Distinct size distribution of endogeneous 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-DEPENDENT 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-of-function 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 approximately 22-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.

Fig. 1.

Small RNA sizes and abundances in wild-type and mop1-1 or rdr2 libraries. (A) Plot comparing the total abundance of maize small RNA sequences versus their size for wild type and mop1-1. The dashed gray line indicates the abundances of the mop1-1 sample adjusted downward based on the average 5.3-fold enrichment of miRNAs observed in C. (B) Plot comparing the total abundance of Arabidopsis small RNA sequences versus their size for wild-type and rdr2 inflorescences. (C) (Left) RNA gel blot analysis of miRNA abundances in wild type and mop1-1. Total RNA was normalized based on levels of the U6 spliceosomal RNA. Zm-small1 is a putative new miRNA candidate described in Results. The Zm-siRNA is a randomly selected 24-nt small RNA from a high-density siRNA cluster. (Right) Original SBS abundance of each miRNA. Measured across the known maize miRNAs, these raw abundances were 5.3-fold higher in the mop1-1 data.

Fig. 2.

Chromosomal distribution of small RNAs from immature ears. (A) Small RNAs were matched to a segment of maize chromosome 1, as described in Results. The x axis indicates the location on the contig, and the y axis indicates the abundance of the small RNA in TP5M, using miRNA-normalized abundances. The lines are representative of a moving average of 10 windows of 10,000 bp. The curly brace indicates the region expanded in B and C. (B and C) Sliding-window calculation of the sum of abundance of small RNAs matched to each location in a 250-kb region of the contig. The x axis indicates the location on the contig, and the y axis indicates the sum of abundance in TP5M. The LTR retrotransposon is marked, including the terminal repeats (black arrowheads). The values were calculated separately for wild type (B) and mop1-1 (C), and for small RNAs ≤22 nt and ≥23 nt.

Fig. 3.

Sizes and hits for repeat- or contig-mapped maize small RNAs. (A) Size distribution of maize small RNAs mapping to LTR retrotransposons, predominantly high-copy elements, on the Chr. 1 and 9 contigs. The LTR retrotransposons were identified by RepeatMasker as gypsy- or copia-type elements and spanned 6,670,685 bp on the two contigs. The y axis indicates the sum of the hits-normalized-abundance (HNA), calculated by dividing the normalized abundance (in TP5M) for each small RNA by the number of genomic locations to which the small RNA maps (the “hits”). The mop1-1 abundances have been reduced by 5.3-fold (normalized by miRNA levels). (B) Same calculations as in A, but performed for predominantly low-copy DNA transposons identified by RepeatMasker, including hAT, hAT-Ac, En-Spm, Harbinger, or MuDR elements. These spanned 85,149 bp on the two contigs. (C) Plots of distinct, contig-matching small RNAs versus their hits. Data represent only small RNAs matched to the Chr. 1 and Chr. 9 maize contigs. The values were calculated separately for wild type (Upper) and mop1-1 (Lower) and for small RNAs ≤22 nt and ≥23 nt. A comparison of the ratios using different ranges for low versus high hits is in Table S5.

Fig. 4.

Size distribution of small RNAs from diverse monocot species. A plot comparing the total abundance of small RNA sequences versus their size for monocot species was sequenced as part of a comparative sequencing project (http://smallrna.udel.edu).