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.

PRH75, a new nucleus-localized member of the DEAD-box protein family from higher plants.

The putative RNA helicases of the DEAD-box protein family are involved in pre-mRNA splicing, rRNA maturation, ribosome assembly, and translation. Members of this protein family have been identified in organisms from Escherichia coli to humans, but except for the translation initiation factor 4A, there have been no reports on the characterization of other DEAD-box proteins from plants. Here we report on a novel member of the DEAD-box protein family, the plant RNA helicase 75 (PRH75). PRH75 is localized in the nucleus and contains two domains for RNA binding. One is located at the C terminus and is similar to RGG RNA-binding domains of nucleus-localized RNA-binding proteins. The other one is located between amino acids 308 and 622, a region containing the conserved motif VI characteristic of DEAD-box proteins and known as the RNA-binding site of eIF-4A. The N-terminal 81 amino acids are sufficient for nuclear targeting of the protein. Northern and Western blot analyses show that PRH75 is mainly expressed in young and rapidly developing tissues. The purified recombinant PRH75 has a weak ATPase activity which is barely stimulated by RNA ligands. The fractionation of spinach whole-cell extracts by glycerol gradient centrifugation and gel filtration on a Superdex 200 column shows that the protein exists in a complex of about 500 kDa. Possible biological functions of PRH75 as well as structure-function relationships in the context of its modular primary structure are discussed.

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.

Use of forward genetic screens to identify genes required for RNA-directed DNA methylation in Arabidopsis thaliana.

RNA-directed DNA methylation is a small RNA-mediated epigenetic modification that contributes to transcriptional silencing of transposons and repetitive sequences in plants. We have conducted several forward genetic screens to identify factors required for RNA-directed DNA methylation and transcriptional gene silencing in Arabidopsis thaliana. Here, we review the findings from these screens and report on two new mutants, dms12 and dms13, that are defective in Pol V-specific subunits NRPE5 and NRPE9b. Cumulative results from genetic screens performed in our laboratory and those of other investigators have revealed that RNA-directed DNA methylation requires a complex transcriptional machinery comprising a number of plant-specific factors, many of which were functionally uncharacterized before being implicated in this pathway. Future challenges include unraveling the detailed mechanism and full range of functions of RNA-directed DNA methylation.

Multiple forms of the U2 small nuclear ribonucleoprotein auxiliary factor U2AF subunits expressed in higher plants.

Requirements for intron recognition during pre-mRNA splicing in plants differ from those in vertebrates and yeast. Plant introns contain neither conserved branch points nor distinct 3' splice site-proximal polypyrimidine tracts characteristic of the yeast and vertebrate introns, respectively. However, they are strongly enriched in U residues throughout the intron, property essential for splicing. To understand the roles of different sequence elements in splicing, we are characterizing proteins involved in intron recognition in plants. In this work we show that Nicotiana plumbaginifolia, a dicotyledonous plant, contains two genes encoding different homologs of the large 50-65-kDa subunit of the polypyrimidine tract binding factor U2AF, characterized previously in animals and Schizosaccharomyces pombe. Both plant U2AF65 isoforms, referred to as NpU2AF65a and NpU2AF65b, support splicing of an adenovirus pre-mRNA in HeLa cell nuclear extracts depleted of the endogenous U2AF factor. Both proteins interact with RNA fragments containing plant introns and show affinity for poly(U) and, to a lesser extend, poly(C) and poly(G). The branch point or the 3' splice site regions do not contribute significantly to intron recognition by NpU2AF65. The existence of multiple isoforms of U2AF may be quite general in plants because two genes expressing U2AF65 have been identified in Arabidopsis, and different isoforms of the U2AF small subunit are expressed in rice.

RBP45 and RBP47, two oligouridylate-specific hnRNP-like proteins interacting with poly(A)+ RNA in nuclei of plant cells.

