Splicing signals and factors in plant intron removal.

Constitutive splicing of the potato invertase mini-exon 2 (9 nt long) requires a branchpoint sequence positioned around 50 nt upstream of the 5' splice site of the adjacent intron and a U(11) element found just downstream of the branchpoint in the upstream intron [Simpson, Hedley, Watters, Clark, McQuade, Machray and Brown (2000) RNA 6, 422-433]. The sensitivity of this in vivo plant splicing system has been used to demonstrate exon scanning in plants, and to characterize plant intronic elements, such as branchpoint and poly-pyrimidine tract sequences. Plant introns differ from their vertebrate and yeast counterparts in being UA- or U-rich (up to 85% UA). One of the key differences in splicing between plants and other eukaryotes lies in early intron recognition, which is thought to be mediated by UA-binding proteins. We are adopting three approaches to studying the RNA-protein interactions in plant splicing. First, overexpression of plant splicing factors and, in particular, UA-binding proteins, in conjunction with a range of mini-exon mutants. Secondly, the sequences of around 65% of vertebrate and yeast splicing factors have high-quality matches to Arabidopsis proteins, opening the door to identification and analysis of gene knockouts. Finally, to discover plant-specific proteins involved in splicing and in, for example, rRNA or small nuclear RNA processing, green fluorescent protein-cDNA fusion libraries in viral vectors are being screened.

Expression of intron-containing GUS constructs is reduced due to activation of a cryptic 5' splice site.

An intron-containing beta-glucuronidase (GUS) gene has been used widely in promoter analyses and as a plant transformation marker. Maximal plant gene expression requires accurate and efficient removal of the intron from the expressed pre-mRNA transcripts by splicing. Detailed analysis of splicing of potato ST-LS1 and pea legumin introns from GUS constructs revealed the activation of a cryptic 5' splice site in the GUS coding sequence 4 nt upstream from the authentic intron 5' splice site. About 40% of transcripts utilised the cryptic 5' splice site in tobacco protoplasts, reducing the translational potential of expressed pre-mRNA. The same cryptic splicing event was evident in transgenic tobacco leaves but at reduced levels. Mutations that removed the cryptic 5' splice site are associated with a two-fold enhancement in GUS activity in tobacco protoplasts, highlighting the need for careful examination of introns and their sites of insertion into gene constructs to minimise variability in gene activity and maximise gene expression.

Multiple snoRNA gene clusters from Arabidopsis.

Small nucleolar RNAs (snoRNAs) are involved in precursor ribosomal RNA (pre-rRNA) processing and rRNA base modification (2'-O-ribose methylation and pseudouridylation). In all eukaryotes, certain snoRNAs (e.g., U3) are transcribed from classical promoters. In vertebrates, the majority are encoded in introns of protein-coding genes, and are released by exonucleolytic cleavage of linearized intron lariats. In contrast, in maize and yeast, nonintronic snoRNA gene clusters are transcribed as polycistronic pre-snoRNA transcripts from which individual snoRNAs are processed. In this article, 43 clusters of snoRNA genes, an intronic snoRNA, and 10 single genes have been identified by cloning and by computer searches, giving a total of 136 snoRNA gene copies of 71 different snoRNA genes. Of these, 31 represent snoRNA genes novel to plants. A cluster of four U14 snoRNA genes and two clusters containing five different snoRNA genes (U31, snoR4, U33, U51, and snoR5) from Arabidopsis have been isolated and characterized. Of these genes, snoR4 is a novel box C/D snoRNA that has the potential to base pair with the 3' end of 5.8S rRNA and snoR5 is a box H/ACA snoRNA gene. In addition, 42 putative sites of 2'-O-ribose methylation in plant 5.8S, 18S, and 25S rRNAs have been mapped by primer extension analysis, including eight sites novel to plant rRNAs. The results clearly show that, in plants, the most common gene organization is polycistronic and that over a third of predicted and mapped methylation sites are novel to plant rRNAs. The variation in this organization among gene clusters highlights mechanisms of snoRNA evolution.

Requirements for mini-exon inclusion in potato invertase mRNAs provides evidence for exon-scanning interactions in plants.

Invertases are responsible for the breakdown of sucrose to fructose and glucose. In all but one plant invertase gene, the second exon is only 9 nt in length and encodes three amino acids of a five-amino-acid sequence that is highly conserved in all invertases of plant origin. Sequences responsible for normal splicing (inclusion) of exon 2 have been investigated in vivo using the potato invertase, invGF gene. The upstream intron 1 is required for inclusion whereas the downstream intron 2 is not. Mutations within intron 1 have identified two sequence elements that are needed for inclusion: a putative branchpoint sequence and an adjacent U-rich region. Both are recognized plant intron splicing signals. The branchpoint sequence lies further upstream from the 3' splice site of intron 1 than is normally seen in plant introns. All dicotyledonous plant invertase genes contain this arrangement of sequence elements: a distal branchpoint sequence and adjacent, downstream U-rich region. Intron 1 sequences upstream of the branchpoint and sequences in exons 1, 2, or 3 do not determine inclusion, suggesting that intron or exon splicing enhancer elements seen in vertebrate mini-exon systems are absent. In addition, mutation of the 3' and 5' splice sites flanking the mini-exon cause skipping of the mini-exon, suggesting that both splice sites are required. The branchpoint/U-rich sequence is able to promote splicing of mini-exons of 6, 3, and 1 nt in length and of a chicken cTNT mini-exon of 6 nt. These sequence elements therefore act as a splicing enhancer and appear to function via interactions between factors bound at the branchpoint/U-rich region and at the 5' splice site of intron 2, activating removal of this intron followed by removal of intron 1. This first example of splicing of a plant mini-exon to be analyzed demonstrates that particular arrangement of standard plant intron splicing signals can drive constitutive splicing of a mini-exon.

