Sliced microRNA targets and precise loop-first processing of MIR319 hairpins revealed by analysis of the Physcomitrella patens degradome

Abstract

Expression profiling of the 5' ends of uncapped mRNAs ("degradome" sequencing) can be used to empirically catalog microRNA (miRNA) targets, to probe patterns of miRNA hairpin processing, to examine mRNA decay, and to analyze accumulation of endogenous short interfering RNA (siRNA) precursors. We sequenced and analyzed the degradome of the moss Physcomitrella patens, an important model system for functional genomic analyses in plant evolution. A total of 52 target mRNAs of 27 different Physcomitrella miRNA families were identified. Many targets of both more conserved and less conserved miRNA families encoded putative regulatory proteins. Remnants of MIRNA hairpin processing also populated the degradome data and indicated an unusual "loop-first" mode of precise processing for the MIR319 gene family. Precise loop-first processing was confirmed for native Physcomitrella, rice, and Arabidopsis MIR319 hairpins, as well as an Arabidopsis artificial MIRNA (aMIRNA) based upon a MIR319 backbone. MIR319 is thus a conserved exception to the general rule of loop-last processing of MIRNA hairpins. Loop-first MIR319 processing may contribute to the high efficacy of a widely used MIR319-based strategy for aMIRNA production in plants.

FIGURE 1.

Mapping and analysis of the Physcomitrella degradome. (A) Proportional Venn diagram showing number of processed degradome tags mapping to the genome and/or the sense strand of the transcriptome. (B,C) Examples of confidently identified sliced miRNA targets. The frequencies of degradome tags with 5′ ends at the indicated positions are shown in black, with the frequency at position 10 of the inset miRNA-target alignment highlighted in red. Full details of all confidently identified miRNA targets are in Supplemental Table 1.

FIGURE 2.

Degradome tags mapping to Physcomitrella MIRNA hairpins. (A) Schematic of a typical MIRNA hairpin showing the position of the lower DCL processing site (blue arrowheads), upper DCL processing site (red arrowheads), and the positions of potential AGO-directed cleavage by the mature miRNA or miRNA* (gray and white arrowheads). Hollow arrowheads represent expected positions of corresponding degradome tags. Degradome tags at the filled arrowheads are not due to canonical DCL processing. (B) Frequency, position, and quality of degradome tags mapping to Physcomitrella MIRNA hairpins. Category one indicates tags at that position were the most abundant relative to other positions on the hairpin, category two indicates tag abundances below the maximum but above the median, and category three indicates all others. Supporting data are in Supplemental Tables 4 and 5.

FIGURE 3.

Precise and conserved “loop-first” processing of MIR319 hairpins. (A) Schematic of a MIR319 hairpin precursor. The position of mature miR319 or Arabidopsis MIR319a-based amiRNAs is indicated; the regions modified for construction of Arabidopsis MIR319a-based amiRNAs is shown in black. Arrowheads are as described in Figure 2. (B) Frequency of degradome tags (green-solid) and gene-specific (GSP) RLM-5′-RACE products (purple-dashed) mapping to the Physcomitrella MIR319b hairpin. For brevity, only the 3′ arm of the hairpin is shown—degradome data on the 5′ arm were negligible and there were no gene-specific RLM-5′-RACE products isolated from that region. See Supplemental Tables 4 and 5 for full details. (C) As in B for Physcomitrella MIR319d. (D) Gene-specific RLM-5′-RACE products for Physcomitrella MIR319 hairpins. Arrowheads indicate the expected sizes of the corresponding processing remnants. The sizes of dsDNA molecular weight standards (10 bp and 1 kb+) are indicated at right. (E) As in B for Arabidopsis MIR319a in wild-type inflorescences. See Supplemental Tables 6 and 7 for full details. (F) Gene-specific RLM-5′-RACE products for Arabidopsis MIR319a and MIR319a-based aMIRNA hairpins. Labels as in D. Asterisks indicate nonspecific artifact bands which also appeared in control reactions lacking a gene-specific primer. (G) As in B for Oryza MIR319b. (H) As in B for Arabidopsis MIR319a-based aMIRNA over-expressing plants.