True and false alternative transcripts of calcium-dependent protein kinase CPK9 and CPK3a genes in Vitis amurensis
- 157 Downloads
Calcium-dependent protein kinases (CDPKs) are major Ca2+ sensors in plants playing important roles in plant development and stress responses. In the present study, RT-PCR analysis revealed that VaCPK3a and VaCPK9 cDNAs lacking extensive regions in the kinase, autoinhibitory, and Ca2+-binding domains were numerous in probes derived from wild grapevine Vitis amurensis. Most of the VaCPK3a and VaCPK9-modified transcripts lacked canonical splice sites and possessed short direct repeated sequences (SDRs) instead. Three VaCPK9 transcript variants were generated using canonical 5′GT and 3′AG splice sites and lacked several subdomains in the kinase domain, including ATP-binding site, which is known to be indispensable for kinase activities. These observations indicate that post-transcriptional mRNA processing might lead to production of CDPKs with abolished phosphorylation activities. Recombinant proteins VaCPK3aSF2, lacking autoinhibitory and Ca2+-binding domain, and VaCPK3aSF3, lacking VIII–XI kinase subdomains and autoinhibitory domain, phosphorylated exogenous substrate in a Ca2+-independent manner. However, reverse transcription at +65 °C using heat-stable reverse transcriptase (RT) markedly lowered abundance of the unusual VaCPK3a and VaCPK9 cDNAs with SDRs, while it did not eliminate VaCPK9 cDNAs generated using canonical splice sites. The results show that VaCPK9 gene undergoes unproductive alternative splicing mediated by canonical splice sites to generate three mRNA isoforms lacking important catalytic kinase subdomains. The unusual VaCPK3a and VaCPK9 transcripts with SDRs are likely to be false alternative transcripts generated by RT template switching in vitro. The data demonstrate that using non-thermostable RTs for studying alternative splicing are not appropriate.
KeywordsCalcium-dependent protein kinases Alternative splicing Splice sites Short direct repeated sequences Vitis amurensis
Short direct repeated sequences
Premature stop codon
We sincerely thank Dr. E.A. Lysenko for helpful advice and discussions on the experimental work. This work was supported by grants from the Dynasty Foundation (DP-B-09/13), the Russian Foundation for Basic Research (12-04-31110-mol_a), and from the Far East Division of the Russian Academy of Sciences.
- Kiselev KV, Dubrovina AS, Shumakova OA, Karetin YA, Manyakhin AY (2013) Structure and expression profiling of a novel calcium-dependent protein kinase gene, CDPK3a, in leaves, stems, grapes, and cell cultures of wild-growing grapevine Vitis amurensis Rupr. Plant Cell Rep 32:431–442PubMedCrossRefGoogle Scholar
- Koo SC, Yoon HW, Kim CY, Moon BC, Cheong YH, Han HJ, Lee SM, Kang KY, Kim MC, Lee SY, Chung WS, Cho MJ (2007) Alternative splicing of the OsBWMK1 gene generates three transcript variants showing differential subcellular localizations. Biochem Biophys Res Commun 360:188–193PubMedCrossRefGoogle Scholar
- Luo D, Niu X, Wang Y, Zheng W, Chang L, Wang Q, Wei X, Yu G, Lu BR, Liu Y (2007) Functional defect at the rice choline monooxygenase locus from an unusual post-transcriptional processing is associated with the sequence elements of short-direct repeats. New Phytol 175:439–447PubMedCrossRefGoogle Scholar
- Nishiyama R, Mizuno H, Okada S, Yamaguchi T, Takenaka M, Fukuzawa H, Ohyama K (1999) Two mRNA species encoding calcium-dependent protein kinases are differentially expressed in sexual organs of Marchantia polymorpha through alternative splicing. Plant Cell Physiol 40:205–212PubMedCrossRefGoogle Scholar