Zdravko J. Lorković

Pre-mRNA splicing in higher plants

Most plant mRNAs are synthesized as precursors containing one or more intervening sequences (introns) that are removed during the process of splicing. The basic mechanism of spliceosome assembly and intron excision is similar in all eukaryotes. However, the recognition of introns in plants has some unique features, which distinguishes it from the reactions in vertebrates and yeast. Recent progress has occurred in characterizing the splicing signals in plant pre-mRNAs, in identifying the mutants affected in splicing and in discovering new examples of alternatively spliced mRNAs. In combination with information provided by the Arabidopsis genome-sequencing project, these studies are contributing to a better understanding of the splicing process and its role in the regulation of gene expression in plants.

Role of plant RNA-binding proteins in development, stress response and genome organization

RNA-binding proteins (RBPs) in eukaryotes have crucial roles in all aspects of post-transcriptional gene regulation. They are important governors of diverse developmental processes by modulating expression of specific transcripts. The Arabidopsis (Arabidopsis thaliana) genome encodes for more than 200 different RBPs, most of which are plant specific and are therefore likely to perform plant-specific functions. Indeed, recent identification and analysis of plant RBPs clearly showed that, in addition to the important role in diverse developmental processes, they are also involved in adaptation of plants to various environmental conditions. Clearly, they act by regulating pre-mRNA splicing, polyadenylation, RNA stability and RNA export, as well as by influencing chromatin modification.

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. Loss-of-function mutations in the RDM1 gene impair the accumulation of 24-nucleotide siRNAs, reduce DNA methylation, and release transcriptional gene silencing at RdDM target loci. RDM1 encodes a small protein that seems to bind single-stranded methyl DNA, and associates and co-localizes with RNA polymerase II (Pol II, also known as NRPB), AGO4 and DRM2 in the nucleus. Our results indicate that RDM1 is a component of the RdDM effector complex and may have a role in linking siRNA production with pre-existing or de novo cytosine methylation. Our results also indicate that, although RDM1 and Pol V (also known as NRPE) may function together at some RdDM target sites in the peri-nucleolar siRNA processing centre, Pol II rather than Pol V is associated with the RdDM effector complex at target sites in the nucleoplasm.

Site-Specific Phosphorylation Profiling of Arabidopsis Proteins by Mass Spectrometry and Peptide Chip Analysis

An estimated one-third of all proteins in higher eukaryotes are regulated by phosphorylation by protein kinases (PKs). Although plant genomes encode more than 1000 PKs, the substrates of only a small fraction of these kinases are known. By mass spectrometry of peptides from cytoplasmic- and nuclear-enriched fractions, we determined 303 in vivo phosphorylation sites in Arabidopsis proteins. Among 21 different PKs, 12 were phosphorylated in their activation loops, suggesting that they were in their active state. Immunoblotting and mutational analysis confirmed a tyrosine phosphorylation site in the activation loop of a GSK3/shaggy-like kinase. Analysis of phosphorylation motifs in the substrates suggested links between several of these PKs and many target sites. To perform quantitative phosphorylation analysis, peptide arrays were generated with peptides corresponding to in vivo phosphorylation sites. These peptide chips were used for kinome profiling of subcellular fractions as well as H 2O 2-treated Arabidopsis cells. Different peptide phosphorylation profiles indicated the presence of overlapping but distinct PK activities in cytosolic and nuclear compartments. Among different H 2O 2-induced PK targets, a peptide of the serine/arginine-rich (SR) splicing factor SCL30 was most strongly affected. SRPK4 (SR protein-specific kinase 4) and MAPKs (mitogen-activated PKs) were found to phosphorylate this peptide, as well as full-length SCL30. However, whereas SRPK4 was constitutively active, MAPKs were activated by H 2O 2. These results suggest that SCL30 is targeted by different PKs. Together, our data demonstrate that a combination of mass spectrometry with peptide chip phosphorylation profiling has a great potential to unravel phosphoproteome dynamics and to identify PK substrates.

Network of Interactions of a Novel Plant-specific Arg/Ser-rich Protein, atRSZ33, with atSC35-like Splicing Factors

Arg/Ser-rich (RS) proteins play a crucial role in splicing and are implicated in splice site selection in metazoa. In plants, intron recognition seems to differ from the one in animals due to specific factor requirements. Here we describe a new plant-specific RS-rich protein, atRSZ33, with a unique domain structure consisting of an RNA recognition motif (RRM), two zinc knuckles embedded in a basic RS region, and an acidic C-terminal domain. atRSZ33 was found to be a phosphoprotein that concentrates in nuclear speckles and is predominantly present in roots and flowers. In a yeast two-hybrid screen, atRSZ33 interacted with splicing factors atSRp34/SR1, anArabidopsis ortholog of human SF2/ASF; atRSZp21 and atRSZp22, which are similar to the human 9G8; and three novel SC35-like splicing factors termed atSCL28, atSCL30, and atSCL33/SR33. Two further members of the SCL family, namely SCL30a and the ortholog of mammalian SC35, atSC35, were also found to interact with atRSZ33. These interactions were verified by in vitro binding assays; furthermore, the transcriptional activity of atRSZ33 was found to overlap with the ones of its interacting partners. These specific interactions coupled with the many similarities of atRSZ33 to SR proteins suggest that its main activity is in spliceosome assembly. Mapping of regions necessary for protein-protein interaction between atRSZ33 and atSCL33/SR33 revealed that both zinc knuckles together with a small part of the RS and the RRM domain are required for efficient binding. However, the interacting domain is relatively small, allowing binding of additional proteins, a feature that is consistent with the proposed role of atRSZ33 in spliceosome assembly.

