Gillian P. Clark

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.

Aberrant mRNA Transcripts and the Nonsense-Mediated Decay Proteins UPF2 and UPF3 Are Enriched in the Arabidopsis Nucleolus

The eukaryotic nucleolus is multifunctional and involved in the metabolism and assembly of many different RNAs and ribonucleoprotein particles as well as in cellular functions, such as cell division and transcriptional silencing in plants. We previously showed that Arabidopsis thaliana exon junction complex proteins associate with the nucleolus, suggesting a role for the nucleolus in mRNA production. Here, we report that the plant nucleolus contains mRNAs, including fully spliced, aberrantly spliced, and single exon gene transcripts. Aberrant mRNAs are much more abundant in nucleolar fractions, while fully spliced products are more abundant in nucleoplasmic fractions. The majority of the aberrant transcripts contain premature termination codons and have characteristics of nonsense-mediated decay (NMD) substrates. A direct link between NMD and the nucleolus is shown by increased levels of the same aberrant transcripts in both the nucleolus and in Up-frameshift (upf) mutants impaired in NMD. In addition, the NMD factors UPF3 and UPF2 localize to the nucleolus, suggesting that the Arabidopsis nucleolus is therefore involved in identifying aberrant mRNAs and NMD.

Determinants of Plant U12-Dependent Intron Splicing Efficiency

Factors affecting splicing of plant U12-dependent introns have been examined by extensive mutational analyses in an in vivo tobacco (Nicotiana tabacum) protoplast system using introns from three different Arabidopsis thaliana genes: CBP20, GSH2, and LD. The results provide evidence that splicing efficiency of plant U12 introns depends on a combination of factors, including UA content, exon bridging interactions between the U12 intron and flanking U2-dependent introns, and exon splicing enhancer sequences (ESEs). Unexpectedly, all three plant U12 introns required an adenosine at the upstream purine position in the branchpoint consensus UCCUURAUY. The exon upstream of the LD U12 intron is a major determinant of its higher level of splicing efficiency and potentially contains two ESE regions. These results suggest that in plants, U12 introns represent a level at which expression of their host genes can be regulated.

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.

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.