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Acta Botanica Sinica 植 物 学 报 2004, 46 (4): 497-504 http://www.chineseplantscience.com Non-coding RNA for ZM401, a Pollen-specific Gene of Zea mays DAI Xiao-Yan, YU Jing-Juan* , ZHAO Qian, ZHU Deng-Yun, AO Guang-Ming (State Key Laboratory for Agricultural Biotechnology, College of Biology, China Agricultural University, Beijing 100094, China) Abst r act : In our previous study, a cDNA library to poly(A) RNA isolated from mature pollen of Zea mays L. was constructed. One cDNA fragment, designated ZM401 (Z. mays), was obtained from maize mat ure pollen cDNA library by differential scree ning and cold-plaque screening method. It was specifically or preferentially expressed in mature pollen. According to the ZM401 cDNA fragment sequence, full length of ZM401 cDNA was generated by 5' and 3' RACE methods. ZM401 cDNA was 1 149 bp in length. Within the full-length cDNA sequence, a clear open reading frame (ORF) was undectable by OMEGA 2.0 and DNAMAN softwares. The longest potential ORF was 269 nucleotides (791–1 060), coding 89 AA, had a poor consensus sequence for translation initiation. But it had a poly(A) tail. All these results suggested that ZM401 was one of a growing number of non-coding mRNA-like RNA transcripts that exerted their cellular functions directly as RNA. RT-PCR and Northern blotting analyses showed the ZM401 was transcribed from tetrad stage of microspore development and increased in concentration up to mature pollen, which suggested that ZM401 belongs to late gene in pollen development. Two transcripts of the ZM401 gene were detected by Northern blotting analysis (1.2 kb, 2.0 kb). Southern hybridisation showed that the ZM401 in corn was present in one or a very few copes in the maize genome. Ke y wo rds: Zea mays ; pollen; non-coding RNA; 5' RACE; 3' RACE; overlapping PCR; developmental expression pattern There were several reports of transcripts without a long open reading frame (ORF) in various eucaryotes (Brannan et al., 1990; Brockdorff et al., 1992; Brown et al. 1992; Askew et al., 1994; Crespi et al., 1994; Velleca et al., 1994; Watanabe and Yamamoto, 1994; Yoshida et al., 1994), and it has been suggested that they function without being translated into proteins. Some genes encode RNAs, rather than proteins, as their final products. tRNA, rRNA, and the small nuclear RNAs and nucleolar RNAs have been studied extensively, and were relatively straightforward to identify by homology searches or wit h sp ecialized algorithms (Lo we and Eddy, 1997; 1999). It has become apparent recently that in addition to th ese structural RNAs, other mRNA-like noncoding RNAs (ncRNAs) exist, which lack protein -coding capacity and exert their action mainly or exclusively at the RNA level (Eddy, 1999; Erdmann et a l., 1999; 2000; 2001; Caprara and Nilsen, 2000; Storz, 2002). Analyses of the properties and functions of ncRNAs indicated that they could act as gene reg ulators, as part of biotic and abiotic stress signals, or as part of RNA-protein complexes with various enzymat ic and structu ral activities. A number of ncRNAs were processed in an mRNA-like manner. Consequently, t hey u n dergo sp licing an d h ave po ly(A ) t ails and , presumably, caps (Erdmann et a l., 2001). The presence of ncRNAs has been described in several systems, for example, in prokaryotic and eukaryotic sys tems (Willard and Salz, 1997; Panning and Janeisch, 1998; Akhtar et al., 2000). Only a few ncRNAs from plants have been report ed. One of the first transcripts identified as an ncRNA in plants was CR2 0 (Teramoto et al ., 1996), a gene iso lated from cucumber (Cucumis satirus) that repressed by cytokinins and b y stress or developmental condition s (Teramoto et al., 1995). This gene was part of a family o f ncRNA with members in several plant species (Taylor and Green, 1995; Teramoto et a l., 1996; v an Hoof et a l., 1997). GUT15 was ano ther characterized member o f this family (Taylor and Green, 1995). The fact that transcripts o f this family were hormonally regu lated and had unst able t ranscripts s uggested that they might play a role in regulatory processes, althoug h their true functions were unknown. Anot her interesting family of ncRNAs present in plants was typified by Mt4 in Medica go truncat ula (Burleigh an d Harrison, 1998) and TPSI1 in tomato (Lycopersicon esculentum) (Liu et al., 1997). As with the GUT15/CR20 family, these genes were regulated by biotic (cytokinins) and abiotic (phosphate starvation) signals. Severalshort non-conserved ORFs were presen t in Mt4 /TPS 11 family, an d all o f t he transcrip ts showed regions of absolute identity at the nucleotide level Received 17 Apr. 2003 Acc epted 14 Sept. 2003 Supported by the National Natural Science Foundation of China (30100014). * Author for correspondence. Tel: +86 (0)10 62893462; E-mail: <[email protected]>. 