Original Paper
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Acta Biochim Biophys
Sin 2007, 39: 829834
doi:10.1111/j.1745-7270.2007.00346.x
Cloning of novel repeat-associated
small RNAs derived from hairpin precursors
in Oryza sativa
Chengguo Yao, Botao Zhao, Wei Li,
Yang Li, Wenming Qin, Bing Huang, and Youxin Jin*
State
Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell
Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences; Graduate School of Chinese Academy of Sciences, Shanghai 200031,
China
Received: April 4,
2007
Accepted: July 1,
2007
This work was
supported by the National Natural Science Foundation of China (No. 30430210),
the National Key Basic Research and Development Program (No. 2005CB724602) and
Chinese Academy of Science (KSCXI-YW-R-64)
*Corresponding
author: Tel, 86-21-54921222; Fax, 86-21-54921011; E-mail, [email protected]
Abstract Plant small non-coding RNAs including microRNAs (miRNAs),
small interfering RNAs (siRNAs) and trans-acting siRNAs, play important
roles in modulating gene expression in cells. Here we isolated 21 novel
endogenous small RNA molecules, ranging from 18 to 24 nucleotides, in Oryza
sativa that can be mapped to 111 hairpin precursors. Further analysis
indicated that most of these hairpin sequences originated from putative
miniature inverted-repeat transposable elements, a major type of DNA
transposon. Considering that miRNA is characteristic of hairpin-like precursor
and plant endogenous siRNAs are often located at transposon regions, we hypothesized
that our cloned small RNAs might represent the intermediate product in the
evolutionary process between siRNAs and miRNAs. Northern blot analysis
indicated that five of them were much more abundantly expressed in flower
compared to other tissues, implying their potential function in inflorescence.
In conclusion, our results enrich rice small RNA data and provide a meaningful
perspective for small RNA annotation in plants.
Keywords small RNA; microRNA; small interfering RNA; hairpin; Oryza
sativa
MicroRNAs (miRNAs), small
interfering RNAs (siRNAs), and trans-acting siRNAs have emerged in
recent years as small but mighty gene attenuators in plants [1]. As small
non-coding RNAs, they are almost indistinguishable in chemical composition and
length, and their classification mainly depends on their biogenesis and mode of
action [2]. miRNA is processed to a ~22 nucleotide (nt) mature sequence from
an incompletely base-paired hairpin-like RNA transcript and then acts in
trans on target messenger RNA [3]. siRNAs are primarily processed from long
double-stranded RNAs, but trans-acting siRNAs act in trans just
like miRNAs, whereas other siRNAs mainly target the transcripts that originate
from siRNAs themselves [4].
Given their relatively
detailed characterization, a number of open questions remain concerning the
differentiation of these small RNAs. For example, siRNAs are well known to be
related to transposable elements (TEs), as opposed to them, miRNAs are thought
to derive from loci different from other genes or TEs [5]. However, several
examples of miRNA genes have been identified to be located at TEs in animals
[6], and recent bioinformatics analysis shows that 55 experimentally
characterized human miRNA genes are derived from TEs, and these TE-derived
miRNAs have the potential to regulate thousands of genes [7]. Miniature
inverted-repeat transposable elements (MITEs) are notable types of TE
associated with miRNA, as they have palindromic structures with terminal
inverted repeats (TIRs) that flank short internal regions. Their expression as
RNA results in the formation of the kinds of hairpins seen for pre-miRNAs [8].
Indeed, MITEs have previously been shown to contribute miRNA genes in human and
Arabidopsis genomes [8,9].
The rice genome contains
approximately 90,000 MITEs, constituting approximately 26% of the genome
sequence and highest-copy-number TEs in rice [10]. Previous sequence analyses
of cloned rice small RNAs ignored the correlation between hairpin-derived small
RNA and TEs [11–13]. We constructed several
rice small RNA libraries from typical organs at different development stages,
and were able to obtain 3003 sequences. Subsequent analysis allowed us to pick
out 21 novel small RNA sequences mapped to 111 hairpin precursors that are
encoded by putative transposons. These small RNAs share characteristics with
both miRNA and siRNA, and might represent the evolutionary link between both.
