Determinants of Plant U12-Dependent Intron Splicing Efficiency

Abstract

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. INTRODUCTIONPre-mRNA splicing in eukaryotes is a fundamental step in gene expression and represents an important level at which the expression of protein-coding genes can be regulated (Reed, 2000;Smith and Valcá rcel, 2000;Graveley, 2001;Hastings and Krainer, 2001;Cartegni et al., 2002;Black, 2003). In higher eukaryotes, there are two classes of nuclear pre-mRNA introns. The most abundant class consists of U2-dependent introns (U2 introns), whereas the second rarer class (<0.4% of introns) consists of U12-dependent introns (U12 introns). U12 introns have been found in the nuclear genomes of vertebrates, plants, and insects (Hall and Padgett, 1994;Sharp and Burge, 1997;Tarn and Steitz, 1997; Krainer, 1998, 1999;Burge et al., 1998;Levine and Durbin, 2001;Patel and Steitz, 2003). Introns belonging to these two distinct classes are spliced by two different spliceosomes: the major U2-type spliceosome and the less abundant U12-type spliceosome (Hall and Padgett, 1996;Tarn and Steitz 1996a. Although the first U12 introns to be described had AT-AC-terminal dinucleotides, the majority of U12-type introns contain GT-AG, and a small number contain other noncanonical terminal dinucleotides, such as AT-AA, AT-AG, AT-AT, GT-AT, or GT-GG (Jackson, 1991;Hall and Padgett, 1994;Dietrich et al., 1997Dietrich et al., , 2001aSharp and Burge, 1997;Burge et al., 1998;Wu and Krainer, 1999;Levine and Durbin, 2001;Zhu and Brendel, 2003). Moreover, functional analyses have shown that AT-AC-terminal dinucleotides are not a defining feature of U12 introns (Dietrich et al., 1997(Dietrich et al., , 2001a. Instead, U12 introns contain highly conserved sequences in the 59 splice site (exon:G/ATATCCTY) and branchpoint region (TCCTTRAY) (Hall and Padgett, 1994;Sharp and Burge, 1997;Burge et al., 1998), which are both required for prespliceosome complex formation (Frilander and Steitz, 1999). The 39 splice site consensus sequence of U12 introns (YAC/G:exon) is less informative than their 59 splice site and branchpoint sequences. U12 introns also lack a polypyrimidine tract and have a short distance between the branchpoint and 39 splice si...

Identification and molecular characterization of a novel monopartite geminivirus associated with mulberry mosaic dwarf disease

“…Incidentally, our study showed that NGS data can be exploited for further exploration of the expression strategy of geminiviruses. The splicing signals found at the border of the MMDaV intron are identical to those conserved in the characteristic U2 plant introns (Lewandowska et al, 2004), but they differ slightly from those reported in all the other geminiviruses known to bear an intron in the complementary-sense transcript (Fig. S3).…”

“…4a). This intron contains the consensus 59 and 39 splice sites and branchpoints characteristic of the U2 plant introns (Lewandowska et al, 2004) and has an RNA U+A content (59.4%) close to the minimum needed for its efficient splicing (Goodall & Filipowicz, 1989;Simpson & Brown, 1993). Interestingly, an intron with similar features can also be predicted in a similar position of the complementary-sense strand of the CCDaV genome.…”

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.…”

The rise and falls of introns

“…The common U2-type splices GT-AG introns, socalled because they have GT and AG dinucleotides at the 5 0 and 3 0 end, respectively. The second is known as the U12 type, and it splices the very rare -and misleadingly named -AT-AC introns, which have a range of dinucleotides at their ends (confusingly, most often GT-AG) (Lewandowska et al, 2004); however, the two types are sufficiently similar for us to treat them as a single entity in this review. Eukaryotes vary considerably in the length and abundance of their introns (Deutsch and Long, 1999), yet most seem to have them and even unicellular eukaryotes have extremely complex spliceosomes for their removal (Jurica and Moore, 2003;Nilsen, 2003).…”