Shu-Hsing Wu

22-nucleotide RNAs trigger secondary siRNA biogenesis in plants

The effect of RNA silencing in plants can be amplified if the production of secondary small interfering RNAs (siRNAs) is triggered by the interaction of microRNAs (miRNAs) or siRNAs with a long target RNA. miRNA and siRNA interactions are not all equivalent, however; most of them do not trigger secondary siRNA production. Here we use bioinformatics to show that the secondary siRNA triggers are miRNAs and transacting siRNAs of 22 nt, rather than the more typical 21-nt length. Agrobacterium-mediated transient expression in Nicotiana benthamiana confirms that the siRNA-initiating miRNAs, miR173 and miR828, are effective as triggers only if expressed in a 22-nt form and, conversely, that increasing the length of miR319 from 21 to 22 nt converts it to an siRNA trigger. We also predicted and validated that the 22-nt miR771 is a secondary siRNA trigger. Our data demonstrate that the function of small RNAs is influenced by size, and that a length of 22 nt facilitates the triggering of secondary siRNA production.

Bioinformatic prediction and experimental validation of a microRNA-directed tandem trans-acting siRNA cascade in Arabidopsis

Small RNAs play pivotal roles in regulating gene expression in higher eukaryotes. Among them, trans-acting siRNAs (ta-siRNAs) are a class of small RNAs that regulate plant development. The biogenesis of ta-siRNA depends on microRNA-targeted cleavage followed by the DCL4-mediated production of small RNAs phased in 21-nt increments relative to the cleavage site on both strands. To find TAS genes, we have used these characteristics to develop the first computational algorithm that allows for a comprehensive search and statistical evaluation of putative TAS genes from any given small RNA database. A search in Arabidopsis small RNA massively parallel signature sequencing (MPSS) databases with this algorithm revealed both known and previously unknown ta-siRNA-producing loci. We experimentally validated the biogenesis of ta-siRNAs from two PPR genes and the trans-acting activity of one of the ta-siRNAs. The production of ta-siRNAs from the identified PPR genes was directed by the cleavage of a TAS2-derived ta-siRNA instead of by microRNAs as was reported previously for TAS1a, -b, -c, TAS2, and TAS3 genes. Our results indicate the existence of a small RNA regulatory cascade initiated by miR173-directed cleavage and followed by the consecutive production of ta-siRNAs from two TAS genes.

The Arabidopsis B-Box Zinc Finger Family

The Arabidopsis thaliana genome encodes >1500 transcription factors, and ∼45% of these belong to families specific to plants ([Riechmann et al., 2000][1]). Comparison of the entire complement of transcription factors of Arabidopsis , Drosophila melanogaster , Caenorhabditis elegans , and

Natural selection on cis and trans regulation in yeasts

Gene expression is regulated both by cis elements, which are DNA segments closely linked to the genes they regulate, and by trans factors, which are usually proteins capable of diffusing to unlinked genes. Understanding the patterns and sources of regulatory variation is crucial for understanding phenotypic and genome evolution. Here, we measure genome-wide allele-specific expression by deep sequencing to investigate the patterns of cis and trans expression variation between two strains of Saccharomyces cerevisiae. We propose a statistical modeling framework based on the binomial distribution that simultaneously addresses normalization of read counts derived from different parents and estimating the cis and trans expression variation parameters. We find that expression polymorphism in yeast is common for both cis and trans, though trans variation is more common. Constraint in expression evolution is correlated with other hallmarks of constraint, including gene essentiality, number of protein interaction partners, and constraint in amino acid substitution, indicating that both cis and trans polymorphism are clearly under purifying selection, though trans variation appears to be more sensitive to selective constraint. Comparing interspecific expression divergence between S. cerevisiae and S. paradoxus to our intraspecific variation suggests a significant departure from a neutral model of molecular evolution. A further examination of correlation between polymorphism and divergence within each category suggests that cis divergence is more frequently mediated by positive Darwinian selection than is trans divergence.

