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.

Evidence That Ion-Based Signaling Initiating at the Cell Surface Can Potentially Influence Chromatin Dynamics and Chromatin-Bound Proteins in the Nucleus.

We have developed tools and performed pilot experiments to test the hypothesis that an intracellular ion-based signaling pathway, provoked by an extracellular stimulus acting at the cell surface, can influence interphase chromosome dynamics and chromatin-bound proteins in the nucleus. The experimental system employs chromosome-specific fluorescent tags and the genome-encoded fluorescent pH sensor SEpHluorinA227D, which has been targeted to various intracellular membranes and soluble compartments in root cells of . We are using this system and three-dimensional live cell imaging to visualize whether fluorescent-tagged interphase chromosome sites undergo changes in constrained motion concurrently with reductions in membrane-associated pH elicited by extracellular ATP, which is known to trigger a cascade of events in plant cells including changes in calcium ion concentrations, pH, and membrane potential.

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.

Expression and testing in plants of ArcLight, a genetically-encoded voltage indicator used in neuroscience research.

Background: It is increasingly appreciated that electrical controls acting at the cellular and supra-cellular levels influence development and initiate rapid responses to environmental cues. An emerging method for non-invasive optical imaging of electrical activity at cell membranes uses genetically-encoded voltage indicators (GEVIs). Developed by neuroscientists to chart neuronal circuits in animals, GEVIs comprise a fluorescent protein that is fused to a voltage-sensing domain.

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.

De novo transcriptome sequence assembly from coconut leaves and seeds with a focus on factors involved in RNA-directed DNA methylation.

Coconut palm (Cocos nucifera) is a symbol of the tropics and a source of numerous edible and nonedible products of economic value. Despite its nutritional and industrial significance, coconut remains under-represented in public repositories for genomic and transcriptomic data. We report de novo transcript assembly from RNA-seq data and analysis of gene expression in seed tissues (embryo and endosperm) and leaves of a dwarf coconut variety.

Distinct and concurrent pathways of Pol II- and Pol IV-dependent siRNA biogenesis at a repetitive trans-silencer locus in Arabidopsis thaliana.

Short interfering RNAs (siRNAs) homologous to transcriptional regulatory regions can induce RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS) of target genes. In our system, siRNAs are produced by transcribing an inverted DNA repeat (IR) of enhancer sequences, yielding a hairpin RNA that is processed by several Dicer activities into siRNAs of 21-24 nt. Primarily 24-nt siRNAs trigger RdDM of the target enhancer in trans and TGS of a downstream GFP reporter gene.

The ability to form homodimers is essential for RDM1 to function in RNA-directed DNA methylation.

RDM1 (RNA-DIRECTED DNA METHYLATION1) is a small plant-specific protein required for RNA-directed DNA methylation (RdDM). RDM1 interacts with RNA polymerase II (Pol II), ARGONAUTE4 (AGO4), and the de novo DNA methyltransferase DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) and binds to methylated single stranded DNA. As the only protein identified so far that interacts directly with DRM2, RDM1 plays a pivotal role in the RdDM mechanism by linking the de novo DNA methyltransferase activity to AGO4, which binds short interfering RNAs (siRNAs) that presumably base-pair with Pol II or Pol V scaffold transcripts synthesized at target loci.

Complete sequence and comparative analysis of the chloroplast genome of coconut palm (Cocos nucifera).

Coconut, a member of the palm family (Arecaceae), is one of the most economically important trees used by mankind. Despite its diverse morphology, coconut is recognized taxonomically as only a single species (Cocos nucifera L.).

Membrane "potential-omics": toward voltage imaging at the cell population level in roots of living plants.

Genetically encoded voltage-sensitive fluorescent proteins (VSFPs) are being used in neurobiology as non-invasive tools to study synchronous electrical activities in specific groups of nerve cells. Here we discuss our efforts to adapt this "light-based electrophysiology" for use in plant systems. We describe the production of transgenic plants engineered to express different versions of VSFPs that are targeted to the plasma membrane and internal membranes of root cells.

