Canonical and mirtronic miRNAs. (A) The distribution of reads across the mir-296 hairpin. Y-axis indicates the read count at each nucleotide position from the indicated library, normalized to the number of t/sn/sc/srpRNA-derived reads from that library. The predicted secondary structure is shown below, along with the 5′ pre-miRNA-flanking reads from the wild-type library (see the text). For each RNA species, the number of reads that were obtained with that sequence and the number of genomic loci to which the sequence maps are indicated at the right. See Supplemental Figure S9 for PhastCons scores. (B) The Dicer and DGCR8 dependencies of annotated miRNA genes (miRBase 10.0), plotted as in Figure 1D. (C) The normalized read frequency from each library, predicted secondary structure, and wild-type reads mapping to the mirtron mir-702, presented as in A. The boundaries of host gene exons are indicated at the bottom. See Supplemental Figure S9 for PhastCons scores. (D) The DGCR8 and Dicer dependencies of all introns <500 bp, plotted as in Figure 1D. (E) The length distribution of intron (blue) and canonical pre-miRNAs (orange). The lengths of the three mirtrons described here are indicated by pink asterisks. (F) The distribution is shown for the percentage of nucleotides that are base paired in the predicted secondary structures of 60- to 120-nt introns (blue) and canonical pre-miRNAs (orange). The value for the three mirtrons described here are indicated by pink asterisks. (G) mir-1982, a tailed-mirtron, was processed from Oaz1. Shown in magenta is the 5′ 11-bp tail that must be removed prior to Dicer processing. Plotted as in C. See Supplemental Figure S9 for PhastCons scores. (H) The secondary structure of mir-1982. Arrows indicate the 5′ ends of the two most abundant Dicer products. (I) A comparison of the levels of the mirtron small RNAs (red) to those of their parent mRNAs (blue). Changes in small RNA levels were determined as the ratio of t/sn/sc/srpRNA-normalized read counts between the wild-type and mutant libraries with a single pseudocount added. Changes in mRNA expression were determined by microarrays.

SINE-derived hp-siRNAs. (A) Sources of Dicer-dependent small RNAs. See Supplemental Table 6 for tabular representation. (B) Small RNAs from the wild-type library that derived from the indicated genomic locus, which contains two inverted B1/Alu SINE elements. Presented as in Figure 3A. (C) Predicted secondary structure of a transcript corresponding to the locus from B, with the most abundantly sequenced small RNA from each register indicated. (D) A second hp-siRNA SINE locus, presented as in B. (E) Predicted secondary structure of a transcript corresponding to the locus from D, with the most abundantly sequenced small RNA from each register indicated.

DGCR8 and Dicer dependence of mouse small RNAs. (A) Classification of small RNAs based on UCSC mouse genome annotations (mm8). See Supplemental Table 1 for a tabular representation of the data. (B) Read counts from the wild-type versus dicer1Δ/Δ libraries for genomic loci with at least 10 reads from the wild-type library, colored as in D. (C) Read counts from the wild-type versus dgcr8Δ/Δ libraries for genomic loci defined in B, colored as in D. (D) Dependencies of the small RNAs derived from genomic loci defined in B. Each axis indicates the following quotient: read count from mutant library/read count from wild-type library, with read counts normalized to the number of t/sn/sc/srpRNA-derived reads from that library. Ratios <0.1 defined enzyme dependence (red hashed line) and ratios >0.5 defined enzyme independence (gold hashed line). Loci were categorized in consideration of their dependencies on both Dicer and DGCR8, and are color coded according to the key. (E) The number of reads from each dependency category from D in the wild-type library. See Supplemental Table 3 for tabular representation.

Small RNAs that are Dicer-dependent and DGCR8-independent and derive from highly conserved shRNA. (A) Small RNAs from mir-320 were DGCR8-independent, Dicer-dependent. (Top) The normalized read count from each library, plotted above the genomic sequence and predicted secondary structure, as in Figure 2A. RNA species are represented below as lines, with thickness/color representing the number of reads (see the key). Below that is the mean locus confidence for small RNA reads at each position; 100% represents a unique match to the genome; percentages are then divided by the mean number of genomic loci to which the sequences map. (Bottom) PhastCons score at each nucleotide position. The arrow represents the 5′ end as determined by RACE in dicer1Δ/Δ and the number above represents the number of clones observed. (B) Predicted secondary structure of the mir-320 pre-miRNA. (C) Small RNAs mapping to a tRNA-Ile gene, shown as in A. Green boxed area represents reads that do not match the genome due to tRNA processing, and red lines represent untemplated CCA addition. mir-tRNA Ile has been annotated as mir-1983. (D) Manually predicted secondary structures for the tRNA cloverleaf structure and alternative shRNA fold. (E) Small RNAs mapping to mir-1980, presented as in A. (F) Predicted secondary structure of the presumed mir-1980 precursor.

Alternative biogenesis pathways of Dicer-dependent small RNAs. (A) Canonical and noncanonical pathways for miRNA biogenesis. DGCR8-independent, Dicer-dependent (noncanonical) miRNAs arise from splicing (mirtrons) or direct transcription (shRNA). Some mirtrons and shRNAs have an additional tail that is removed by an unknown nuclease. (B) Endogenous siRNA biogenesis. Inverted repeats can form long hairpins, which are processed by Dicer into siRNAs (hp-siRNAs). dsRNA duplexes can also arise from convergent transcription, or gene/pseudogene pairs. Note that convergent transcription and gene/pseudogene pairs have only been identified in mouse oocytes and were not detected in ES cells.