Molecular Analyses of the Arabidopsis TUBBY-Like Protein Gene Family

In mammals, TUBBY-like proteins play an important role in maintenance and function of neuronal cells during postdifferentiation and development. We have identified a TUBBY-like protein gene family with 11 members in Arabidopsis, named AtTLP1-11. Although seven of the AtTLP genes are located on chromosome I, no local tandem repeats or gene clusters are identified. Except for AtTLP4, reverse transcription-PCR analysis indicates that all these genes are expressed in various organs in 6-week-old Arabidopsis. AtTLP1, 2, 3, 6, 7, 9, 10, and 11 are expressed ubiquitously in all the organs tested, but the expression of AtTLP5 and 8 shows dramatic organ specificity. These 11 family members share 30% to 80% amino acid similarities across their conserved C-terminal tubby domains. Unlike the highly diverse N-terminal region of animal TUBBY-like proteins, all AtTLP members except AtTLP8 contain a conserved F-box domain (51–57 residues). The interaction between AtTLP9 and ASK1 (Arabidopsis Skp1-like 1) is confirmed via yeast (Saccharomyces cerevisiae) two-hybrid assays. Abscisic acid (ABA)-insensitive phenotypes are observed for two independent AtTLP9 mutant lines, whereas transgenic plants overexpressing AtTLP9 are hypersensitive to ABA. These results suggest that AtTLP9 may participate in the ABA signaling pathway.

LZF1, a HY5-regulated transcriptional factor, functions in Arabidopsis de-etiolation

We surveyed differential gene expression patterns during early photomorphogenesis in both wild-type and mutant Arabidopsis defective in HY5, an influential positive regulator of the responses of gene expression to a light stimulus, to identify light-responsive genes whose expression was HY5 dependent. These gene-expression data identified light-regulated zinc finger protein 1 (LZF1), a gene encoding a previously uncharacterized C2C2-CO B-box transcriptional regulator. HY5 has positive trans-activating activity toward LZF1 and binding affinity to LZF1 promoter in vivo. HY5 is needed but not sufficient for the induction of LZF1 expression. Anthocyanin content is significantly diminished in lzf1 under far red, which is the most efficient light for the induction of LZF1. The expression of PAP1/MYB75 is elevated in plants overexpressing LZF1, which leads to the hyperaccumulation of anthocyanin in transgenic Arabidopsis. The transition from etioplast to chloroplast and the accumulation of chlorophyll were notably compromised in the lzf1 mutant. We provide molecular evidence that LZF1 influences chloroplast biogenesis and function via regulating genes encoding chloroplast proteins. In the absence of HY5, mutation of LZF1 leads to further reduced light sensitivity for light-regulated inhibition of hypocotyl elongation and anthocyanin and chlorophyll accumulation. Our data indicate that LZF1 is a positive regulator functioning in Arabidopsis de-etiolation.

Widespread translational control contributes to the regulation of Arabidopsis photomorphogenesis

Environmental ‘light’ has a vital role in regulating plant growth and development. Transcriptomic profiling has been widely used to examine how light regulates mRNA levels on a genome‐wide scale, but the global role of translational regulation in the response to light is unknown. Through a transcriptomic comparison of steady‐state and polysome‐bound mRNAs, we reveal a clear impact of translational control on thousands of genes, in addition to transcriptomic changes, during photomorphogenesis. Genes encoding ribosomal protein are preferentially regulated at the translational level, which possibly contributes to the enhanced translation efficiency. We also reveal that mRNAs regulated at the translational level share characteristics of longer half‐lives and shorter cDNA length, and that transcripts with a cis‐element, TAGGGTTT, in their 5′ untranslated region have higher translatability. We report a previously neglected aspect of gene expression regulation during Arabidopsis photomorphogenesis. The identities and molecular signatures associated with mRNAs regulated at the translational level also offer new directions for mechanistic studies of light‐triggered translational enhancement in Arabidopsis.

