Mutation of putative branchpoint consensus sequences in plant introns reduces splicing efficiency

Abstract

Intron lariat formation between the 5' end of an intron and a branchpoint adenosine is a fundamental aspect of the first step in animal and yeast nuclear pre-mRNA splicing. Despite similarities in intron sequence requirements and the components of splicing, differences exist between the splicing of plant and vertebrate introns. The identification of AU-rich sequences as major functional elements in plant introns and the demonstration that a branchpoint consensus sequence was not required for splicing have led to the suggestion that the transition from AU-rich intron to GC-rich exon is a major potential signal by which plant pre-mRNA splice sites are recognized. The role of putative branchpoint sequences as an internal signal in plant intron recognition/definition has been re-examined. Single nucleotide mutations in putative branchpoint adenosines contained within CUNAN sequences in four different plant introns all significantly reduced splicing efficiency. These results provide the most direct evidence to date for preferred branchpoint sequences being required for the efficient splicing of at least some plant introns in addition to the important role played by AU sequences in dicot intron recognition. The observed patterns of 3' splice site selection in the introns studied are consistent with the scanning model described for animal intron 3' splice site selection. It is suggested that, despite the clear importance of AU sequences for plant intron splicing, the fundamental processes of splice site selection and splicing in plants are similar to those in animals.

TheElm1(ZmHy2) Gene of Maize Encodes a Phytochromobilin Synthase

“…6A) is the best candidate for the BPS used in conjunction with the type 3 AG. However, the normally observed distance between the BPS and 3# splice site AG is 18 to 40 nt (Simpson et al, 1996(Simpson et al, , 2002 and therefore larger than the 16 nt between BPS5 and the type 3 AG. The suboptimal placement of the intron IV type 3 AG relative to BPS5 may promote the use of the additional type 1 and type 2 sites.…”

“…If multiple AGs lie in close proximity to each other, sequence context can affect selection of the preferred AG (Smith et al, 1993). The position of an upstream branch point sequence (BPS) further influences the selection of the 3# splice site (Simpson et al, 1996). The BPS is defined by the consensus sequence CURAY (most highly conserved underlined) and is generally located 18 to 40 nt upstream of the 3# splice site.…”

Natural variation in ARF18 gene simultaneously affects seed weight and silique length in polyploid rapeseed

“…By comparing the sixth intron of the ARF18 alleles, extensive variations, including six SNPs and an 11-bp insertion, were detected in the region upstream of the 3′ splicing site in the nonfunctional sixth intron. In plants, although individual introns exhibit extensive variation around highly conserved dinucleotides (GU and AG) at the 5′ and 3′ splice sites (29), branchpoint sequences (YUNAN consensus) that usually are located 18-40 nt upstream of the 3′ splice site of introns play an important role in splicing efficiency (30). Introns are removed via a two-step cleavage-ligation reaction in which the first step involves cleavage at the 5′ splice site with formation of an intron lariat at the branchpoint (31).…”

“…Previous reports have shown that Q-rich MRs in ARFs function as activation domains (29). This finding suggests that ARF18 might function as a repressor because it does not possess a Q-rich MR. To confirm our inference, we detected the transcriptional activity of ARF18 by using a protoplast assay system.…”

A stable tRNA-like molecule is generated from the long noncoding RNA GUT15 in Arabidopsis

“…The intron sequences together with the fragments (>55 bp) from the original 5′- and 3′-exons were isolated from Arabidopsis genomic DNA by PCR and introduced to the plant expression vector pDH515 within an intronless zein gene using the unique Bam HI restriction site [42,43]. For the mutant intron construct generation (with different tRNA types), a two-step PCR-based approach was used.…”