Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 56 (2023)
RIKEN
Accel. Prog. Rep. 56 (2023)

Analysis of splicing patterns in Arabidopsis egy1-4 carrying
argon-induced mutations in the intron 3-exon 4 region
A. Sanjaya,∗1,∗2 Y. Fujii,∗1 M. Asano,∗1 T. Abe,∗3 Y. Kazama,∗2,∗3 and M. Fujiwara∗1,∗3
Eukaryotic gene expression involves the splicing ofprecursor messenger RNAs before the translation. TheU2 spliceosome, a nuclear ribonucleoprotein complex,
is important in the process of splicing, and recognizes
certain conserved “canonical” sequences in introns, including a GU at the 5’ end, an adenine known as the“branchpoint” located 20–50 bases upstream of the 3’
end, and an AG at the 3’ end. Based on the mechanismof intron excision discovered, the intron 5’ splice andintron 3’ splice sites are referred to as the splice donorsite and the splice acceptor site, respectively. Splicing
begins at the 5’ splice site and involves the formation of
an intron lariat at the branchpoint as the cleaved 5’ endof the intron becomes attached to it. The excision of theintron lariat from the 3’ splice site then allows for theligation of the exons.
Mutations at such canonical intron sites typicallycause ineﬃcient and/or cryptic splicings.1) In Arabidopsis thaliana, mutations of the intron 3’ splice site, either“AG to AA” or “AG to GG,” are known to activate
only cryptic 3’ splice sites but not 5’ splice sites.1,2) A
3’ splice site mutation resulting in cryptic 5’ splice site
activation has never been reported.
In A. thaliana, the Ethylene-dependent Gravitro-pismdeﬁcient and Yellow-green 1 (EGY1 ) gene (AGI code:At5g35220) encodes a chloroplast-targeted metalloprotease and is composed of 10 exons and 9 introns. During its functional analysis, an argon-ion-induced allele,egy1-4 (Ar-28-pg13) ), has been noted as it contains an
AG to AC transition at the 3’-end of intron 3, in addition to 4-bp substitutions and a 5-bp deletion in thedownstream exon 4.4) To determine whether these mutations in the intron 3-exon 4 junction region of egy1-4
aﬀect the splice site selection in EGY1, the total RNAs
were extracted from 2-week-old seedlings of wild-type
and egy1-4, and analyzed using RT-PCR with primers
binding to the EGY1 5’-untranslated region and exon
5. The ampliﬁed PCR products were ligated to a vector
and transformed into an E. coli DH5α strain. The resulting colonies were subsequently cultured for the propagation of plasmids, which were then Sanger-sequenced.
The sequence of EGY1 cDNAs around intron 3 was thencompared between the wild-type and egy1-4.
Only one splicing pattern of intron 3 was observed in
the wild-type by sequencing 30 clones (Fig. 1; Table 1).
However, among 58 cDNA clones of egy1-4 examined,42 exhibited no splicing of intron 3 (unspliced pattern).
The rest exhibited distinct splicing patterns from thewild-type ones: 5 and 8 clones exhibited activation of∗1
∗2
∗3

Graduate School of Science and Technology, Sophia University
Graduate School of Bioscience and Biotechnology, Fukui Prefectural University
RIKEN Nishina Center

Fig. 1. Schematic comparison of EGY1 transcript splicing
patterns between the wild-type and egy1-4. Sole splicingpattern (wt) was observed in the wild-type. Cryptic 3’
splice sites were activated without (patterns 1 and 2) orwith (pattern 3) the activation of cryptic 5’ splice site inegy1-4. The nucleotide letters in red denote that they are
uniquely produced in egy1-4 by argon-ion-irradiation.3)

Table 1. RT-PCR and cloning analysis of intron 3 splicing
patterns in wild-type and egy1-4.

Plantline
Wildtype
egy1-4Intron 3
splicing
patternwt

Total:(unspliced)
123
Total:

Number ofcDNA clones
sequenced
3030
4258358

Relativefrequency
100 %100 %72.4 %8.6 %13.8 %
5.2 %100 %upstream and downstream cryptic 3’ splice sites, respectively. Moreover, a new splicing pattern, exhibiting activation of cryptic 5’ splice site alongside activation of
3’ splice site, was found in 3 clones (Fig. 1; Table 1).
As sole splicing patterns were observed in all the otherintrons for both wild-type and egy1-4 (unpublished results), the pale green phenotype of egy1-4 plants4) was
attributed to the expression of truncated forms of EGY1.
This is due to premature stop codons in the mutant exon
3 or exon 4 in any spliced/unspliced variants. Our ﬁnding demonstrates that mutations induced by heavy-ion
irradiation signiﬁcantly impact on the terminal phenotype of plant bodies via interference of splicing.
References
1) J. W. S. Brown, Plant J. 10, 771 (1996).
2) M. A. Schuler, in Splice Site Requirements and Switches
in Plants, edited by A. S. N. Reddy and M. Golovkin(Springer, Berlin, 2008), p. 39.
3) T. Hirano et al., Mutat. Res. 735, 19 (2012).
4) A. Sanjaya et al., Plants 10, 1254 (2021).

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