Translational Landscape of Photomorphogenic Arabidopsis

This work describes translational activity at single-nucleotide resolution in deetiolating Arabidopsis. Light selectively triggers the increase or decrease of ribosome density of specific transcripts. This work also reports the global effects of upstream open reading frames and microRNAs in negatively regulating the translation of annotated open reading frames in deetiolating Arabidopsis. Translational control plays a vital role in regulating gene expression. To decipher the molecular basis of translational regulation in photomorphogenic Arabidopsis thaliana, we adopted a ribosome profiling method to map the genome-wide positions of translating ribosomes in Arabidopsis etiolated seedlings in the dark and after light exposure. We found that, in Arabidopsis, a translating ribosome protects an ∼30-nucleotide region and moves in three-nucleotide periodicity, characteristics also observed in Saccharomyces cerevisiae and mammals. Light enhanced the translation of genes involved in the organization and function of chloroplasts. Upstream open reading frames initiated by ATG but not CTG mediated translational repression of the downstream main open reading frame. Also, we observed widespread translational repression of microRNA target genes in both light- and dark-grown Arabidopsis seedlings. This genome-wide characterization of transcripts undergoing translation at the nucleotide-resolution level reveals that a combination of multiple translational mechanisms orchestrates and fine-tunes the translation of diverse transcripts in plants with environmental responsiveness.

Two New Clock Proteins, LWD1 and LWD2, Regulate Arabidopsis Photoperiodic Flowering

The “light” signal from the environment sets the circadian clock to regulate multiple physiological processes for optimal rhythmic growth and development. One such process is the control of flowering time by photoperiod perception in plants. In Arabidopsis (Arabidopsis thaliana), the flowering time is determined by the correct interconnection of light input and signal output by the circadian clock. The identification of additional clock proteins will help to better dissect the complex nature of the circadian clock in Arabidopsis. Here, we show LIGHT-REGULATED WD1 (LWD1)/LWD2 as new clock proteins involved in photoperiod control. The lwd1lwd2 double mutant has an early-flowering phenotype, contributed by the significant phase shift of CONSTANS (CO), and, therefore, an increased expression of FLOWERING LOCUS T (FT) before dusk. Under entrainment conditions, the expression phase of oscillator (CIRCADIAN CLOCK ASSOCIATED1 [CCA1], LATE ELONGATED HYPOCOTYL [LHY], TIMING OF CAB EXPRESSION1 [TOC1], and EARLY FLOWERING4 [ELF4]) and output (GIGANTEA, FLAVIN-BINDING, KELCH REPEAT, F-BOX1, CYCLING DOF FACTOR1, CO, and FT) genes in the photoperiod pathway shifts approximately 3 h forward in the lwd1lwd2 double mutant. Both the oscillator (CCA1, LHY, TOC1, and ELF4) and output (COLD, CIRCADIAN RHYTHM, AND RNA BINDING2 and CHLOROPHYLL A/B-BINDING PROTEIN2) genes have a short period length in the lwd1lwd2 double mutant. Our data imply that LWD1/LWD2 proteins function in close proximity to or within the circadian clock for photoperiodic flowering control.

COP1-mediated degradation of BBX22/LZF1 optimizes seedling development in Arabidopsis

Light regulates multiple aspects of growth and development in plants. Transcriptomic changes govern the expression of signaling molecules with the perception of light. Also, the 26S proteasome regulates the accumulation of positive and negative regulators for optimal growth of Arabidopsis (Arabidopsis thaliana) in the dark, light, or light/dark cycles. BBX22, whose induction is both light regulated and HY5 dependent, is a positive regulator of deetiolation in Arabidopsis. We found that during skotomorphogenesis, the expression of BBX22 needs to be tightly regulated at both transcriptional and posttranslational levels. During photomorphogenesis, the expression of BBX22 transiently accumulates to execute its roles as a positive regulator. BBX22 protein accumulates to a higher level under short-day conditions and functions to inhibit hypocotyl elongation. The proteasome-dependent degradation of BBX22 protein is tightly controlled even in plants overexpressing BBX22. An analysis of BBX22 degradation kinetics shows that the protein has a short half-life under both dark and light conditions. COP1 mediates the degradation of BBX22 in the dark. Although dispensable in the dark, HY5 contributes to the degradation of BBX22 in the light. The constitutive photomorphogenic development of the cop1 mutant is enhanced in cop1BBX22ox plants, which show a short hypocotyl, high anthocyanin accumulation, and expression of light-responsive genes. Exaggerated light responsiveness is also observed in cop1BBX22ox seedlings grown under short-day conditions. Therefore, the proper accumulation of BBX22 is crucial for plants to maintain optimal growth when grown in the dark as well as to respond to seasonal changes in daylength.