Systematic identification of factors involved in post-transcriptional processes in wheat grain
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Post-transcriptional processing of primary transcripts can significantly affect both the quantity and the structure of mature mRNAs and the corresponding protein products. It is an important mechanism of gene regulation in animals, yeast and plants. Here we have investigated the interactive networks of pre-mRNA processing factors in the developing grain of wheat (Triticum aestivum), one of the world’s major food staples. As a first step we isolated a homologue of the plant specific AtRSZ33 splicing factor, which has been shown to be involved in the early stages of embryo development in Arabidopsis. Real-time PCR showed that the wheat gene, designated TaRSZ38, is expressed mainly in young, developing organs (flowers, root, stem), and expression peaks in immature grain. In situ hybridization and immunodetection revealed preferential abundance of TaRSZ38 in mitotically active tissues of the major storage organ of the grain, the endosperm. The protein encoded by TaRSZ38 was subsequently used as a starting bait in a two-hybrid screen to identify additional factors in grain that are involved in pre-mRNA processing. Most of the identified proteins showed high homology to known splicing factors and splicing related proteins, supporting a role for TaRSZ38 in spliceosome formation and 5′ site selection. Several clones were selected as baits in further yeast two-hybrid screens. In total, cDNAs for 16 proteins were isolated. Among these proteins, TaRSZ22, TaSRp30, TaU1-70K, and the large and small subunits of TaU2AF, are wheat homologues of known plant splicing factors. Several, additional proteins are novel for plants and show homology to known pre-mRNA splicing, splicing related and mRNA export factors from yeast and mammals.
KeywordsGrain development Pre-mRNA processing factors Protein–protein interaction Splicing Yeast two-hybrid system
Days after pollination
GenBank expressed sequence tag database
Nuclei extraction buffer
Nuclear localization signal
Nonsense mediated decay
RNA recognition motif
Arginine/serine proteins with one or two zinc fingers
- SCL proteins
- SD domains
Serine/aspartic acid rich domains
Small nuclear ribonucleoprotein particles
- SR domains
Serine/arginine rich domains
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We thank Ursula Langridge for assistance with growing plants in the glasshouse. This work was supported by the Australian Grain Research and Development Corporation (grant no. UA00083 to P.L.).
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