Download Non-coding RNA for ZM401, a Pollen

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]>.
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(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
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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.
Askew D S, Li J, Ihle J N. 1994. Retrovial ins tert ions in the
murine His-1 locus activate t he expression of a novel RNA
that lacks an extensive open reading frame. Mol Cell Biol, 14:
1743－1751.
Brannan C I, Dees E C, Ingram R S, Tilghman S M . 1990. The
product of the H19-gene may function as RNA. Mol Cell Biol,
10:28－36.
Brockdorff N, Ashworth A, Kay G F, M cCabe V M , Norris D P,
Cooper P J , Sw ift S, Rastan S. 1992. The product of t he
mouse Xist gene is a 15 kb inavtive X-specific transcript containing no conserved ORF and located in the nucleus. Cell, 71:
515－526.
Brown C J, Hendrich B D, Rupert J L, Lafrenière R G, Xing Y,
Lawrence J, Willard H F. 1992. The human XIST gene: analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus. Cell,
71:527－542.
Burleigh S M , Harrison M J. 1998. Characterization of the Mt4
gene from Medicago truncatula. Gene, 216:47－53.
Caprara M G, Nilsen T W. 2000. RNA: versatility in form and
function. Nat Struct Biol, 7:831－833.
Crespi M D , Jurkevitch E, Poiret M , d’Aubenton-Carafa Y,
Petrovics G, Kondorosi A. 1994. enod40, a gene expressed
duringnodule organ genesis, codes for a non-translatable RNA
involved in plant growth. EMBO J, 13:5099－5112.
Eddy S R. 1999. NoncodingRNA genes. Curr Opin Genet Dev, 9:
695－699.
Erdman V A. 1999. Collection of mRNA-like non-coding RNAs.
Nucleic Acids Res, 27:192－195.
Erdmann VA, Barcizewska M Z, Szymanski M , HochbergA, de
Groot N , Barcisz ewski J. 2001. The noncoding RN As as
riboregulators. Nucleic Acids Res, 29:189－193.
Erdmann V A , Szy manski M , H ochberg A, de Groot N ,
Barciszewski J. 2000. Non-coding, mRNAs-like RNAs database Y2k. Nucleic Acids Res, 28:197－200.
Hao Y, Crenshaw T, M oulton T, Newcomb E, Tycko B. 1993.
Tumour-suppressor activity of H19 RNA. Nature, 365:764－
767.
Lee J T, Davidow L S, Warshawsky D. 1999. Tsix, a gene antisense
to Xist at the X-inactivation center. Nat Genet, 21:400－404.
Li C-X , Liu J-Q , Yu J-J , Zhao Q , Ao G-M . 2001. Cloning and
expression analysis of pollen-specific cD NA ZM401 from
Zea Mays. J Agr Biotechnol , 4:374－377. (in Chinese with
５０４
Ａｃｔａ　Ｂｏｔａｎｉｃａ　Ｓｉｎｉｃａ　植物学报　Ｖｏｌ．４６　Ｎｏ．４　２００４
English abstract)
Liu C, M uchhal U S, Raghothama K G. 1997. Differential expres-
of genes with unstable transcripts (GUTs) in tobacco. Plant
Mol Biol, 28:27－38.
sion of TPSI1, a phosphate stawation-induced gene in tomato.
Teramoto H, Toyama T, Takeba G, Tsuji H. 1995. Changes in
Plant Mol Biol, 33:867－874.
Lowe T M , Eddy S R. 1997. tRNAscan-SE: a program for im-
expression of two cytokinin-repressed genes, CR9 and CR20,
in relation to aging, greening and wounding in cucumber. Planta,
proved detection of transfer RNA genes in genomic sequence.
Nucleic Acids Res, 25:955－964.
Lowe T M , Eddy S R. 1999.A computational screen for methylation guide snoRNAs in yeast. Science, 283:1168－1171.
M acIntosh G C, Wilkerson C, Green P J. 2001. Identification and
analysis of arabidopsis expressed sequence tags characterisic
of non-coding RNAs. Plant Physiol, 127:765－776.
196:387－395.
Teramoto H , Toyama T, Takeba G, Ts uji H. 1996. Noncoding
RNA for CR20, a cytokinin-repressed gene of cucumber. Plant
Mol Biol, 32:797－808.
van Hoof A, Kastenmayer J P, Taylor C B, Green P J. 1997.
GUT15 cDNAs from tobacco (accession No. U84972) and
Arabidopsis (accession No. U84973) correspond to transcripts
M artin A C, Pozo J C, Iglesias J , Rubio V, Solano R, de La Pena
A, Leyva A, Paz-Ares J. 2000. Influence of cytokinins on the
with unusual metabolism and a short conserved ORF. Plant
Physiol, 113:1004.
express ion of p hos phate st arvat ion resp ons ive genes in
Velleca M A, Wallace M C, Merlie J P. 1994. A novel
Arabidopsis. Plant J, 24:559－567.
Pachnis V, Brannan C I, Tighman S M . 1988. The structure and
synapseassociated noncoding RNA. Mol Cell Biol, 14:70957104.
exp res s ion of a novel gene act ivat ed in early mous e
Wat anabe Y, Yamamoto M . 1994. S pombe mei2+ encodes an
embryogenesis. EMBO J, 7:673－681.
Panning B, Janeisch R. 1998. RNA and epigenetic regulation of X
RNA-binding protein essential for premeiotic DNA synthesis and meiosisⅠ, which cooperates with a novel RNA spe-
chromosome inactivation. Cell, 93:305－308.
Sambrook J, Fris ch E F, M aniatis T. 1989. M oleular Cloning: A
Laboratory M anual. 2nd ed. New York: Cold Spring Harbor
Laboratory Press.
St orz G. 2002. An expanding univers e of noncoding RNA s.
Science, 296:1260－1262.
cies mei RNA. Cell, 78:487－498.
Willard H F, Salz H K. 1997. Remodelling chromatin with RNA.
Nature, 386:228－229.
Yoshida H, Kumimoto H , Okamot o K. 1994. dutA RN A functions as an untrans latable RN A in the development of
Dictyostelium discoideum. Nucleic Acids Res, 22:41－46.
Taylor C B, Green P J. 1995. Identification and characterization
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