO donor, S-nitrosoglutathione. An in vitro S-nitrosylation assay revealed that VND7 can be S-nitrosylated at Cys264 and Cys320 located near the transactivation activity-related domains, which were shown to be important for transactivation activity of VND7 by transient reporter assay. Our results suggest crucial roles for GSNOR1-regulated protein S-nitrosylation in xylem vessel cell differentiation, partly through the post-translational modification of VND7.