A GFP splicing reporter in a coilin mutant background reveals links between alternative splicing, siRNAs, and coilin function in Arabidopsis thaliana.

Coilin is a scaffold protein essential for the structure of Cajal bodies, which are nucleolar-associated, nonmembranous organelles that coordinate the assembly of nuclear ribonucleoproteins (RNPs) including spliceosomal snRNPs. To study coilin function in plants, we conducted a genetic suppressor screen using a coilin (coi1) mutant in Arabidopsis thaliana and performed an immunoprecipitation-mass spectrometry analysis on coilin protein. The coi1 mutations modify alternative splicing of a GFP reporter gene, resulting in a hyper-GFP phenotype in young coi1 seedlings relative to the intermediate wild-type level.

Loss of Dok-3 in Non-tumor Cells Induces Malignant Transformation of Benign Epithelial Tumor Cells of the Intestine.

Unlabelled: The fundamental difference between benign and malignant tumors lies in their invasive ability. It is believed that malignant conversion of benign tumor cells is induced by a tumor cell-intrinsic accumulation of driver gene mutations. Here, we found that disruption of the tumor suppressor gene led to malignant progression in the intestinal benign tumor model ApcMin/+ mice.

A Collection of Pre-mRNA Splicing Mutants in .

To investigate factors influencing pre-mRNA splicing in plants, we conducted a forward genetic screen using an alternatively-spliced reporter gene in This effort generated a collection of sixteen mutants impaired in various splicing-related proteins, many of which had not been recovered in any prior genetic screen or implicated in splicing in plants. The factors are predicted to act at different steps of the spliceosomal cycle, snRNP biogenesis pathway, transcription, and mRNA transport. We have described eleven of the mutants in recent publications.

Global impacts of chromosomal imbalance on gene expression in and other taxa.

Changes in dosage of part of the genome (aneuploidy) have long been known to produce much more severe phenotypic consequences than changes in the number of whole genomes (ploidy). To examine the basis of these differences, global gene expression in mature leaf tissue for all five trisomies and in diploids, triploids, and tetraploids of was studied. The trisomies displayed a greater spread of expression modulation than the ploidy series.

PRP4KA, a Putative Spliceosomal Protein Kinase, Is Important for Alternative Splicing and Development in .

Splicing of precursor messenger RNAs (pre-mRNAs) is an essential step in the expression of most eukaryotic genes. Both constitutive splicing and alternative splicing, which produces multiple messenger RNA (mRNA) isoforms from a single primary transcript, are modulated by reversible protein phosphorylation. Although the plant splicing machinery is known to be a target for phosphorylation, the protein kinases involved remain to be fully defined.

A Genetic Screen Identifies PRP18a, a Putative Second Step Splicing Factor Important for Alternative Splicing and a Normal Phenotype in .

Splicing of pre-mRNA involves two consecutive -esterification steps that take place in the spliceosome, a large dynamic ribonucleoprotein complex situated in the nucleus. In addition to core spliceosomal proteins, each catalytic step requires step-specific factors. Although the genome encodes around 430 predicted splicing factors, functional information about these proteins is limited.

A Genetic Screen for Pre-mRNA Splicing Mutants of Identifies Putative U1 snRNP Components RBM25 and PRP39a.

In a genetic screen for mutants showing modified splicing of an alternatively spliced reporter gene in , we identified mutations in genes encoding the putative U1 small nuclear ribonucleoprotein (snRNP) factors RBM25 and PRP39a. The latter has not yet been studied for its role in pre-messenger RNA (pre-mRNA) splicing in plants. Both proteins contain predicted RNA-binding domains and have been implicated in 5' splice site selection in yeast and metazoan cells.

A genetic screen implicates a CWC16/Yju2/CCDC130 protein and SMU1 in alternative splicing in .

To identify regulators of pre-mRNA splicing in plants, we developed a forward genetic screen based on an alternatively spliced reporter gene in In wild-type plants, three major splice variants issue from the gene but only one represents a translatable mRNA. Compared to wild-type seedlings, which exhibit an intermediate level of expression, mutants identified in the screen feature either a "GFP-weak" or "Hyper-GFP" phenotype depending on the ratio of the three splice variants. GFP-weak mutants, including previously identified and , contain a higher proportion of unspliced transcript or canonically spliced transcript, neither of which is translatable into GFP protein.

Identification of Coilin Mutants in a Screen for Enhanced Expression of an Alternatively Spliced GFP Reporter Gene in Arabidopsis thaliana.

