Regulation of flowering time by RNA processing.

Plants control the time at which they flower by integrating environmental cues such as day length and temperature with an endogenous program of development. Flowering time is a quantitative trait and a model for how precision in gene regulation is delivered. In this review, we reveal that flowering time control is particularly rich in RNA processing-based gene regulatory phenomena. We review those factors which function in conserved RNA processing events like alternative 3' end formation, splicing, RNA export and miRNA biogenesis and how they affect flowering time. Likewise, we review the novel plant-specific RNA-binding proteins identified as regulators of flowering time control. In addition, we add to the network of flowering time control pathways, information on alternative processing of flowering time gene pre-mRNAs. Finally, we describe new approaches to dissect the mechanisms which underpin this control.

RNA processing and Arabidopsis flowering time control.

Plants control their flowering time in order to ensure that they reproduce under favourable conditions. The components involved in this complex process have been identified using a molecular genetic approach in Arabidopsis and classified into genetically separable pathways. The autonomous pathway controls the level of mRNA encoding a floral repressor, FLC, and comprises three RNA-binding proteins, FCA, FPA and FLK. FCA interacts with the 3'-end RNA-processing factor FY to autoregulate its own expression post-transcriptionally and to control FLC. Other components of the autonomous pathway, FVE and FLD, regulate FLC epigenetically. This combination of epigenetic and post-transcriptional control gives precision to the control of FLC expression and flowering time.

UBP1, a novel hnRNP-like protein that functions at multiple steps of higher plant nuclear pre-mRNA maturation.

Efficient splicing of higher plant pre-mRNAs depends on AU- or U-rich sequences in introns. Moreover, AU-rich sequences present in 3'-untranslated regions (3'-UTRs) may play a role in 3' end processing of plant mRNAs. Here, we describe the cloning and characterization of a Nicotiana plumbaginifolia nuclear protein that can be cross-linked to U-rich intron and 3'-UTR sequences in vitro, and associates with nuclear poly(A)(+) RNA in vivo. The protein, UBP1, strongly enhances the splicing of otherwise inefficiently processed introns when overexpressed in protoplasts. It also increases the accumulation of reporter mRNAs that contain suboptimal introns or are intronless. The enhanced accumulation is apparently due to UBP1 interacting with the 3'-UTR and protecting mRNA from exonucleolytic degradation. The effect on mRNA accumulation but not on mRNA splicing was found to be promoter specific. The fact that these effects of UBP1 can be separated suggests that they represent two independent activities. The properties of UBP1 indicate that it is an hnRNP protein that functions at multiple steps to facilitate the nuclear maturation of plant pre-mRNAs.

Environmental-dependent acceleration of a developmental switch: the floral transition.

The transition from vegetative growth to reproductive growth in plants in which flowers are produced requires the activation of specific genes. Simpson and Dean discuss two recent reports that characterize the FLOWERING LOCUS T (FT) gene in Arabidopsis, which is part of the floral transition pathway. Unlike many of the known genes that initiate flower production, the FT gene appears to encode a membrane-associated protein that could function in signaling from the cell surface.

When to switch to flowering.

At a certain stage in their life cycle, plants switch from vegetative to reproductive development. This transition is regulated by multiple developmental and environmental cues. These ensure that the plant switches to flowering at a time when sufficient internal resources have been accumulated and the environmental conditions are favorable. The use of a molecular genetic approach in Arabidopsis has resulted in the identification and cloning of many of the genes involved in regulating floral transition. The current view on the molecular function of these genes, their division into different genetic pathways, and how the pathways interact in a complex regulatory network are summarized.

Splicing of precursors to mRNA in higher plants: mechanism, regulation and sub-nuclear organisation of the spliceosomal machinery.

