Phased, secondary, small interfering RNAs in posttranscriptional regulatory networks

Abstract

Plant genomes are the source of large numbers of small RNAs, generated via a variety of genetically separable pathways. Several of these pathways converge in the production of phased, secondary, small interfering RNAs (phasiRNAs), originally designated as trans-acting small interfering RNAs or tasiRNAs. PhasiRNA biogenesis requires the involvement of microRNAs as well as the cellular machinery for the production of siRNAs. PhasiRNAs in Arabidopsis thaliana have been well described for their ability to function in trans to suppress target transcript levels. Plant genomic data from an expanding set of species have demonstrated that Arabidopsis is relatively sparing in its use of phasiRNAs, while other genomes contain hundreds or even thousands of phasiRNA-generating loci. In the dicots, targets of those phasiRNAs include several large or conserved families of genes, such as those encoding disease resistance proteins or transcription factors. Suppression of nucleotide-binding, leucine-rich repeat (NB-LRR) disease resistance genes by small RNAs is particularly unusual because of a high level of redundancy. In this review, we discuss plant phasiRNAs and the possible mechanistic significance of phasiRNA-based regulation of the NB-LRRs.

Figure 1.

Pathways for the Biogenesis of PhasiRNAs Modeled on Arabidopsis. (A) As the first step in secondary siRNA biogenesis, mRNA targets are cleaved by a miRNA. In the one-hit model, exemplified in Arabidopsis by TAS1, TAS2, and TAS4, a 22-nucleotide miRNA targets a single site. In the two-hit model, there are two target sites for a 21-nucleotide miRNA, exemplified in Arabidopsis by TAS3 transcripts cleaved by an AGO7-loaded miR390. Activity of the trigger miRNA recruits RDR6 and SGS3, resulting in production of a second strand of the target mRNA. (B) The dsRNA is successively processed by DCL4 and other components to generate 21-nucleotide tasiRNAs; the direction of processing depends on the miRNA trigger mechanism. The secondary siRNAs are loaded onto an AGO protein and go on to function against other mRNAs.

Figure 2.

Triggers and Processing Mechanisms of PhasiRNAs The primary mechanisms of processing for plant phasiRNAs are described along with prototypical loci and the miRNAs that trigger siRNA biogenesis at these loci. Red arrows indicate cleavage sites, and orange arrows indicate the direction of precursor processing into phasiRNAs, which are indicated by gray lines in the double-stranded black/blue precursors. (A) The one-hit pathway is typified by a single target site for a 22-nucleotide miRNA that results in downstream processing of the target transcript into ∼21-nucleotide phasiRNAs. This is denoted as a 1₂₂ locus. There are at least three notable variations on the one-hit model, including (1) the reproductive lncRNAs of monocots that are processed by DCL5 into 24-nucleotide phasiRNAs, triggered by miR2275 and thus also 1₂₂ loci, but with different biogenesis components; (2) and (3) are both 2₂₂ loci, but the 3′ site can be either cleaved or not cleaved. (B) The two-hit pathway is typified by two target sites of a 21-nucleotide miRNA that results in processing upstream of the 3′ site. This is denoted as a 2₂₁ locus, and the best characterized examples are TAS3 and related loci, although a few other examples have been described. The 5′ site may be cleaved, which may result in processing from both directions, or the 5′ site may be noncleaved, as originally described for the Arabidopsis TAS3 locus.

Figure 3.

miRNAs Target Nucleotides Encoding Conserved Protein Motifs of Several Gene Families. In each case, miRNAs, designated by a black arrowhead, target sites that encode protein motifs or domains as indicated in each panel. (A) PPR genes encoding the P subclass of PPRs are targeted by both miRNAs and tasiRNAs. Each gray box represents one degenerate repeat of ∼35 amino acids. PPR proteins have a widely varying number of these repeat units (indicated by the broken repeat unit). Gray or outlined arrowheads indicate that miRNA or tasiRNA target sites may exist at varying levels or may not exist at all in some repeats, due to the degeneracy of the repeat sequences. (B) miRNAs target nucleotides encoding H3 motifs in the conserved R3 domains of MYB transcription factors in plants. (C) Numerous miRNAs target nucleotides encoding conserved motifs of NB-LRRs in many plant species. The NB domain has five conserved motifs indicated by colored boxes; other conserved domains and motifs characterize these proteins, as indicated. Considering many plant species, multiple encoded motifs of NB-LRRs are targeted, including the TIR1, TIR2, P-loop, kinase-2, and MHDV motifs. miR472 and miR482 are nearly identical (see Figure 4A) and indicated parenthetically for TNLs, as CNLs are the preferential targets (with TNLs as less frequent targets). miR825* is indicated in gray, as it is observed to target an encoded TIR2 only in Arabidopsis.

Figure 4.

miRNAs Target Conserved Sequences in Members of the NB-LRR Gene Families. (A) The miR482/miR2118 superfamily of miRNAs is a relatively diverse group (above) that typically targets nucleotides encoding the P-loop motif of NB-LRR proteins (below). Consensus sequences of either the miRNAs or their targets demonstrate a high degree of conservation (illustrated by WebLogo). In this figure, for illustrative purposes, we’ve randomly selected a diverse set of miRNA superfamily members (listed by their names in miRBase) and ∼16 targets from the same source species. In this case, the targets were predominantly CNLs, but miR2118 also targets many TNLs. For ease of alignment, the miRNAs are shown 5′ to 3′ in the consensus, the mRNA targets are shown 3′ to 5′, and the translated protein motif is inverted relative to the target mRNAs (indicated by the curved arrow). (B) Redundancy in miRNA targeting at the encoded TIR-1 motif of NB-LRRs. miR6019 and miR2109 aligned to their TNL-encoding targets show they target the same encoded motif (blue bar at top), but at adjacent, nonoverlapping sites. The consensus at the top is from Meyers et al. (1999). Red letters indicate the amino acids encoded by the target region. Example targets from tomato (“Soly…”) and Medicago (“Medtr…”) are indicated aligned to the miRNA sequences.