Introns in plant nuclear pre-mRNAs are highly enriched in U or U + A residues and this property is essential for efficient splicing. Moreover, 3'-untranslated regions (3'-UTRs) in plant pre-mRNAs are generally UA-rich and contain sequences that are important for the polyadenylation reaction. Here, we characterize two structurally related RNA-binding proteins (RBPs) from Nicotiana plumbaginifolia, referred to as RBP45 and RBP47, having specificity for oligouridylates. Both proteins contain three RBD-type RNA-binding domains and a glutamine-rich N-terminus, and share similarity with Nam8p, a protein associated with U1 snRNP in the yeast Saccharomyces cerevisiae. Deletion analysis of RBP45 and RBP47 indicated that the presence of at least two RBD are required for interaction with RNA and that domains other than RBD do not significantly contribute to binding. mRNAs for RBP45 and RBP47 and mRNAs encoding six related proteins in Arabidopsis thaliana are constitutively expressed in different plant organs. Indirect immunofluorescence and fractionation of cell extracts showed that RBP45 and RBP47 are localized in the nucleus. In vivo UV crosslinking experiments demonstrated their association with the nuclear poly(A)+ RNA. In contrast to UBP1, another oligouridylate-binding nuclear three-RBD protein of N. plumbaginifolia (Lambermon et al., EMBO J, 2000, 19:1638-1649), RBP45 and RBP47 do not stimulate mRNA splicing and accumulation when transiently overexpressed in protoplasts. Properties of RBP45 and RBP47 suggest they represent hnRNP-proteins participating in still undefined steps of pre-mRNA maturation in plant cell nuclei.

The low molecular mass PsbW protein is involved in the stabilization of the dimeric photosystem II complex in Arabidopsis thaliana.

Arabidopsis thaliana plants have been transformed with an antisense gene to the psbW of photosystem II (PSII). Eight transgenic lines containing low levels of psbW mRNA have been obtained. Transgenic seedlings with low contents of PsbW protein (more than 96% reduced) were selected by Western blotting and used for photosynthetic functional studies. There were no distinct differences in phenotype between the antisense and wild type plants during vegetative period under normal growth light intensities. However, a sucrose gradient separation of briefly solubilized thylakoid membranes revealed that no dimeric PSII supracomplex could be detected in the transgenic plants lacking the PsbW protein. Furthermore, analysis of isolated thylakoids demonstrated that the oxygen-evolving rate in antisense plants decreased by 50% compared with the wild type. This was found to be due to up to 40% of D1 and D2 reaction center proteins of PSII disappearing in the transgenic plants. The absence of the PsbW protein also altered the contents of other PSII proteins to differing extents. These results show that in the absence of the PsbW protein, the stability of the dimeric PSII is diminished and consequently the total number of PSII complexes is greatly reduced. Thus the nuclear encoded PsbW protein may play a crucial role in the biogenesis and regulation of the photosynthetic apparatus.

Molecular characterization of PsbW, a nuclear-encoded component of the photosystem II reaction center complex in spinach.

We describe the isolation and characterization of cDNAs encoding the precursor polypeptide of the 6.1-kDa polypeptide associated with the reaction center core of the photosystem II complex from spinach. PsbW, the gene encoding this polypeptide, is present in a single copy per haploid genome. The mature polypeptide with 54 amino acid residues is characterized by a hydrophobic transmembrane segment, and, although an intrinsic membrane protein, it carries a bipartite transit peptide of 83 amino acid residues which directs the N terminus of the mature protein into the chloroplast lumen. Thylakoid integration of this polypeptide does not require a delta pH across the membrane, nor is it azide-sensitive, suggesting that the polypeptide chain inserts spontaneously in an as yet unknown way. The PsbW mRNA levels are light regulated. Similar to cytochrome b559 and PsbS, but different from the chlorophyll-complexing polypeptides D1, D2, CP43, and CP47 of photosystem II, PsbW is present in etiolated spinach seedlings.