498 (Mart in et al ., 2000). Th e high d egree of nucleot ide sequence conserv ation an d low lev el of ORF conserv ation suggested that the final product of these genes was RNA and not protein. We previo usly isolated so me cDNAs for pollen-s pecific genes in Z. mays by differential screening (Li et a l., 2001). One o f cDNA fragments, named ZM4 01, was s elected to be analyzed. The length of ZM4 01 cDNA fragmen t was 663 bp. Two trans crip ts (1.2 kb, 2.0 kb) of the ZM401 were detect ed by Nort hern blot ting analysis. In this study, we began our molecular analysis of ZM401 by using 5' RACEand 3' RACEto clone a full-length cDNA for the 1.2 kb RNA. We dis cuss t he pos sibility that ZM4 01 was a non-coding RNA. Using RT-PCR and Northern blotting analysis, we studied th e developmental expres s pattern of ZM401 in Z. mays pollen. 1 Materials and Methods 1.1 Plant material Corn (Zea mays L.) po llen and ant hers from vario us stages of microspore development were collected from field grown plants of the cultivar (Nongda 108). These were immediately frozen in liquid N2 and stored at –70 ℃ until used for RNA isolation. 1.2 3' and 5' RACE-based cloning of ZM401 cDNA To isolate a full-length cDNA from total RNA extracted from Z. mays pollen, we employed 3' RACE and 5' RA CE. ZM401-specific primers were designed based on the ZM401 cDNA fragments, to amplify the 3' end of ZM401 mRNA, an anchored oligo (dT) primed single-strand cDNA was synthesized from 2 µg of total RNA using AMV reverse transcriptase followed by PCR with an ZM401-specific primer (NGSP2) 5'-GGTGAGGCGTCA ATTTATAGGG-3' and an anchor primer according to the manufacturer’s protocol (3' RACE System For Rapid Amplification of cDNA Ends, Version 2.0, Gibco, BRL No. 18373-019). 5' RACE was performed according to the manufacture’s protocol (5' RACE System For Rapid Amplification of cDNA Ends, Version 2.0,Gibco, BRL NO. 18374-058). In brief, single-strand cDNA was generated by rev ers e trans crip tion with a primer GSP1: 5' TAGCCATACTCCGGTGAC-3' . A homopoly meric dC tail was add ed t o t h e 5'-end o f t h e cDNA b y termin al deoxynucleotidyltransferase followed by PCR with a nested primer, GSP4: 5' -CTCACCGCCGTAAGCTCAATCTTGC-3', and an abridged anchor primer was included in the kit. PCR was performed under conditions of 30 cycles of 1 min at 94 ℃, 1 min at 64 ℃ and 1 min at 72 ℃ followed by 10 min at 72 ℃ in a t hermal cycle. Amplified PCR frag ment s were clo n ed in t o p M D18-T an d s eq u en ced . ZCF1 (5'- Acta Botanica Sinica 植物学报 Vol.46 No.4 2004 TTGCGAGCACATGGACGGAGGCGC-3' ) and ZCF2 (5' TTTTTTTTTTTTTTTGATCACGAACCTTATAATAAAGTTGAAGCTGG3' ) primers were designed for splicing 5' RACEand 3' RACE amplified product. 1.3 Isolation of total RNA and reverse transcripti onpolymerase chain reaction (RT-PCR) analysis Total RNA was isolated from mature pollen and anthers of various stages of microspore development,RNA was iso lat ed u s in g t he ho t p heno l meth od ,and su b sequen tly treated with RNase-free DNase Ⅰ(Promega Co. Ltd.). Total RNAs (1 µg) were reverse-transcribed by AMV reverse transcriptase (Promega Co. Ltd.) using oligo(dT)12-18 as a primer followed by PCR with ZM401-specific internal primers (forward, P1 5' -ATCGGGAGCGAGTGGTGC-3' and reverse P3 5' -CGTCACATGAAAACCGAAGAAC-3'). PCR react ion cond itions were as follows: 94 ℃ 1 min , 56 ℃ 1 min , 72 ℃ 1 min , 72 ℃ exten sio n 10 min, 30 cycle. PCR products were separated by electrophoresis in a 0.8% agaros e g el fo llo wed b y b lo t ting to a ny lon memb ran e (Amersham Pharmacia Biotech). The ZM401 products were detected by hybridization with a 32 P-labeled ZM401 cDNA. In control experiments, RNA was treated as above but without revers e transcriptase. In all cases, these con trol reactio ns gav e no signal after hybridization. All t he RT-PCR experiments presented in this st udy were repeated using two or more independent RNA preparations. 