Interplay among RNA polymerases II, IV and V in RNA-directed DNA methylation at a low copy transgene locus in Arabidopsis thaliana.

RNA-directed DNA methylation (RdDM) is an epigenetic process whereby small interfering RNAs (siRNAs) guide cytosine methylation of homologous DNA sequences. RdDM requires two specialized RNA polymerases: Pol IV transcribes the siRNA precursor whereas Pol V generates scaffold RNAs that interact with siRNAs and attract the methylation machinery. Recent evidence also suggests the involvement of RNA polymerase II (Pol II) in recruiting Pol IV and Pol V to low copy, intergenic loci.

Unusual case of apparent hypermutation in Arabidopsis thaliana.

The dms4 (defective in meristem silencing 4) mutant of Arabidopsis thaliana is unique in having defects in both RNA-directed DNA methylation (RdDM) and plant development. DMS4 is an evolutionarily conserved, putative transcription factor of the Iwr1 (interacts with RNA polymerase II) type. DMS4 interacts with Pol II and also with RNA polymerases IV and V, which function in RdDM.

Involvement of a GHKL ATPase in RNA-directed DNA methylation in Arabidopsis thaliana.

RNA-directed DNA methylation (RdDM) is a small interfering RNA (siRNA)-mediated epigenetic modification that contributes to transposon silencing in plants. RdDM requires a complex transcriptional machinery comprising two RNA polymerase II-related RNA polymerases, called Pol IV and Pol V, as well as chromatin remodelers, transcription factors, and other novel proteins whose roles in the RdDM mechanism remain poorly understood. We have identified a new component of the RdDM machinery, DMS11 (defective in meristem silencing 11), which has a GHKL (gyrase, Hsp90, histidine kinase, MutL) ATPase domain.

Atypical DNA methylation of genes encoding cysteine-rich peptides in Arabidopsis thaliana.

Background: In plants, transposons and non-protein-coding repeats are epigenetically silenced by CG and non-CG methylation. This pattern of methylation is mediated in part by small RNAs and two specialized RNA polymerases, termed Pol IV and Pol V, in a process called RNA-directed DNA methylation. By contrast, many protein-coding genes transcribed by Pol II contain in their gene bodies exclusively CG methylation that is independent of small RNAs and Pol IV/Pol V activities.

AGO6 functions in RNA-mediated transcriptional gene silencing in shoot and root meristems in Arabidopsis thaliana.

RNA-directed DNA methylation (RdDM) is a small interfering RNA (siRNA)-mediated epigenetic modification that contributes to transposon silencing in plants. RdDM requires a complex transcriptional machinery that includes specialized RNA polymerases, named Pol IV and Pol V, as well as chromatin remodelling proteins, transcription factors, RNA binding proteins, and other plant-specific proteins whose functions are not yet clarified. In Arabidopsis thaliana, DICER-LIKE3 and members of the ARGONAUTE4 group of ARGONAUTE (AGO) proteins are involved, respectively, in generating and using 24-nt siRNAs that trigger methylation and transcriptional gene silencing of homologous promoter sequences.

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.

An RNA polymerase II- and AGO4-associated protein acts in RNA-directed DNA methylation.

DNA methylation is an important epigenetic mark in many eukaryotes. In plants, 24-nucleotide small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4), can direct de novo DNA methylation by the methyltransferase DRM2 (refs 2, 4-6). Here we report a new regulator of RNA-directed DNA methylation (RdDM) in Arabidopsis: RDM1.

Ion channels at the nucleus: electrophysiology meets the genome.

The nuclear envelope is increasingly viewed from an electrophysiological perspective by researchers interested in signal transduction pathways that influence gene transcription and other processes in the nucleus. Here, we describe evidence for ion channels and transporters in the nuclear membranes and for possible ion gating by the nuclear pores. We argue that a systems-level understanding of cellular regulation is likely to require the assimilation of nuclear electrophysiology into molecular and biochemical signaling pathways.