Splicing-independent processing of plant box C/D and box H/ACA small nucleolar RNAs.

Small nucleolar RNAs (snoRNAs) are involved in various aspects of ribosome biogenesis and rRNA maturation. Plants have a unique organisation of snoRNA genes where multiple, different genes are tightly clustered at a number of different loci. The maize gene clusters studied here include genes from both of the two major classes of snoRNAs (box C/D and box H/ACA) and are transcribed as a polycistronic pre-snoRNA transcript from an upstream promoter. In contrast to vertebrate and yeast intron-encoded snoRNAs, which are processed from debranched introns by exonuclease activity, the particular organisation of plant snoRNA genes suggests a different mode of expression and processing. Here we show that single and multiple plant snoRNAs can be processed from both non-intronic and intronic transcripts such that processing is splicing-independent and requires endonucleolytic activity. Processing of these different snoRNAs from the same polycistronic transcript suggests that the processing machineries needed by each class are not spatially separated in the nucleolus/nucleus.

U14snoRNAs of the fern, Asplenium nidus, contain large sequence insertions compared with those of higher plants.

Northern analyses of U14snoRNAs in different plant species showed the expected hybridising band of approximately 120 nt in monocotyledonous and dicotyledonous angiosperms. In the lower plant, Bird's nest fern (Asplenium nidus), U14s were larger and three hybridising RNAs of approximately 190, 210 and 250 nt were observed. RT-PCR cloning of all three size variants using primers to the conserved 5' and 3' ends of higher plant U14snoRNAs showed large insertions in one of the plant-specific regions corresponding in position to the yeast U14-specific Y-domain. The insertions are pyrimidine-rich in their 5' halves and purine-rich in their 3' halves and are likely to be sequestered in stem structures consistent with the proposed model of U14snoRNA secondary structure. The 5' flanking regions of one of the fern U14 variants was generated by PCR and lacked classical plant snRNA promoter elements.

Processing of vertebrate box C/D small nucleolar RNAs in plant cells.

The recent isolation of a number of plant box C/D small nucleolar (sno)RNAs demonstrates the conservation in plants of sequence and structural elements of processed box C/D snoRNAs. Boxes C and D, and terminal inverted repeats are known to be essential for accumulation and processing in vertebrates and yeast. Processing of vertebrate box C/D snoRNAs was examined by expression of various mouse hsc70 intron 5-U14 constructs in tobacco protoplasts. Full-length U14 and internally deleted U14 accumulated in the plant cells. Human U3 and U8 fragments, consistent with processing to internal box C/C' sequences, also accumulated in the plant cells. The similarity of processing behaviour of the vertebrate box C/D constructs in tobacco protoplasts and Xenopus oocytes suggests the mechanism of processing, involving recognition and association of proteins, is conserved in plants.

Clusters of multiple different small nucleolar RNA genes in plants are expressed as and processed from polycistronic pre-snoRNAs.

Small nucleolar RNAs (snoRNAs) are involved in many aspects of rRNA processing and maturation. In animals and yeast, a large number of snoRNAs are encoded within introns of protein-coding genes. These introns contain only single snoRNA genes and their processing involves exonucleolytic release of the snoRNA from debranched intron lariats. In contrast, some U14 genes in plants are found in small clusters and are expressed polycistronically. An examination of U14 flanking sequences in maize has identified four additional snoRNA genes which are closely linked to the U14 genes. The presence of seven and five snoRNA genes respectively on 2.05 and 0.97 kb maize genomic fragments further emphasizes the novel organization of plant snoRNA genes as clusters of multiple different genes encoding both box C/D and box H/ACA snoRNAs. The plant snoRNA gene clusters are transcribed as a polycistronic pre-snoRNA transcript from an upstream promoter. The lack of exon sequences between the genes suggests that processing of polycistronic pre-snoRNAs involves endonucleolytic activity. Consistent with this, U14 snoRNAs can be processed from both non-intronic and intronic transcripts in tobacco protoplasts such that processing is splicing independent.

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.

Complementary deletions in expressed potato U2snRNA gene variants support the hypothesis that stem-loop IIb is dispensable for splicing.

A polymerase chain reaction (PCR) strategy designed to amplify DNA sequences between closely linked U2snRNA genes has generated extensive coding and 5' regulatory sequence information on the potato U2snRNA multigene family. Two of the U2snRNA coding sequences isolated differed substantially from normal U2snRNAs by containing both complementary deletions and regions of novel sequence. However, sequences such as Sm-binding sites and loops of stem-loops III and IV, which are some of the most highly conserved regions in U2snRNA, remain highly conserved in these genes. The complementary deletions would effectively remove stem-loop IIb which has been shown in yeast to be unnecessary for pre-mRNA splicing. Transcripts from one of the genes have been detected by reverse transcriptase-PCR (RT-PCR) in total RNA. These novel U2snRNA genes represent the first reported example of naturally occurring structural variants and provide support for the proposed non-essential role of U2snRNA stem-loop IIb.

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.

Chickenpox pneumonia, its complications and management. A report of three cases, including the use of extracorporeal membrane oxygenation.

We report three cases of chickenpox pneumonia in adults, all of whom required intermittent positive pressure ventilation. One patient developed a variety of complications, and another, a pregnant woman, required extracorporeal membrane oxygenation.