Distinct size distribution of endogenous siRNAs in maize: Evidence from deep sequencing in the mop1-1 mutant

Abstract

Small RNAs from plants are known to be highly complex and abundant, with this complexity proportional to genome size. Most endogenous siRNAs in Arabidopsis are dependent on RNA-DEPEN-DENT RNA POLYMERASE 2 (RDR2) for their biogenesis. Recent work has demonstrated that the maize MEDIATOR OF PARAMUTATION1 (mop1) gene is a predicted ortholog of RDR2. The mop1 gene is required for establishment of paramutation and maintenance of transcriptional silencing of transposons and transgenes, suggesting the potential involvement of small RNAs. We analyzed small RNAs in wild-type maize and in the isogenic mop1-1 loss-offunction mutant by using Illumina's sequencing-by-synthesis (SBS) technology, which allowed us to characterize the complement of maize small RNAs to considerable depth. Similar to rdr2 in Arabidopsis, in mop1-1, the 24-nucleotide (nt) endogenous heterochromatic short-interfering siRNAs were dramatically reduced, resulting in an enrichment of miRNAs and transacting siRNAs. In contrast to the Arabidopsis rdr2 mutant, the mop1-1 plants retained a highly abundant heterochromatic Ϸ22-nt class of small RNAs, suggesting a second mechanism for heterochromatic siRNA production. The enrichment of miRNAs and loss of 24-nt heterochromatic siRNAs in mop1-1 should be advantageous for miRNA discovery as the maize genome becomes more fully sequenced.miRNA ͉ mop1 ͉ rdr2 ͉ small RNA T he small RNAs found in a typical plant cell include a small number of highly abundant, mainly 21-nt microRNAs (miRNAs) and a large number of small interfering RNAs (mainly 24 nt heterochromatic siRNAs, or simply siRNAs) recognizing many diverse sequences. In addition, several additional subclasses of varying and in some cases overlapping functional importance have been described, including the transacting siRNAs (ta-siRNAs) (1-3), natural antisense siRNAs, a type thus far observed only under stress conditions (4, 5), and natural antisense miRNAs (6).MicroRNAs have a variety of regulatory roles in development and stress responses (for review, see ref. 7). In addition, work in Arabidopsis has led to the hypothesis that transcription of repeats is performed by the plant-specific RNA polymerase IV (pol IV) followed by reverse transcription and cleavage by RDR2 and DCL3, respectively. These repeated sequences include transposons and retrotransposons in plants (8), and this series of events produces a complex set of heterochromatic siRNAs (for review, see ref. 9). The genome size of plants varies substantially among species mainly caused by variation in content of repeated DNA. The complexity of siRNAs is correspondingly greater in rice than in Arabidopsis (10), consistent with larger numbers of repeated sequences in rice.The maize b1 locus is an excellent model for paramutation, a phenomenon in which alleles communicate in trans, resulting in meiotically heritable gene expression changes (11). Molecular work, combined with fine-structure recombination mapping, has demonstrated that this activity is mediated by tandem repeats at b1 that are required to e...

RNA-mediated trans -communication can establish paramutation at the b1 locus in maize

“…2A). The reduction in b1TR-siRNAs in mop1-1 was consistent with the global reduction in 24-nt siRNAs previously reported in the mop1-1 mutant (14). SNPs are present in four of the seven repeats, and they allowed us to map distinct siRNAs to more than one repeat (Table S1), indicating that multiple repeats are transcribed and processed into siRNAs in B′.…”

“…2A). A total of 33 and 3 b1TR-siRNAs were present in the WT and mop1-1 libraries, respectively, from a total of 13 million reads (14). These small RNAs had characteristics of siRNAs because they were predominantly 24 nt in size and examples were found that matched to both strands ( Fig.…”

“…MiRNAs play key roles during plant development and mutations, altering the biogenesis or action of miRNAs result in numerous developmental defects (34). Based on the observation that raw abundances for perfect matches to known miRNAs were different in WT vs. mop1-1 small RNA libraries (14) and on a report showing that levels of miR156 were down-regulated in feminized tassels of mop1-1 (35), we hypothesized that other developmental phenotypes observed in the mop1-1 mutant might be related to alterations in levels of additional miRNAs. To investigate this, we examined the levels of nine highly conserved miRNAs (36) in mop1-1 homozygous and WT plants by RNA blot analysis.…”

“…Activity of mop1 is required for paramutation at the b1 locus and other loci (5,6,12), and it is required to maintain the silent B′ state (5). Similar to Arabidopsis RDR2, mop1 is required for the accumulation of the vast majority of 24-nt siRNAs (13)(14)(15), and it is involved in regulating the expression of a subset of transposable elements (TEs), transgenes, and several non-TE genes (7,(13)(14)(15)(16)(17)(18).…”

Revisiting Criteria for Plant MicroRNA Annotation in the Era of Big Data

“…Endogenous hc-siRNAs comprise a major portion of the regulatory small RNA pool in key angiosperm model organisms including A. thaliana (Lu et al, 2006), O. sativa (Jeong et al, 2011), and Zea mays (Nobuta et al, 2008). Most hc-siRNAs are 24 nucleotides in length and are thought to function in RNAdirected DNA methylation, which couples 24-nt siRNA production from heterochromatic regions to targeting of chromatin-associated nascent non-coding RNA transcripts.…”