Common functions for diverse small RNAs of land plants

Abstract

Endogenous small RNAs, including microRNAs (miRNAs) and short interfering RNAs (siRNAs), are critical components of plant gene regulation. Some abundant miRNAs involved in developmental control are conserved between anciently diverged plants, while many other less-abundant miRNAs appear to have recently emerged in the Arabidopsis thaliana lineage. Using large-scale sequencing of small RNAs, we extended the known diversity of miRNAs in basal plants to include 88 confidently annotated miRNA families in the moss Physcomitrella patens and 44 in the lycopod Selaginella moellendorffii. Cleavage of 29 targets directed by 14 distinct P. patens miRNA families and a trans-acting siRNA (ta-siRNA) was experimentally confirmed. Despite a core set of 12 miRNA families also expressed in angiosperms, weakly expressed and apparently lineage-specific miRNAs accounted for the majority of miRNA diversity in both species. Nevertheless, the molecular functions of several of these lineage-specific small RNAs matched those of angiosperms, despite dissimilarities in the small RNA sequences themselves, including small RNAs that mediated negative feedback regulation of the miRNA pathway and miR390-dependent ta-siRNAs that guided the cleavage of AUXIN RESPONSE FACTOR mRNAs. Diverse, lineage-specific, small RNAs can therefore perform common biological functions in plants.

Figure 1.

Properties of miRNA Expression in P. patens. (A) Schematic depictions of representative clustered P. patens miRNAs that are potentially processed from polycistronic pri-miRNAs, indicated by black lines. In total, 48 out of the 205 P. patens miRNA loci were contained in 21 clusters containing two or three miRNA stem loops each (see Supplemental Table 1 online). Distances refer to the number of nucleotides separating the regions annotated as miRNA foldbacks. (B) Mapping of mature P. patens miRNAs with respect to annotated protein-coding genes. The numbers of mature miRNAs that overlapped various annotations of the P. patens Phypa1_1 genome assembly are indicated.

Figure 2.

Validated P. patens miRNA and ta-siRNA Targets. (A) Cleaved targets of conserved P. patens miRNAs. Schematics of target mRNAs along with the relative positions of the 5′ residues of all sequenced uncapped cDNAs are shown. The positions of the miRNA complementary sites are indicated with red vertical lines. Gray boxes indicate the positions of regions coding for conserved protein domains, whose abbreviated names are listed below. Boxes correspond to open reading frames and horizontal black lines to untranslated regions. Shaded pixels indicate the positions of 5′ residues of individual cDNA clones, which are shaded as indicated in the key below to indicate their proximity to the site predicted by miRNA-mediated cleavage between the tenth and eleventh nucleotide of complementarity. An asterisk indicates that Pp SBP3 was included as a positive control (Arazi et al., 2005). Alignments of target sites with small RNAs are provided in Supplemental Table 3 online. nt, nucleotides. (B) Cleaved targets of nonconserved P. patens miRNAs as in (A). Hatch marks in Phypa1_1 109598 indicate a region where the splicing of the observed cDNA fragments differed from the annotation.

Figure 3.

Analogous miRNA-Mediated Feedback Loops Despite Divergent Small RNA Sequences. (A) Schematic of the Pp DCL1a pre-mRNA, which contains an expressed miRNA within intron seven. Thick rectangles represent predicted coding exons, thin rectangles represent untranslated regions, and thin lines represent predicted introns. Colored exonic regions correspond to conserved protein domains as indicated. Gray indicates the location of the MIR1047 hairpin within intron seven. (B) Cleavage at miR1047 in intron seven of the Pp DCL1a pre-mRNA. The predicted secondary structure of MIR1047 is shown, with the miRNA/miRNA* duplex highlighted in red. Fractions indicate the number of 5′-RACE products observed to terminate at the indicated positions over the total number of 5′-RACE clones sequenced from three different experiments from the indicated combinations of RNA samples and RT primers. (C) An unrooted phylogenetic reconstruction of relationships among Arabidopsis, O. sativa, and P. patens DCL proteins using C. reinhardtii DCL1 as an outgroup. The known miRNA-mediated feedback loops between At DCL1 pre-mRNA, mRNA, protein, and DCL1-dependent miRNAs are indicated in blue. The hypothesized feedback loop between Pp DCL1a pre-mRNA, protein, and ppt-MIR1047 is indicated in red. Nodes with <100% bootstrap support are noted with the percentage of supporting replicates. Accession numbers are given in Methods. (D) An unrooted phylogenetic reconstruction of relationships among Arabidopsis, O. sativa, and P. patens AGO proteins using C. reinhardtii AGO1 and AGO2 as outgroups as displayed in (B). miRNA-mediated control of Arabidopsis AGO genes is shown in blue, and miRNA-mediated control of P. patens AGO genes by ppt-miR904 is shown in red. Accession numbers are given in Methods.

Figure 4.

Similar ARF Targets for Diverse ta-siRNAs and miRNAs. (A) T-COFFEE alignment of the regions of Pp TAS3a-d between the two miR390 complementary sites. Two ∼21-nucleotide blocks conserved between the four paralogs are indicated. (B) Alignments of the block 1 (+) siRNAs and the block 2 (-) RNAs with their predicted and validated targets. Lowercase letters indicate positions that cannot form either Watson-Crick or G-U pairs with the target. Arrows indicate cleavage-validated target sites. The asterisk indicates the cleavage site of the AP2 domain–containing gene Phypa1_1 129196 by Pp TAS3a-d block 1 (+)–derived siRNAs as demonstrated by Talmor-Neiman et al. (2006b). (C) An unrooted phylogenetic reconstruction of the relationships between Arabidopsis and P. patens ARF proteins. Nodes with <100% bootstrap support are noted with the percentage of supporting replicates. ARF genes known or predicted to be under small RNA–mediated regulation are indicated. Accession numbers are given in Methods.

Figure 5.

Small RNA Populations in P. patens and S. moellendorffii. (A) Categorization of 561,102 P. patens small RNA reads that matched at least one WGS trace. Reads were categorized as miRNAs (matching one or more of the 205 annotated miRNA hairpins), ta-siRNAs (from PpTAS3a-d), unknown (corresponding to 10 or fewer loci in the Phypa1_1 genome assembly), unknown-repetitive (corresponding to 11 or more loci in the Phypa1_1 genome assembly), or those not matching any loci in the Phypa1_1 genome assembly. Histograms display the length distributions of each of these small RNA populations. Reads that corresponded to the nuclear rRNAs and the chloroplast genome were not included. (B) Categorization of 149,586 S. moellendorffii small RNA reads that matched at least one WGS trace. Reads are categorized as miRNAs (matching one or more of the 58 annotated miRNA hairpins), unknown (matching 100 or fewer WGS traces), or unknown-repetitive (corresponding to 101 or more WGS traces). Histograms display the length distributions of each of these small RNA populations. Reads that corresponded to nuclear rRNA were not included.

Figure 6.

The 3′-Most Residue of P. patens miR160 Is Modified. The indicated RNA samples were oxidized with periodate, blotted, and probed for miR160. Markers (M) from top to bottom are 24, 21, and 18 nucleotides, respectively.