Materials and methods
Cloning of small RNA sequences
from rice
Whole rice plants (Oryza
sativa L. ssp. japonica) were grown under natural conditions. Different
plant tissues at various development stages (root, leaf, flower, and stem) were
collected, washed with double distilled H2O, and frozen in liquid
nitrogen. Total RNAs from O. sativa were prepared using the Trizol
method (Invitrogen, Carlsbad, USA) in which the isopropanol precipitation was
replaced by ethanol precipitation. In brief, small RNAs from 18 to 28 nt were
size-fractionated, purified, and ligated sequentially to the 5‘ DNA
adapter (ACCGAATTCACAGTCA-GACC, EcoRI site) and 3‘
adapter (GCAGATCGTCAGA-ATTCCAG, EcoRI site) with T4 RNA ligase
from New England Biolabs (Beverly, USA). The 3‘ adapter was blocked by
ddA with terminal transferase (NEB) at its 3‘ terminus and
phosphorylated by T4 polynucleotide kinase (NEB) at the 5‘ terminus. The
ligated RNA was reverse transcribed into cDNA by the Access Quick reverse
transcription-polymerase chain reaction (RT-PCR) system (Promega, Madison,
USA) with the 5‘ adapter sequence and another primer complementary to
the 3‘ adapter. The RT-PCR product was amplified by PCR with the same
primers. After purification, the PCR product was digested with EcoRI
(NEB) and concatemerized with T4 DNA ligase (NEB) followed by purification
with the kit (Bioasia, Shanghai, China). PCR was carried out with the previous
primers and the DNA of approximately 800 bp was cloned into pGEM-T vector
(Promega) for sequencing.
To avoid losing the cDNAs
containing the EcoRI site, the adaptors with a SalI site but not
an EcoRI site were also used to generate some cDNA libraries.
Sequence analysis and
prediction of fold-back structures
Using BLASTN on the National
Center for Biotechnology Information (NCBI) website (http:www.ncbi.nlm.nih.gov/blast),
cloned sequences shorter than 16 nt and possible ribosomal RNA, messenger RNA,
transfer RNA, and small nucleolar RNA fragments were discarded. This step
allowed the removal of most non-siRNA and non-miRNA species and identification
of cloned known miRNAs that could be confirmed in the miRNA registry (http://microrna.sanger.ac.uk/sequences/).
After redundancy analysis and exclusion of previously reported rice small RNAs
that were also cloned through construction of libraries, the remaining
sequences were compared with the latest version of the rice genome at the Rice
Genome Database (RGP; http://riceblast.dna.affrc.go.jp/). The
surrounding sequences of 200 nt in both orientations were extracted and,
together with the perfectly matched sequences in the RGP, were submitted for
RNA folding using the Mfold program [14]. Folding results were inspected, and
fold-back structures with small RNA in the stem were considered hairpin
precursors.
Next, we ran BLAST search
against the TIGR rice repeat database (http://tigrblast.tigr.org/euk-blast/index.cgi?project=osa1)
with the hairpin precursors. Sequences with at least 80% similarity, with one
hit including the mature region, were regarded as candidates for rice repeat
genome data [7], and some of them were perfect matches. Genomic annotation was
examined using the Rice Genome Automated Annotation System (http://ricegaas.dna.affrc.go.jp/).
Northern blot analysis of
small RNAs
Approximately 10 mg total RNA was isolated from different
rice samples (embryo, seedling, leaf, stem, flower, and fruit), loaded and
resolved on the denaturing 15% polyacrylamide gel, and transferred to a Hybond
N+
nylon membrane (Amersham, Piscataway, USA). The membrane was ultraviolet
cross-linked. DNA probes complementing miRNA sequences were end-labeled with [g–32P]ATP (3000 Ci/mM; Amersham)
using T4 polynucleotide kinase (MBI, Vilnius, Lithuania). Unincorporated [g–32P]ATP was removed using a
Sephadex G-25 column (Pharmacia, Uppsala, Sweden). Methylene blue staining of
membranes prior to hybridization was used to detect ribosomal RNA. The membrane
was pre-hybridized and hybridized using ExpressHyb Hybridization Solution
(Clontech, Palo Alto, USA) then washed according to the user manual. The
membrane was briefly air-dried then exposed to a PhosphorImager (Amersham).
Results
Novel small RNA gene families
identified in rice show features of both miRNA and siRNA
Construction of a small RNA
library is a practical and effective way to study small RNAs. In fact, many
rice miRNAs, endogenous siRNAs and other uncharacterized tiny non-coding RNAs
were identified through this starting point. We constructed six small RNA
libraries ranging from embryo to flower in rice plant (O. sativa L. ssp.
japonica). Small RNAs of 18–26 nt in length were isolated
by size fractionation and ligated to 5‘ and 3‘ adapters, then
cloned and sequenced. A total of ~460 clones comprising 3003 small cDNA
insertion sequences were collected. After removal of sequences shorter than 16
nt, we blasted the remained
sequences against the rice genome in the NCBI database to discard the query
sequences bearing no hits, and those that could be degradation products of
ribosomal RNA, transfer RNA, small nucleolar RNA, or small nuclear RNA that
constituted approximately half of the overall sequences. The BLAST results in
the NCBI database and miRNA registry (http://www.sanger.ac.uk/Software/Rfam/mirna/search.shtml)
also showed that we had cloned many known miRNAs and endogenous siRNAs. A total
of 1416 small RNA sequences were matched to the rice genome (japonica genome
database at RGP). One hundred and eleven loci corresponding to 21 small RNAs
were predicted to hold hairpin-like fold-backs (Table 1), characteristic
of miRNAs. However, sequences blasted
against the rice repeat database indicated that they all showed high sequence
similarity with one or more hits. Most of them are putative MITEs, and others
are putative unclassified transposons or putative retrotransposons (data not
shown), which are obviously features of siRNA-generating sequences.
