Craig Simpson

Invertase mini-exon splicing

One consideration of exon bridging is that when exons are very small (less than 50 nucleotides) steric hindrance inhibits assembly of complexes at each end of the exon. There are a number of very small exons in both plants and animals. In animals these require specific elements in the flanking introns (Intron Splicing Enhancers or ISEs) which bind factors that activate splicing at one or other splice site.

Plant invertase genes contain a nine nucleotide mini-exon. For splicing, two elements: a branchpoint and adjacent U-rich region in the upstream intron, are required. The distance of the branchpoint from the mini-exon (50-80 nt) is important. Our model is that factors binding to the branchpoint/U-rich region interact, via bridging, with a complex at the 5' splice site, to activate splicing of the downstream intron. This is followed by splicing of the upstream intron to give the final mRNA.

Invertase mini-exon diagram

Simpson et al. (2000) RNA 6, 422-433.

Intron splicing mutants in Arabidopsis

Up to 15% of human mutations are splice site mutations. A survey of Arabidopsis mutants, which carry mutations to splice sites and affect splicing provided the initial suggestion that exon definition occurs in plant pre-mRNA splicing. The various mutants exhibited different splicing behaviours: activation of cryptic 5' and 3' splice sites, exon skipping and intron retention, described for human genetic mutations affecting splice sites.

Intron mutants diagram

Brown (1996) Plant J. 10, 771-780.
Simpson et al. (1998) Plant J. 15, 125-131.

AT-AC introns

Higher eukaryotes contain a minor class of introns which are spliced by a novel spliceosome (the U12-dependent spliceosome) which differs from the U2-dependent spliceosome, responsible for removal of the majority of introns. The minor class of introns are called U12-dependent introns or AT-AC introns as many of them contain AT and AC as 5' and 3' splice sites respectively, although some contain the normal GT and AG combination. AT-AC introns also differ in the sequence and position of their branchpoints.

The U12-dependent spliceosome contains U11, U12, U4atac and U6atac snRNPs instead of the normal U1, U2, U4 and U6 snRNPs. U5snRNP is common to both U2- and U12-dependent spliceosomes.

In collaboration with Professor Artur Jarmolowski, University of Poznan, Poland, we analysed the splicing efficiency of plant pre-mRNAs containing AT-AC introns (CBP20, GSH and Ld). We found evidence that the 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. A major determinant for high splicing efficiency in the LD U12 intron was the presence of the upstream exon sequence. We are investigating the possible exon splicing enhancers located within this exon.

AT-AC Intron diagram

U-rich binding proteins

Plant introns differ from vertebrate and yeast introns as they are U-rich with many dicot introns containing up to 85% UA. Plant intron splicing signals (5' and 3' splice sites, branchpoint sequences and polypyrimidine tracts) are similar to those of vertebrate introns. It is expected that the UA-rich intron sequences are recognised and bound by U-rich binding proteins which aid in spliceosome assembly.

One of the key questions in plant intron splicing is which proteins recognise and interact with the UA-rich intron sequences. RNA-binding proteins with affinity for oligoU have been cloned from plants in the lab of Professor Witek Filipowicz, Basel. We are analysing the effects of over-expression of these proteins on splicing of mutants of the potato invertase mini-exon system. Further characterisation of these proteins will be carried out in collaboration with Professor Andrea Barta, University of Vienna.

Spliceosome components

We have isolated over forty U1, U2, U5 and U6snRNA genes from potato and maize. In many cases, the genes are linked. Genes for the SmG core protein and the U1snRNP-, U2snRNP- and U5snRNP-specific proteins, U1A, U2B" and PRP8 have been isolated from various plants. In RNA binding studies, potato U2B" interacted with human U2A' to confer specific and stable binding to U2snRNA, highlighting the degree of conservation of these RNA-binding proteins.

In collaboration with Professor David Meinke, Oklahoma State University, the properties of PRP8 genes in maize and Arabidopsis have been examined. An Arabidopsis PRP8 gene was isolated as a T-DNA insertion mutant, sus2-1. The genes are of interest because they differ in the presence (maize) and absence (Arabidopsis) of proline-rich regions in the N-terminal end, known to be required for function in yeast. In addition, the maize gene contains 23 introns while the Arabidopsis gene contains only 11. In both plants there are 3-4 PRP8 genes, while there is only one in yeast and human.

Spliceosome Components diagram

Simpson et al. (1995) EMBO J. 14, 4540-4550.
Brown and Simpson (1998) Ann. Rev. Plant Physiol. & Plant Mol. Biol. 49, 77-95.

Splicing signals

Vertebrate intron signals comprise the 5' and 3' splice site, a branchpoint sequence and a polypyrimidine tract downstream of the branchpoint. Plant introns contain 5' and 3' splice sites similar to those of vertebrate and yeast introns. However, plant introns are different in being U-rich.

To analyse intron splicing signals, mutations are cloned into an expression cassette, transfected into protoplasts, RNA isolated and analysed by RT-PCR. By using one fluorescently labelled primer, products are separated on automatic DNA sequencing gels.

We have demonstrated that branchpoints are required for efficient intron splicing through mutational analysis. We have also used the sensitive potato invertase mini-exon system to systematically mutate the sequence surrounding the branchpoint and the polypyrimidine tract. This analysis has defined a plant branchpoint and polypyrimidine tract for the first time. The sequences (below) are similar to vertebrate sequences:

Diagram showing intron sequences

Brown (1996) Plant J. 10, 771-780.
Brown et al. (1996) Plant Mol. Biol. 32, 531-535.
Simpson et al. (1996) Plant J. 9, 369-380.
Simpson et al. (2002) RNA 8, 47-56.
Simpson et al. (2004) Plant J. 37, 82-91.

Plant pre-mRNA splicing

The majority of plant protein-coding genes contain introns which must be removed from pre-mRNAs to produce mRNAs for translation into proteins. Introns contain conserved splicing signals which ensure fidelity of splicing.

Introns are removed in a large complex called the spliceosome. The major spliceosomal components are the U1, U2, U4/U6 and U5snRNPs (small nuclear ribonucleoprotein particles). SnRNPs contain one or two snRNAs (small nuclear RNAs) complexed with core Sm proteins and snRNP-specific proteins. In addition, a number of transiently associated proteins (for example, hnRNP and SR proteins, RNA helicases) are required for spliceosome assembly.

Plant introns differ from those of vertebrates and yeast by being U-rich, probably requiring the action of U-rich binding proteins for early recognition and spliceosome assembly.

In eukaryotes a minor spliceosome with different but related snRNAs exists to splice a minor class of introns called AT-AC introns.

Genes and Development

Biochemical and developmental processes determine how plants grow and respond to external stimuli. We are interested in the molecular basis of particular plant developmental and metabolic pathways. In particular, we address general aspects of post-transcriptional regulation at the level of alternative splicing and non-coding RNAs and how such regulation determines flowering time.

The biochemical pathway of lignin biosynthesis is an extremely important and topical area of research due to the opportunities in biofuels and bioenergy. The potential for biotechnological applications to address areas like biofuels and biotic and abiotic stress tolerance in the face of climate change requires research on technologies to express multiple genes or pathways and to target modification of specific genes.

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