All RNA sequences extracted from Illumina reads as described in methods were deposited in the Gene Expression Omnibus with the following accession number: (GSE11738). Included under this accession number are the following data sets: developmental timecourse/mixed stage, 5´ monophosphate-dependent; prg-1(tm872) and
fog-2(q71) mutant analysis, 5´ monophosphate-dependent; prg-1(tm872) mutant analysis,
5´ monophosphate-independent; PRG-1 co-IP analysis. 21U-RNA sequences are provided as a supplemental Fasta-formatted text file (Supplemental Table III-1). Tools for scoring 21U-RNA loci trained using data from (Ruby et al., 2006) and applied here are available for anonymous download at http://web.wi.mit.edu/bartel/pub/.
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Supplemental Results and Supplemental Figures
Analysis of 21U-RNA length variants
Shortened 21U-RNA variants were far more abundant than longer variants (supplemental Figure III-1A; Ruby et al., 2006). The strength of 5´ U preference in excess of the preference for 21nt length (supplemental Figure III-1B) suggested that 21U- RNA 3´ heterogeneity was more common than 5´ heterogeneity. To test this possibility, we evaluated the upstream motif score distributions of all 20mers identified in our data sets, assuming them to be the product of a single nucleotide excision from either their 3´ or 5´ ends. Consistent with the idea that 3´ heterogeneity is more common, we found that 20mers assumed to arise from a 3´ excision exhibited a much higher motif score distribution than did 20mers assumed to arise via a 5´ truncation (supplemental Figure III- 1C).
Re-classification of two annotated miRNAs as 21U-RNAs
Comparison of miRNA sequences from these libraries showed that prg-1 was not required for miRNA biogenesis. We observed a very good correlation between miRNA read counts in the wild type versus prg-1 mutant samples. The only two exceptions were miR-78 (8 reads from prg-1; 214 reads from wt) and miR-798 (no reads in the prg-1 library and 79 reads in the wild type library). Both of these RNAs are 21nt, have 5´ U’s, and derive from genomic regions rich in 21U-RNA loci. These two RNAs were supported by 1,392 reads (miR-78) and 171 reads (miR-798) across our full datasets, but despite that abundance, the miRNA star strands (a hallmark of processing from an
miRNA-like hairpin precursor (Ruby et al., 2006)) were not observed for either. Moreover, both sequences had high-scoring 21U-RNA upstream motif matches (21.6 for miR-78; 23.6 for miR-798), indicating that they should be re-classified as 21U-RNAs.
A particular small RNA mapping to Tc3 was previously annotated as a 21U-RNA on the basis of its length, 5´ nucleotide identity, and genomic position (21U-RNA-139; Ruby et al, 2006). Its sequence was also recovered only 6 times in our combined data sets but was not recovered in the PRG-1 immune complex. Its sequence mapped to 20 Tc3 genomic positions, all of which exhibit very poor motif scores, suggesting that this small RNA was likely mis-annotated as a 21U-RNA.
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Supplemental Figure III-1. Analysis of small RNA reads associated with the conserved 21U-RNA motif.
(A) The abundance of high-scoring reads whose lengths are less than (blue), equal to
(green), or greater than (orange) 21nt. Plotted as in Figure III-1B. (B) The abundance of high-scoring reads whose 5´ nucleotide is a U (red) or is an A, C, or G (blue). All read lengths were included, and the 5´ nucleotide position of the scoring matrix was omitted. Plotted as in Figure III-1B. (C) The 21U-RNA upstream motif score distributions for sequenced 20mers, assuming them to be the degradation products of 21mers. Scores were calculated using either the true 5´ end of the read (assuming degradation from the 3´ end; red) or the 5´ end shifted one nt upstream (assuming degradation from the 5´ end; blue). Plotted as in Figure III-1B. (D) The frequency of sense (orange) and antisense (blue) exon overlap across all 21U-RNA loci. Most common core motif positions (green) and 21U-RNA span (pink) are highlighted. (E) The frequency, by position, of nucleotide conservation in whole-genome alignments. Blue, orange, and green lines indicate per- position conservation of C. elegans nucleotide identity with C. brenneri, C. remanei, and
C. briggsae, respectively. Most common core motif positions (green) and 21U-RNA
Supplemental Figure III-2. Proteins involved in the RNAi are not required for the accumulation of 21U-RNAs.