Genomic annotation of new rice
small RNAs
Non-coding small RNAs are
usually located in the genomic segments that are distinct from protein-coding
regions. Examination results of the corresponding loci with the Rice Genome
Automated Annotation System (http://ricegaas.dna.affrc.go.jp/) conformed
to the traditional knowledge, in that almost all of the loci were in the
intergenic or intron, non-coding expressed sequence tag regions, except for a
few in the exon of a hypothetical protein and cDNA region, which could be
pseudo genes. The proteins specified by the intron region are nearly all
hypothetical or putative, indicating that they might also be pseudo genes. For
those small RNAs encoded by multiple loci, we could not assign their exact
origin unambiguously, and some of them might not be genuine genes, as referred
to above.
Cloning of putative
influorescence-associated small RNAs
To validate the cloning
results, we carried out Northern blot analysis to examine the existence of
these small RNAs using total RNA samples isolated from diverse organs from different
development stages. Interestingly, five of them were exclusively abundant in
flower compared to other tissues (Fig. 1), suggesting that they might
specially function in the process of inflorescence. However, their targets and
the mechanisms through which they play the role are yet to be discovered. As a
control, miR159c was also examined, and showed ubiquitous expression in the
samples listed in our experimental conditions. Some small RNAs were not
detected by Northern blot analysis with our available rice tissues. They might
be expressed at very low levels or confined to some specific tissues or cell
types, therefore the amount of these miRNAs was inappreciable in the limited
total RNA samples.
Discussion
As more and more detailed
information about plant small RNAs, including miRNAs, endogenous siRNAs, and
trans-acting siRNAs are being proffered [15–17],
the differences between them seem to be much clearer. However, some aspects
still remain controversial, such as their conservation, origin, and length
[18], which convinced us of the view that the diversity of plant small RNAs is
more complex than previously expected. Recent cloning and analysis of rice
endogenous small non-coding RNAs corroborate this point [13]. To date, most
reports have emphasized their distinction and classification, but ignored the
correlation and possible evolutionary intermediates. Here we present data to
provide a link between plant siRNAs and miRNAs. A full-length DNA-type
transposable element contains an open reading frame flanking two TIRs. When it
becomes a non-autonomous MITE, it is deprived of an open reading frame, and the
hairpin structure would be formed by base-pair interaction of MITE TIRs.
Previously, an inverted duplication model for miRNA gene evolution in plants
was proposed [19], however, the role of MITEs was not discussed in that the
researchers used known miRNA fold-back sequences. There is a postulation that
miRNAs could have evolved from TE-encoded siRNAs in the way that the TIRs that
are processed from longer RNAs to form siRNAs could be similarly processed to
form miRNAs [8]. Thus we assumed that small RNAs that originated from MITEs
were matured by miRNA biogenesis pathways (Fig. 2). However, additional
analysis of these small RNAs in rice mutants relating to the biogenesis of
miRNAs, such as DCL1, is necessary to validate this hypothesis [20,21].
Predictably, the evolutionary relationship and distinction between plant miRNA
and siRNA will become clearer as the biogenesis knowledge about these MITE-derived
small RNAs accumulates.
The average copy number of
rice MITEs ranges from dozens to thousands. Here we only extracted hairpin
precursors perfectly matched to the cloned sequences, thus many homologs might
have been missed. In view of our limited number of small RNA libraries,
predicting the average copy number of the MITE-derived small RNAs is not
practical. However, taking account of the variability of plant fold-back
precursors and 90,000 MITEs in rice, it is a really interesting area of study,
and our cloned small RNA sequences from MITEs just show the tip of the iceberg.
Although plant small RNA target prediction
is convenient for the extensive complementarity between small RNA and its
target according to the accepted principles [22,23], the authentic targets of
the vast majority of plant small RNAs are not characterized, and the phenotypic
consequences of disrupted or altered small RNA regulation are also obscure
because projects often take place over long time periods, and chance events
take place, especially in the study of rice [11,24–26]. Northern blot data
revealed that we have cloned five flower-exclusive small RNAs among the tissues
analyzed, hinting that they might act like miRNAs to get involved in
inflorescence. The molecular scenarios are waiting to be unraveled.
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