The removal of introns from pre-mRNA transcripts and the concomitant ligation of exons is known as pre-mRNA splicing. It is a fundamental aspect of constitutive eukaryotic gene expression and an important level at which gene expression is regulated. The process is governed by multiple cis-acting elements of limited sequence content and particular spatial constraints, and is executed by a dynamic ribonucleoprotein complex termed the spliceosome. The mechanism and regulation of pre-mRNA splicing, and the sub-nuclear organisation of the spliceosomal machinery in higher plants is reviewed here. Heterologous introns are often not processed in higher plants indicating that, although highly conserved, the process of pre-mRNA splicing in plants exhibits significant differences that distinguish it from splicing in yeast and mammals. A fundamental distinguishing feature is the presence of and requirement for AU or U-rich intron sequence in higher-plant pre-mRNA splicing. In this review we document the properties of higher-plant introns and trans-acting spliceosomal components and discuss the means by which these elements combine to determine the accuracy and efficiency of pre-mRNA processing. We also detail examples of how introns can effect regulated gene expression by affecting the nature and abundance of mRNA in plants and list the effects of environmental stresses on splicing. Spliceosomal components exhibit a distinct pattern of organisation in higher-plant nuclei. Effective probes that reveal this pattern have only recently become available, but the domains in which spliceosomal components concentrate were identified in plant nuclei as enigmatic structures some sixty years ago. The organisation of spliceosomal components in plant nuclei is reviewed and these recent observations are unified with previous cytochemical and ultrastructural studies of plant ribonuleoprotein domains.

Molecular characterization of the spliceosomal proteins U1A and U2B" from higher plants.

In addition to their role in pre-mRNA splicing, the human spliceosomal proteins U1A and U2B" are important models of how RNP motif-containing proteins execute sequence-specific RNA binding. Genes encoding U1A and U2B" have been isolated from potato and thereby provide the only evolutionary comparison available for both proteins and represent the only full-length genes encoding plant spliceosomal proteins to have been cloned and characterized. In vitro RNA binding experiments revealed the ability of potato U2B" to interact with human U2A' to enhance sequence-specific binding and to distinguish cognate RNAs of either plant or animal origin. A comparison of the sequence of U1A and U2B" proteins indicated that multiple residues which could affect RNP motif conformation probably govern the specific distinction in RNA binding by these proteins. Since human U1A modulates polyadenylation in vertebrates, the possibility that plant U1A might be exploited in the characterization of this process in plants was examined. However, unlike vertebrate U1A, neither U1A from potato nor Arabidopsis bound their own mRNA and no evidence for binding to upstream efficiency elements in polyadenylation signals was obtained, suggesting that plant U1A is not involved in polyadenylation.

The organization of spliceosomal components in the nuclei of higher plants.

To analyze the organization of spliceosomal snRNPs in plant nuclei, we have used both immunofluorescence labelling with the antibody 4G3, raised against the human snRNP-specific protein U2B", and in situ hybridization with anti-sense probes to conserved regions of U1, U2 and U6 snRNAs. The organization comprises a fibrous interchromatin network, which may include both interchromatin fibrils and granules, and very prominent nuclear and nucleolar-associated bodies. Double labelling with an anti-p80 coilin antibody shows that these are coiled bodies. Dynamic changes in the labelling pattern were observed through the cell cycle, and in response to and on recovery from heat shock. The similarity of this organization to that observed in mammalian nuclei is strong evidence that it is fundamental to the processing of pre-mRNA in eucaryotes in general.

Isolation of a maize cDNA encoding a protein with extensive similarity to an inhibitor of protein kinase C and a cyanobacterial open reading frame.

A full-length cDNA clone, Mz2-12, with a predicted amino acid sequence showing extensive similarity to the sequence of a protein inhibitor of protein kinase C, purified from bovine brain, has been isolated from maize. The sequence of Mz2-12 is also similar to an open reading frame of unknown function on a cyanobacterial dicistronic message. The extensive similarity of the three protein sequences identifies a novel class of evolutionarily conserved proteins.

Plant pre-mRNA splicing and splicing components.

Pre-mRNA splicing or the removal of introns from precursor messenger RNAs depends on the accurate recognition of intron sequences by the plant splicing machinery. The major components of this machinery are small nuclear ribonucleoprotein protein particles (snRNPs) which consist of snRNAs and snRNP proteins. We have analysed various aspects of intron sequence and structure in relation to splice site selection and splicing efficiency and we have cloned snRNA genes and a gene encoding the snRNP protein, U2B". In the absence of an in vitro splicing system for plants, transient expression in protoplasts and stable plant transformations have been used to analyse splicing of intron constructs. We aim to address the function of the UsnRNP-specific protein, U2B", via the production of transgenic plants expressing antisense U2B" transcripts and epitope-tagged U2B" protein. In addition, we have cloned genes encoding other proteins which potentially interact with RNA, such as RNA helicases, and strategies involving transgenic plants are being developed to analyse their function.