Coilin is a marker protein for subnuclear organelles known as Cajal bodies, which are sites of various RNA metabolic processes including the biogenesis of spliceosomal small nuclear ribonucleoprotein particles. Through self-associations and interactions with other proteins and RNA, coilin provides a structural scaffold for Cajal body formation. However, despite a conspicuous presence in Cajal bodies, most coilin is dispersed in the nucleoplasm and expressed in cell types that lack these organelles.

GFP Loss-of-Function Mutations in Arabidopsis thaliana.

Green fluorescent protein (GFP) and related fluorescent proteins are widely used in biological research to monitor gene expression and protein localization in living cells. The GFP chromophore is generated spontaneously in the presence of oxygen by a multi-step reaction involving cyclization of the internal tripeptide Ser65 (or Thr65)-Tyr66-Gly67, which is embedded in the center of an 11-stranded β-barrel structure. Random and site-specific mutagenesis has been used to optimize GFP fluorescence and create derivatives with novel properties.

An Rtf2 Domain-Containing Protein Influences Pre-mRNA Splicing and Is Essential for Embryonic Development in Arabidopsis thaliana.

Alternative splicing is prevalent in plants, but little is known about its regulation in the context of developmental and signaling pathways. We describe here a new factor that influences pre-messengerRNA (mRNA) splicing and is essential for embryonic development in Arabidopsis thaliana. This factor was retrieved in a genetic screen that identified mutants impaired in expression of an alternatively spliced GFP reporter gene.

RNA-Directed DNA Methylation: The Evolution of a Complex Epigenetic Pathway in Flowering Plants.

RNA-directed DNA methylation (RdDM) is an epigenetic process in plants that involves both short and long noncoding RNAs. The generation of these RNAs and the induction of RdDM rely on complex transcriptional machineries comprising two plant-specific, RNA polymerase II (Pol II)-related RNA polymerases known as Pol IV and Pol V, as well as a host of auxiliary factors that include both novel and refashioned proteins. We present current views on the mechanism of RdDM with a focus on evolutionary innovations that occurred during the transition from a Pol II transcriptional pathway, which produces mRNA precursors and numerous noncoding RNAs, to the Pol IV and Pol V pathways, which are specialized for RdDM and gene silencing.

Does the upstream region possessing MULE-like sequence in rice upregulate PsbS1 gene expression?

The genomic nucleotide sequences of japonica rice (Sasanishiki and Nipponbare) contained about 2.7-kb unique region at the point of 0.4-kb upstream of the OsPsbS1 gene.

DNA methylation in plants: relationship to small RNAs and histone modifications, and functions in transposon inactivation.

DNA methylation is a type of epigenetic marking that strongly influences chromatin structure and gene expression in plants and mammals. Over the past decade, DNA methylation has been intensively investigated in order to elucidate its control mechanisms. These studies have shown that small RNAs are involved in the induction of DNA methylation, that there is a relationship between DNA methylation and histone methylation, and that the base excision repair pathway has an important role in DNA demethylation.

siRNA-mediated chromatin maintenance and its function in Arabidopsis thaliana.

Small interfering RNAs (siRNAs) are widespread in various eukaryotes and are involved in maintenance of chromatin modifications, especially those for inert states represented by covalent modifications of cytosine and/or histones. In contrast to mammalian genomes, in which cytosine methylation is restricted mostly to CG dinucleotide sequences, inert chromatin in plants carries cytosine methylation in all sequence contexts, and siRNAs play a major role in directing cytosine methylation through the process of RNA-directed DNA methylation. Recent advances in this field have revealed that siRNA-mediated maintenance of inert chromatin has diverse roles in development as well as in plant responses to the environment.

Genetic evidence that DNA methyltransferase DRM2 has a direct catalytic role in RNA-directed DNA methylation in Arabidopsis thaliana.

RNA-directed DNA methylation (RdDM) is a small RNA-mediated epigenetic modification in plants. We report here the identification of DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) in a forward screen for mutants defective in RdDM in Arabidopsis thaliana. The finding of a mutation in the presumptive active site argues in favor of direct catalytic activity for DRM2.

RNA-directed DNA methylation and plant development require an IWR1-type transcription factor.

RNA-directed DNA methylation (RdDM) in plants requires two RNA polymerase (Pol) II-related RNA polymerases, namely Pol IV and Pol V. A genetic screen designed to reveal factors that are important for RdDM in a developmental context in Arabidopsis identified DEFECTIVE IN MERISTEM SILENCING 4 (DMS4). Unlike other mutants defective in RdDM, dms4 mutants have a pleiotropic developmental phenotype.

