An atlas of soybean small RNAs identifies phased siRNAs from hundreds of coding genes

Abstract

Small RNAs are ubiquitous, versatile repressors and include (1) microRNAs (miRNAs), processed from mRNA forming stem-loops; and (2) small interfering RNAs (siRNAs), the latter derived in plants by a process typically requiring an RNA-dependent RNA polymerase. We constructed and analyzed an expression atlas of soybean (Glycine max) small RNAs, identifying over 500 loci generating 21-nucleotide phased siRNAs (phasiRNAs; from PHAS loci), of which 483 overlapped annotated protein-coding genes. Via the integration of miRNAs with parallel analysis of RNA end (PARE) data, 20 miRNA triggers of 127 PHAS loci were detected. The primary class of PHAS loci (208 or 41% of the total) corresponded to NB-LRR genes; some of these small RNAs preferentially accumulate in nodules. Among the PHAS loci, novel representatives of TAS3 and noncanonical phasing patterns were also observed. A noncoding PHAS locus, triggered by miR4392, accumulated preferentially in anthers; the phasiRNAs are predicted to target transposable elements, with their peak abundance during soybean reproductive development. Thus, phasiRNAs show tremendous diversity in dicots. We identified novel miRNAs and assessed the veracity of soybean miRNAs registered in miRBase, substantially improving the soybean miRNA annotation, facilitating an improvement of miRBase annotations and identifying at high stringency novel miRNAs and their targets.

Figure 1.

Expression Profiling of Novel and Tissue-Preferential miRNAs. (A) Novel miRNAs identified in this study included many with enriched abundances in specific tissues or organs. (B) Analysis of previously described soybean miRNAs also reveals a range of tissue preferences in flower, leaf, and nodule.

Figure 2.

Protein-Coding PHAS Genes. The soybean genome contains more protein-coding loci generating phasiRNAs than has been described for any other plant genome. (A) Categories and numbers of coding PHAS loci. (B) Expression profiling and hierarchical clustering of PHAS genes in the NB-LRR family. (C) Distributions and clusters of phasi-NB-LRR genes in the soybean genome.

Figure 3.

Triggers and Processing Mechanisms of Soybean TAS3 TasiRNAs. (A) The patterns of abundance in flower, leaf, nodule, and seed tissues for the summed total of tasiRNAs from each of the six TAS3 loci present in the soybean genome. TAS3a and TAS3b are identical and thus cannot be measured separately. (B) TasiARFs derived from TAS3a/b/c/d/e/f. The validated targets for all of the TAS3 5′8D(+) and 5′7D(+) siRNAs were all in the family of auxin response factors (ARFs), consistent with their relatively well conserved sequences (data not shown). (C) Two or three miR390 target sites exist at soybean TAS3 loci, and the direction of phasing relative to these targets sites suggests a noncanonical processing direction for siRNAs triggered by a 21-nucleotide miRNA at TAS3e and TAS3f.

Figure 4.

The ARF3 PHAS-Locus Triggered by the TasiARF. (A) The soybean TAS3-derived tasiARFs target ARF3 at two identical sites, a 5′ site for which cleavage was validated by PARE (bottom panel) and a 3′ site for which no cleavage was observed. This two-hit activity of the tasiARFs generated phased siRNAs (middle panel). The y axis is a phasing “score” that is an estimated P value for the significance of phasing (see Methods). The lower two images are our Web browser showing the small RNA (middle) or PARE data (lower), with the orange dashed line indicating the tasiARF cleavage site. Colored spots are small RNAs with abundances indicated on the y axis; light blue spots indicate 21-nucleotide sRNAs, green are 22-nucleotide sRNAs, orange are 24-nucleotide sRNAs, and other colors are other sizes. Red boxes are annotated exons (pink are untranslated regions). Purple lines indicate a k-mer frequency for repeats. (B) The data from panel A suggest a cascade of two-hit phased siRNA biogenesis in which the 21-nucleotide (nt) miR390 triggers 21-nucleotide tasiARF biogenesis, and via a two-hit mechanism, the tasiARFs trigger the production of additional secondary siRNAs from ARF3 and ARF4 (see Supplemental Figure 7 online). The ARF siRNAs may function in cis or trans.

Figure 5.

Anther-Specific PhasiRNAs Derived from Arogenate Dehydrogenase Loci. (A) The biochemical pathway in which arogenate dehydrogenase is involved. (B) Schematic pathway of phasiRNA production from arogenate dehydrogenase-related loci. At left, a gene fragment that forms a hairpin is processed into phasiRNAs. (C) The levels of read abundances for miRNA triggers and phasiRNAs derived from the two arogenate dehydrogenase PHAS genes in different tissues (red bars) and the levels of gene expression (green bars) are shown as values normalized into RP5M (read per five million) and RP25M (read per 25 million), respectively.

Figure 6.

Anther Specificity of Two PHAS Loci. sRNA and PARE reads mapped onto soybean genome indicating noncoding PHAS loci and their phasing patterns. The features of each panel is as described in previous figures. At the top of each panel, the “cleaved site” indicates weak PARE data supporting the specificity of cleavage but strong correspondence to the initiation site of the phasiRNAs. (A) A phased locus on chromosome 2 at ∼9,272,000 bp. (B) A phased locus on chromosome 20 at ∼158,400 bp.

Figure 7.

Pollen-Specific PhasiRNAs Targeting Transposons from the PHAS Locus on Chromosome 20.‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬ (A) ‪A locus on chromosome 20 produces phasiRNAs that are specifically expressed in pollen. These phasiRNAs are triggered by miR4392, the precursor of which located immediately upstream, and also accumulates specifically in pollen. The targets of the most abundant phasiRNAs from the locus were predicted using conventional rules for miRNA target identification. As indicated in the table below, the predicted targets for all of the abundant phasiRNAs belong to a wide variety of transposable elements. The lower table lists phasiRNAs that are abundant and target 10 or more transposon loci; the target data were selected from Supplemental Data Set 1J, which contains more details about these phasiRNAs.‬ The abundance data are the sum of abundance for all the libraries described in this article.‬‬‬‬‬‬‬‬‬ (B) The syntenic region between soybean and common bean. Both MIR4392 and the PHAS locus are located on the chromosome 20 of soybean. The phasiRNA signals detected at the PHAS locus are indicated in the dotted rectangle. The MIR4392 pseudogene located in the duplicated region on the chromosome 9 of soybean is shown. No phasiRNA signal was detected in duplicated region on chromosome 9. The MIR4392 locus is absent in the synthetic region on the chromosome 3 of common bean. No phasiRNA signal was detected in the syntenic region of common bean. Genes located on the sense and antisense strands of the chromosomes are presented in red and blue, respectively.