Detection of antisense transcripts in transgenic plants by RT-PCR.

A reverse transcriptase-polymerase chain reaction (RT-PCR) where one oligonucleotide primer is end-labelled has been used to analyse expression in transgenic plants carrying antisense gene constructs. Specific detection of both sense and antisense RNA transcripts of the spliceosomal protein gene, U2B'', was achieved using the same pair of oligonucleotide primers. To maintain specificity, a reaction step in which reverse transcriptase was inactivated and RNA digested was found to be essential.

Detection of a plant protein analogous to the yeast spliceosomal protein, PRP8.

We have investigated whether a spliceosomal protein analogous to the yeast protein, PRP8, was present in higher plants. A protein with a molecular weight > 200 kDa was detected in Western blots of tobacco (Nicotiana tabacum L.) nuclear extracts with affinity-purified antibodies, raised against four different beta-galactosidase-PRP8 fusion proteins. The < 200 kDa protein was also immunoprecipitated by antibodies against the snRNA-specific trimethylguanosine cap structure and was, therefore, snRNP-associated. The presence of this protein in plants, in addition to yeast, Drosophila and humans, and the conservation of large size and epitopes highlights the importance of PRP8 in pre-mRNA splicing.

Evolutionary conservation of the spliceosomal protein, U2B''.

U1 and U2snRNPs play key roles in pre-mRNA splicing. The interactions between the U1 and U2snRNP-specific proteins, U1A, U2A' and U2B'' and their respective UsnRNAs are of interest both to elucidate their roles in splicing, and as models to study RNA-protein interactions. We have cloned a full-length cDNA, encoding U2B'', from potato. This is the first report of a sequence for a plant UsnRNP protein. The plant U2B'' sequence exhibits extensive similarity with the human U2B'' protein at both the DNA and amino acid levels. The evolutionary conservation at the protein level, particularly in sequences implicated in determining specific binding to U2snRNA, suggests conservation of U2B'' function from plants to man. The significance of amino acid substitutions in the RNP-80 motif with respect to U2snRNA binding in plants is discussed.

Earth history at the century mark of the U.S. Geological Survey.

Earth history involves all aspects of geological and biological evolution, especially paleontology and stratigraphy. Early paleontological exploration of the western United States by and before the U.S. Geological Survey featured the dramatic discoveries and rivalries of the great vertebrate paleontologists Leidy, Cope, Marsh, and Osborn. Invertebrate paleontology and paleobotany in the U.S. Geological Survey blossomed with emphasis on practical missions. The most illuminating and useful earth history, nevertheless, emerges where there is a high degree of interaction with academic scholars. Despite a good knowledge of its broad features, the drama of earth history remains obscure in detail. Whereas it speaks conclusively for the reality of organic evolution, it is less conclusive about mechanisms and many important transitions. Current investigations, however, especially in pre-Phanerozoic, mammalian, and human paleontology, promise improved insights. New techniques in collecting, sample preparation, and research are revealing previously unknown kinds of fossils and exquisite details of preservation. Plate tectonic theory provides a new framework for historical geography and biogeography. Emerging techniques in geochronology-matching paleopolarity sequences, for example-promise to resolve old problems of the synchroneity or heterochroneity of different biotal provinces. As it splits into subfields, the teaching and practice of paleontology expand to cover all of them. The fossils themselves, however, remain the basic objective evidence. All hypotheses about them must answer to this court of appeal. But nature rarely responds in an either-or way. The most probable hypotheses are those that have repeatedly confronted objective reality and survived all opportunity for disproof.

Fossil penguin from the late cenozoic of South Africa.

Spheniscus predemersus, new species, from the late Pliocene of Langebaanweg, Cape Province, is the first fossil penguin to be described from Africa. It is closely related and possibly ancestral to the living species, Spheniscus demnersus, of southern and southwestern Africa.

Ages of fossil penguins in new zealand.

The age of a New Zealand specimen generally believed to represent the oldest known penguin, hitherto considered early Eocene (Heretaungan), has been restudied by the New Zealand Geological Survey and is early Miocene. The oldest known penguins are from the late Eocene. The reported great range of a single species, Palaeeudyptes antarcticus, from late Eocene to late Oligocene or early Miocene (Kaiatan to Waitakian) is not acceptable. Dating of some other specimens is less precise than previously reported.