RNA-mediated chromatin-based silencing in plants.

Plants have evolved an elaborate transcriptional machinery dedicated to eliciting sequence-specific, chromatin-based gene silencing. Two Pol II-related, plant-specific RNA polymerases, named Pol IV and Pol V, collaborate with proteins of the RNA interference machinery to generate long and short noncoding RNAs involved in epigenetic regulation. As revealed by a variety of genetic, molecular, and genomic technologies, these RNAs are used extensively in plants to direct the establishment, spread, and removal of DNA cytosine methylation throughout their genomes.

PolV(PolIVb) function in RNA-directed DNA methylation requires the conserved active site and an additional plant-specific subunit.

Two forms of a plant-specific RNA polymerase (Pol), PolIV(PolIVa) and PolV(PolIVb), currently defined by their respective largest subunits [NRPD1(NRPD1a) and NRPE1(NRPD1b)], have been implicated in the production and activity of 24-nt small RNAs (sRNAs) in RNA-directed DNA methylation (RdDM). Prevailing models support the view that PolIV(PolIVa) plays an upstream role in RdDM by producing the 24-nt sRNAs, whereas PolV(PolIVb) would act downstream at a structural rather than an enzymatic level to reinforce sRNA production by PolIV(PolIVa) and mediate DNA methylation. However, the composition and mechanism of action of PolIV(PolIVa)/PolV(PolIVb) remain unclear.

A stepwise pathway for biogenesis of 24-nt secondary siRNAs and spreading of DNA methylation.

We used a transgene system to study spreading of RNA-directed DNA methylation (RdDM) during transcriptional gene silencing in Arabidopsis thaliana. Forward and reverse genetics approaches using this system delineated a stepwise pathway for the biogenesis of secondary siRNAs and unidirectional spreading of methylation from an upstream enhancer element into downstream sequences. Trans-acting, hairpin-derived primary siRNAs induce primary RdDM, independently of an enhancer-associated 'nascent' RNA, at the target enhancer region.

Pol V transcribes to silence.

In most cases, the functions of long noncoding RNAs remain uncertain. Working in the model plant Arabidopsis, Wierzbicki et al. (2008) provide evidence that transcription of intergenic noncoding regions by RNA polymerase V promotes heterochromatin formation and silencing of nearby genes.

A structural-maintenance-of-chromosomes hinge domain-containing protein is required for RNA-directed DNA methylation.

RNA-directed DNA methylation (RdDM) is a process in which dicer-generated small RNAs guide de novo cytosine methylation at the homologous DNA region. To identify components of the RdDM machinery important for Arabidopsis thaliana development, we targeted an enhancer active in meristems for methylation, which resulted in silencing of a downstream GFP reporter gene. This silencing system also features secondary siRNAs, which trigger methylation that spreads beyond the targeted enhancer region.

Targets of RNA-directed DNA methylation.

RNA-directed DNA methylation contributes substantially to epigenetic regulation of the plant genome. Methylation is guided to homologous DNA target sequences by 24 nt 'heterochromatic' small RNAs produced by nucleolar-localized components of the RNAi machinery and a plant-specific RNA polymerase, Pol IV. Plants contain unusually large and diverse populations of small RNAs, many of which originate from transposons and repeats.

RNA-directed DNA methylation mediated by DRD1 and Pol IVb: a versatile pathway for transcriptional gene silencing in plants.

RNA-directed DNA methylation, which is one of several RNAi-mediated pathways in the nucleus, has been highly elaborated in the plant kingdom. RNA-directed DNA methylation requires for the most part conventional DNA methyltransferases, histone modifying enzymes and RNAi proteins; however, several novel, plant-specific proteins that are essential for this process have been identified recently. DRD1 (defective in RNA-directed DNA methylation) is a putative SWI2/SNF2-like chromatin remodelling protein; DRD2 and DRD3 (renamed NRPD2a and NRPD1b, respectively) are subunits of Pol IVb, a putative RNA polymerase found only in plants.

Endogenous targets of RNA-directed DNA methylation and Pol IV in Arabidopsis.

DRD1 is a SWI/SNF-like protein that cooperates with a plant-specific RNA polymerase, Pol IVb, to facilitate RNA-directed de novo methylation and silencing of homologous DNA. Screens to identify endogenous targets of this pathway in Arabidopsis revealed intergenic regions and plant genes located primarily in euchromatin. Many putative targets are near retrotransposon LTRs or other intergenic sequences that encode short RNAs, which might epigenetically regulate adjacent genes.