Comparative analysis of the small RNA transcriptomes of Pinus contorta and Oryza sativa

Abstract

The diversity of microRNAs and small-interfering RNAs has been extensively explored within angiosperms by focusing on a few key organisms such as Oryza sativa and Arabidopsis thaliana. A deeper division of the plants is defined by the radiation of the angiosperms and gymnosperms, with the latter comprising the commercially important conifers. The conifers are expected to provide important information regarding the evolution of highly conserved small regulatory RNAs. Deep sequencing provides the means to characterize and quantitatively profile small RNAs in understudied organisms such as these. Pyrosequencing of small RNAs from O. sativa revealed, as expected, ∼21-and ∼24-nt RNAs. The former contained known microRNAs, and the latter largely comprised intergenic-derived sequences likely representing heterochromatin siRNAs. In contrast, sequences from Pinus contorta were dominated by 21-nt small RNAs. Using a novel sequence-based clustering algorithm, we identified sequences belonging to 18 highly conserved microRNA families in P. contorta as well as numerous clusters of conserved small RNAs of unknown function. Using multiple methods, including expressed sequence folding and machine learning algorithms, we found a further 53 candidate novel microRNA families, 51 appearing specific to the P. contorta library. In addition, alignment of small RNA sequences to the O. sativa genome revealed six perfectly conserved classes of small RNA that included chloroplast transcripts and specific types of genomic repeats. The conservation of microRNAs and other small RNAs between the conifers and the angiosperms indicates that important RNA silencing processes were highly developed in the earliest spermatophytes. Genomic mapping of all sequences to the O. sativa genome can be viewed at http://microrna.bcgsc.ca/cgi-bin/gbrowse/rice_build_3/.[Supplemental material is available online at www.genome.org.] . The heterochromatin siRNAs are a diverse set of 24-nt-long small RNAs that are processed by DCL3 from double-stranded RNA precursors produced by RDR2 (Xie et al. 2004). These RNAs are involved in heterochromatin formation and maintenance by directing sequencespecific DNA and histone methylation of transposable elements and some larger genomic loci (Pontier et al. 2005). Other 24-nt long siRNAs produced by DCL2 in A. thaliana can direct an initial cleavage of target transcripts, which are further cleaved into 21-nt siRNAs by DCL1 (Borsani et al. 2005). Finally, the trans-acting siRNAs (tasiRNAs), which are 21 nt long, are matured by a poorly understood mechanism involving DCL4. These tasiRNAs perform post-transcriptional gene silencing much like the miRNAs (Xie et al. 2004).Identification of functional small RNAs in other plant species has, until recently, been accomplished by searching for homologous sequences in expressed sequence data (Zhang et al. 2006a) and genomic sequences (Bonnet et al. 2004) and has been, with a few exceptions (Williams et al. 2005; TalmorNeiman et al. 2006), limited to the discovery of the more highly cons...

The Cornucopia of Small RNAs in Plant Genomes

“…SBS sequencing of small RNAs from a wild rice relative, Oryza barthii (Fig. 2, an unpublished experiment recently undertaken in our lab) and 454 sequencing of cultivated rice small RNAs (from O. sativa [67]) have both demonstrated the two major sizes of sRNAs, 21 and 24 nt, consistent with prior reports from Arabidopsis and other species. In general, high-throughput analyses have enabled the explo- …”

“…ration of rice small RNA populations, and many new miRNAs have been discovered recently in rice [34,36,47,67]. This includes a special class of natural antisense transcript miRNAs (nat-miRNAs), which are derived from natural cis-antisense transcripts with exons primarily located antisense to the introns of their target genes; these nat-miRNAs are DCL1-dependent [47].…”

miRNA profiling in leaf and cork tissues of Quercus suber reveals novel miRNAs and tissue-specific expression patterns

“…It is well established that the relative abundance of the 24-nt and the 21-nt length classes may vary depending on the species and the tissue type, among other factors. In fact, previous reports refer that the 24-nt sRNAs is the predominant size class, for instance in Arabidopsis thaliana, Nicothiana benthamiana, Solano lycopersicum, Oriza sativa and Olea europea (Morin et al 2008;Pantaleo et al 2010;Donaire et al 2011;Kasschau et al 2007), while the 21-nt class is more abundant in species such as Eschscholzia californica, Hordeum vulgare, Vitis vinifera, Pinus contorta and Panax gynseng (Pantaleo et al 2010;Morin et al 2008;Schreiber et al 2011;Barakat et al 2007). The tissue-dependent variation in the pools of 24 and 21-nt classes has also been observed in other plant species (Pantaleo et al 2010) and, in a few cases, striking variation has been detected (Slotkin et al 2009) reflecting the sRNA biogenesis pathways operating in such tissues.…”

The Norway spruce genome sequence and conifer genome evolution

“…There has been conflicting evidence about the presence of 24-nucleotide short RNAs (sRNAs) in gymnosperms [26][27][28][29] , a class of sRNA that silence transposable elements by the establishment of DNA methylation 30 . Across 22 samples, we identified numerous 24-nucleotide sRNAs, but these were highly specific to reproductive tissues, largely associated with repeats but present at substantially lower levels than in angiosperms ( Fig.…”