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. The DMS4 protein is similar to yeast IWR1 (interacts with RNA polymerase II), a conserved putative transcription factor that interacts with Pol II subunits. The DMS4 complementary DNA partly complements the K1 killer toxin hypersensitivity of a yeast iwr1 mutant, suggesting some functional conservation. In the transgenic system studied, mutations in DMS4 directly or indirectly affect Pol IV‐dependent secondary short interfering RNAs, Pol V‐mediated RdDM, Pol V‐dependent synthesis of intergenic non‐coding RNA and expression of many Pol II‐driven genes. These data suggest that DMS4 might be a regulatory factor for several RNA polymerases, thus explaining its diverse roles in the plant.

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. siRNAs accumulate in the dms11 mutant, and repressive epigenetic modifications undergo only modest reductions at target sequences. DMS11 interacts physically with another RdDM component, DMS3, an unusual structural maintenance of chromosomes (SMC) hinge domain-containing protein that lacks the ATPase motifs of authentic SMC proteins. The hinge region of DMS3 resembles that of the mammalian epigenetic factor SMCHD1, which also has a GHKL-type ATPase. In vitro, DMS11 has ATPase activity that is stimulated by DMS3. We suggest that DMS11 provides the missing ATPase function for DMS3 and that these proteins cooperate in the RdDM pathway to promote transcriptional repression. GHKL ATPases are thus emerging as new players in epigenetic regulation in plants and mammals.

UBA1 and UBA2, Two Proteins That Interact with UBP1, a Multifunctional Effector of Pre-mRNA Maturation in Plants

ABSTRACT Nicotiana plumbaginifolia UBP1 is an hnRNP-like protein associated with the poly(A)+ RNA in the cell nucleus. Consistent with a role in pre-mRNA processing, overexpression of UBP1 in N. plumabaginifolia protoplasts enhances the splicing of suboptimal introns and increases the steady-state levels of reporter mRNAs, even intronless ones. The latter effect of UBP1 is promoter specific and appears to be due to UBP1 binding to the 3′ untranslated region (3′-UTR) and protecting the mRNA from exonucleolytic degradation (M. H. L. Lambermon, G. G. Simpson, D. A. Kirk, M. Hemmings-Mieszczak, U. Klahre, and W. Filipowicz, EMBO J. 19:1638-1649, 2000). To gain more insight into UBP1 function in pre-mRNA maturation, we characterized proteins interacting with N. plumbaginifolia UBP1 and one of its Arabidopsis thaliana counterparts, AtUBP1b, by using yeast two-hybrid screens and in vitro pull-down assays. Two proteins, UBP1-associated proteins 1a and 2a (UBA1a and UBA2a, respectively), were identified in A. thaliana. They are members of two novel families of plant-specific proteins containing RNA recognition motif-type RNA-binding domains. UBA1a and UBA2a are nuclear proteins, and their recombinant forms bind RNA with a specificity for oligouridylates in vitro. As with UBP1, transient overexpression of UBA1a in protoplasts increases the steady-state levels of reporter mRNAs in a promoter-dependent manner. Similarly, overexpression of UBA2a increases the levels of reporter mRNAs, but this effect is promoter independent. Unlike UBP1, neither UBA1a nor UBA2a stimulates pre-mRNA splicing. These and other data suggest that UBP1, UBA1a, and UBA2a may act as components of a complex recognizing U-rich sequences in plant 3′-UTRs and contributing to the stabilization of mRNAs in the nucleus.

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. AGO4 is the main AGO protein implicated in the RdDM pathway. Here we report the identification of the related AGO6 in a forward genetic screen for mutants defective in RdDM and transcriptional gene silencing in shoot and root apical meristems in Arabidopsis thaliana. The identification of AGO6, and not AGO4, in our screen is consistent with the primary expression of AGO6 in shoot and root growing points.

Plant SR Proteins and Their Functions

SR proteins are a family of splicing factors important for splice site recognition and spliceosome assembly. Their ability to bind to RNA and to interact with proteins as well identifies them as important players in splice site choice and alternative splicing. Plants possess twice as many SR proteins as animals, and some of the subfamilies are plant specific. Arabidopsis SR proteins are involved in different aspects of plant growth and development as well as in responses to environmental cues. The plant-specific subfamilies have been shown to be regulated by alternative splicing events, which are highly conserved in evolution. The tight regulation of splicing factors by alternative splicing might allow coordinated responses of their target genes.