In flowering plants, small RNAs below 30 nucleotides in size are essential and diverse components of the transcriptome [1
]. Since their initial discovery in plants, small RNA systems have become better characterised and various classes of small RNA molecules with different functions are now recognisable. These functions include regulation of gene expression at transcriptional and post-transcriptional levels, repression of transposable element activity and defence against viral pathogens, and directed epigenetic changes in the genome such as DNA methylation and heterochromatin formation [1
MicroRNAs are a distinct class of small RNAs derived from endogenous Pol II-transcribed genes. The transcripts from these genes form fold-back structures or hairpins that are recognised in the nucleus by the protein machinery of the microRNA pathway [1
]. These hairpin transcripts are processed in specific bodies in the nucleus by proteins such as DICER-LIKE1 (DCL1) [3
]. Here, a site-specific excision process removes a ~21 nucleotide duplex RNA from the stem of the hairpin structure. This RNA is then exported to the cytoplasm, where one strand binds the ARGONAUTE1 (AGO1) protein in the RISC complex. Subsequently, this protein-RNA complex interacts with mRNAs with high complementarity to the miRNA. In flowering plants, the Slicer endonuclease activity of the AGO1 protein acts to cleave the mRNA where it binds the miRNA [5
]. Subsequently, the cleaved mRNA is routed into degradation pathways. In this manner, not only is the translation of the mRNA prevented but the levels of the mRNA target are down-regulated. In plants, it has now been shown that microRNAs also act to repress translation without cleavage of the mRNA, a regulatory mechanism that appears to involve not only AGO1 but also AGO10 and probably another member of the ARGONAUTE family [6
]. Over 100 microRNAs have now been described in Arabidopsis thaliana
and these are known to directly regulate a large number of transcripts, in particular those that encode key regulatory proteins such as transcription factors and F-box proteins that regulate entry of other proteins into the ubiquitin-dependent degradation pathway.
Certain miRNAs do not target coding mRNAs but instead recognise long non-coding transcripts from so-called TAS
]. These miRNAs are homologous to one or more typically two different sites on the target TAS
transcript. These sites act as recognition points from which further small RNAs are formed [7
]. After cleavage to generate two TAS
fragments, the TAS
RNA is further processed to generate secondary small RNAs [8
]. An RNA-dependent RNA polymerase enzyme, RDR6, is then recruited to synthesise a complementary strand and the resulting duplex RNA is sequentially cut by the DICER-LIKE 4 DCL4 enzyme to form phased 21 nt small RNAs called trans-acting siRNAs [9
]. The exact mechanism by which a single conventional cleavage event directed by a microRNA recruits RDR6 and DCL4 activity to generate secondary siRNAs from TAS
RNAs remains poorly understood [11
]. Secondary siRNAs are known to be only rarely produced after conventional miRNA cleavage events on coding transcripts [12
In plants, further classes of small RNAs are produced from RNAs derived from non-coding regions of the genome by various mechanisms, from both PolII and PolII-independent sources [13
]. These small RNAs have a major role in establishing and maintaining patterns of DNA methylation and heterochromatin architecture throughout the genome [13
Recent innovations in sequencing technology [15
] have allowed extensive surveys of small RNA populations in flowering plants, notably in the model species Arabidopsis thaliana
. However, until recently much of this work was focused on collectively sequencing the small RNAs from complex aggregates of sporophytic tissues to whole plants from both wild type and mutant lines. Notably absent has been a focus on the reproductive cells and the highly reduced post-meiotic male and female gametophytes (pollen and embryo-sacs). In flowering plants, gametes are not directly formed from the meiotic products but instead arise from a limited number of haploid mitotic divisions. In the male gametophyte, the four products of meiosis each undergo two further mitotic divisions. The first division is highly asymmetric and forms a larger vegetative cell and a smaller generative or germ cell. The germ cell undergoes a further division to form a pair of sperm cells that are suspended within the vegetative cell cytoplasm. In Arabidopsis, mature pollen grains at the point of release from the anthers consist of three cells of these two distinct cell types.
Previous work has questioned whether small RNA pathways are maintained and function as the male gametophyte develops [16
]. However, it is clear from recent investigations that small RNA pathways are not down regulated, but transcript levels of various small RNA pathway genes show dynamic and complex changes during male gametophyte development [17
]. It is evident that cell-specific differences contribute to these dynamic changes as sperm cells are significantly enriched for some transcripts, such as AGO5
, compared to whole mature pollen [18
]. Recent work has shown that silencing of repetitive DNA and transposons is weakened in the vegetative cell leading to the formation of 21 nt small RNAs from their transcripts that are claimed to reinforce silencing in the associated sperm cells [19
]. A functioning microRNA pathway in mature pollen has been confirmed despite suggestions that mature microRNAs may not be produced from their precursors in late pollen development [16
]. Not only have several families of microRNAs with a known function in the somatic phase of development been identified in pollen by in situ
], but many more have been discovered by RT-PCR analysis and the use of a modified 5' RACE protocol that has demonstrated the precise cleavage of transcripts targeted by several different microRNAs [17
]. Thus, microRNAs and other small RNAs are likely to make a major but hitherto unappreciated contribution to pollen gene expression patterns.
We have utilised 454 sequencing to explore the diversity of small RNAs in mature pollen of A. thaliana. We describe the general composition of the small RNA population present in mature pollen and focus on the diversity of microRNAs and tasiRNAs, as these small RNAs have a direct effect on the abundance and translation of target mRNAs. We identify known microRNAs and tasiRNAs and also survey the sequence information for putative novel microRNAs. We independently verify the expression in pollen and relative abundance of selected microRNA sequences identified by 454 sequencing using quantitative RT-PCR. Amongst the candidate novel microRNAs, we confirm that one, named ath-MIR2939, cleaves its predicted target transcript, At3g19890. This corresponds to a pollen-specific F-box family transcript that is significantly enriched in sperm cells. Its discovery supports the assertion that novel miRNAs exist with putative roles in regulating gametophyte-specific transcripts that may be of key importance in reproduction.