1.4 Southern blot Southern blotting analysis was performed as described by Church method with slig ht modification s. Twen ty µg genomic DNA was digest ed with s ix restriction en zymes (Hind Ⅲ, EcoRⅠ, Ba mHⅠ, KpnⅠ, SacⅠ, EcoRⅤ), and electropho resis on an agarose gel, an d transferred to a Nylon membrane (Amersham Pharmacia Biotech) (Sambrook et al., 1989). The membrane was prehybridized at 65 ℃ for 6 h in a prehybridization solution consisting of 1% BSA, 1 mmol/L EDTA, 0.5 mol/LNa2 HPO4 (pH 7.2), 7% SDS. Probes labelled with 32 P were prepared from ZM401 cDNA using a random-primed DNA labelling kit (Promega). Hybridization wa s p e rfo r med wit h 32 p - lab ele d p r o b e s i n t h e prehybridization solution for 18 h at 65 ℃. The membrane was washed at 65 ℃ in 2×SSC plus 0.5% SDS for 30 min, 1 × SSC plus 0.5% SDS for 30 min , 0.5 × SSC plus 0.1% SDS for 30 min, and then, 0.1 × SSC plus 0.1% SDS for 30 min. 1.5 Northern blot Total RNAs (20 µg) from different stages of microspore development an d mature pollen g rains were isolated and resolved by electrophoresis on a 1.2% agarose-formald eh y d e gel. Th e RNAs were t ran sfo rmed t o a n y lo n DAI Xiao-Yan et al.: Non-coding RNA for ZM401, a Pollen-specific Gene of Zea mays 1997). Although the transcripts of most of them, including H1 9 , XIS T, C R2 0 , wer e t h o u g h t t o fu n ct i o n as untranslatable RNAs, t he molecular mech anisms of th eir fu nct ion still remain un clear. Wat anabe and Yamamo to (1994) demonstrated that mei RNA contained no long ORF and formed a complex with Mei2 protein and performed an essential role in the induction of meiosis in fissio n yeast. The amino acid sequences deduced from all possible readin g frames of the ZM4 01 cDNA h ad man y t ermin ation codons through out each reading frame, an d no long ORF was found, but it had poly(A) tail. The nucleotide sequence sho wed no s ignifican t homolog y to non-coding mRNA sequence isolated from plants. Therefore the ZM401 RNA could be a member of a new category of non-coding RNA. Northern blotting analysis revealed ZM401 was pollenspecific gene (Li et al., 2001) and had two transcripts in Z. mays mature pollen. RT-PCR and Northern blotting analysis showed ZM401 began t o express from tetrad st age of microspore development to mature pollen. North ern blotting analysis revealed the presence of two transcripts (2.0 kb and 1.2 kb) of ZM401 in Z. ma ys. There would be little con trib utio n of degradat ion p rodu cts generated during preparation of RNA to the observed t wo RNA ban ds, as indicated by the fact that d egradation of rRNAs was not detected, the results could be due to (1) different members of a gene family, (2) alternative splicing, (3) degradation of the RNAs in the in tact plants, and (4) different tran scription start site. Genomic Southern blotting analysis showed that ZM401 was encoded by a single or a very few copies (Fig.9). Because th ere were at least two ban ds of ZM4 01 transcripts on North ern blot s, and d ifferent patt erns of bands were obtained from the different stages of microspore development, t he hypoth esis that a complex gene family existed and/or degradation of the RNAs in the intact plants occured failed adequately to explain the results. Therefore, two tran scripts o f ZM4 01 might derive from alternat ive splicing of ZM401 gene or different transcription start site if a single copy gene encodes ZM401 cDNA. If a few genes encode ZM401, two trans cripts might derive from homology gene of ZM401. Multiple transcripts, as in the case of ZM401, were generated by alternative splicing for human XIST (not in case of mouse) and cucumber CR20 (Brown et al., 1992; Hao et al ., 1993), unst able mRNA g enerating mult iple t ranscripts for GUT15 (MacIntos h et a l., 2001). However, the significance of multiple transcripts for many non-coding genes remains unknown. Ecotop ic expressio n of ZM40 1 in t obacco or over-expression in maize all res ulted in pollen sterile (data n ot shown), suggesting that ZM401 functions as a non-coding 503 mRNA and plays an important role in pollen development of Z. mays. References: Akhtar A, Zink D, Becker P B. 2000. Chromodomains are protein-RNA interacation modules. Nature, 407:405-409. 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