(A) Northern blot analysis of 21U-RNA-1, and miR-66 expression in wild-type and mutant strains as indicated. The SL1 precursor is a loading control.
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Supplemental Figure III-3. Specificity of the PRG-1 antibody.
(A) Western blot analysis on wild-type and prg-1 mutant worm lysates using an antibody
generated against PRG-1. Anti-tubulin is a loading control. (B) Schematic representation of the mutations in PRG-1 and PRG-2 used in this study. Areas in yellow represent the PAZ domain. The PIWI domain is represented in red. Grey bars represent areas deleted in the genome (if an insertion event has occurred, the sequence is represented on top of the grey bar). Green squares indicate the location of the peptides used to generate the antibody.
Supplemental Methods
Protein and RNA Preparation
Synchronous populations of animals were grown on NGM plates, with OP50 E. coli at a density of approximately 90,000 animals per 15 cm Petri dish, and harvested at specific stages of development. The harvested animals were washed three times with M9 buffer and incubated for 30 minutes in M9 buffer to remove the bacteria from gut. The incubation was followed by three washes with M9 buffer. A last wash was performed with cold protein lysis buffer (30 mM HEPES-KOH [pH7.4], 2mM magnesium acetate, 100 mM potassium acetate) and the pellet frozen in a dry ice and ethanol bath. The frozen pellets were kept at – 80 ºC. The frozen pellet was ressuspended in ice-cold buffer 1:1 (v/v) containing 2mM DTT, 0.1% Igepal CA 630 (Fluka), 4x concentration Complete proteinase inhibitor (Roche) and 1% (v/v) SUPERase-IN (Ambion) and homogenized using a stainless steel Dounce homogenizer (Wheaton Incorporated). The homogenized extract was clarified by a centrifugation at 13,817 x g for 20 min at 4C. For RNA extraction, the last wash was performed using sterile water and animals were frozen in TRI Reagent (MRC, Inc.). Worms were homogenized in a glass dounce and total RNA was isolated accordingly to the TRI Reagent protocol.
Protein Immunoprecipitation
Imunoprecipitation of PRG-1 containing protein complexes was preformed by pre- clearing worm extracts with 20µl of Protein A/G PLUS-Agarose (Santa Cruz Biotechnologies) beads per 5 mg of total protein for 45 minutes at 4C. The cleared extract was then incubated with: 10 µg of monoclonal antibody anti-AFP 3E6 (Qbiogene) or 15 µg of the PRG-1 specific antibody, for 1h at 4OC followed by 50 µl of Protein A/G
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PLUS-Agarose beads. The agarose beads were then washed three times with ice-cold lisys buffer. A sample (5%) was used for Western blot analysis. RNA was extracted from the Protein A/G PLUS-Agarose beads with TRI Reagent.
Western blot analysis
Proteins were resolved by SDS-PAGE on an 8% gel and transferred to Hybond-C membrane (Amersham Biosciences). The membrane was incubated overnight at 4°C with either: (i) affinity purified anti-PRG-1, 1 µg/ml, (ii) Full-Length A.v. Polyclonal Antibody (BD Bioscience), diluted 1:1000, or (iii) anti alpha-tubulin (Accurate Chemical) diluted 1:2000, in PBST-5%milk solution (137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, pH 7.4, and 5% [w/v] dried milk). The membrane was incubated 1 h at room temperature with HRP-conjugated secondary antibody (Jackson Immunoresearch) diluted 1:5,000 in PBST and then visualized by Western Lightening ECL Kit from Perkin Elmer. Images were collected on a LAS-3000 Intelligent Dark-Box (Fujifilm).
Northern Blot Analysis
After total RNA extraction, small RNA was enriched using the mirVana kit (Ambion). Ten to fifteen µg of small RNA were resolved on a 15% Urea-PAGE gel and transferred to Hybond N+ (GE Healthcare) membrane. Starfire probes were 32P-labeled and hybridized in Ultrahyb Oligo buffer (Ambion). Probe sequences are available upon request.
Solexa cloning method
Total RNA was combined with trace amounts of 5’-32P-labeled RNA standards, AGC GUG UAG GGA UCC AAA and GGC AUU AAC GCG GCC GCU CUA CAA UAG