Protection from feed-forward amplification in an amplified RNAi mechanism

Abstract

The effectiveness of RNA interference (RNAi) in many organisms is potentiated through the signal-amplifying activity of a targeted RNA-directed RNA polymerase (RdRP) system that can convert a small population of exogenously-encountered dsRNA fragments into an abundant internal pool of small interfering RNA (siRNA). As for any biological amplification system, we expect an underlying architecture that will limit the ability of a randomly encountered trigger to produce an uncontrolled and self-escalating response. Investigating such limits in Caenorhabditis elegans, we find that feed-forward amplification is limited by biosynthetic and structural distinctions at the RNA level between (1) triggers that can produce amplification and (2) siRNA products of the amplification reaction. By assuring that initial (primary) siRNAs can act as triggers but not templates for activation, and that the resulting (secondary) siRNAs can enforce gene silencing on additional targets without unbridled trigger amplification, the system achieves substantial but fundamentally limited signal amplification.

Figure 1

Characterization of primary and secondary siRNA pools using a mismatched trigger. (A) Mismatched trigger assay. (B) Proportions of distinct small RNA populations from 5’-P-independent small RNA capture from N2 (wild type) animals fed mismatched sel-1 dsRNA. (C) Proportions of distinct small RNA populations from 5’-monoP-enriched small RNA capture from N2 (wild type) animals fed mismatched sel-1 dsRNA. (D) Plot of sel-1 siRNAs from 5’-P-independent capture from N2 animals fed mismatched sel-1 dsRNA. (E) Plot of sel-1 siRNAs from 5’-monoP-enriched capture from N2 animals fed mismatched sel-1 dsRNA. (D, E) grey: sel-1 mRNA, green: region of sel-1 mRNA encompassed in mismatched trigger, black: siRNA matching both target and trigger RNAs, blue: siRNA matching only target RNA, red: siRNA matching only trigger RNA. Lightened shading indicates multiple incidence. (See also Figure S1.)

Figure 2

A doubly mismatched trigger to test for use of trigger RNA as template for synthesis of secondary siRNAs. (A) Scheme for using a double mismatched trigger to distinguish by sequence between primary siRNAs, target derived molecules and RdRP products, and hypothetical products of RdRP copying of trigger sequences. (B) Sequences of target RNA (wild type), sense and antisense strands of the trigger RNA molecule. Asterisks: Class 1 type mismatches where base pairing is maintained between the two strands of the trigger dsRNA. Arrowheads: Class 2 type mismatches where GU wobble pairs are introduced into the trigger dsRNA. (B) Plot of sel-1 siRNAs captured using 5’-P-independent capture protocol. grey: sel-1 mRNA, green: region of sel-1 mRNA encompassed in double mismatched trigger, blue: siRNA matching only target RNA, red: siRNA matching one strand of trigger RNA. Absent were the hypothetical (black) sequences that would have been derived from copying of the primary trigger by RdRP. Lightened shading indicates multiple incidence.

Figure 3

Use of a deletion heterozygote to test for production of tertiary siRNAs. (A) Model for generation of hypothetical tertiary siRNAs in a deletion heterozygote. The ben-1 (cc1934/cc1921) trans-heterozygote contains two populations of ben-1 mRNAs containing either the cc1934 or the cc1921 deletions. dsRNA trigger molecules encompassing the region deleted in the cc1934 allele is introduced when it is subsequently processed into primary siRNAs (red). The primary siRNA guides the Argonaute (presumably RDE-1) to the mRNA derived from the cc1921 allele whence the RdRP is recruited generating a population of secondary siRNAs (blue). Absent from these secondary siRNAs, having been generated on the cc1921 mRNA template, are sequences deleted in the cc1921 allele. If the secondary siRNA, in turn, is capable of recruiting the RdRP to naive mRNAs, the resulting population should include copies made from the cc1934 deletion mRNA, resulting in some siRNAs that would overlap the region deleted in the cc1921 allele. If, however, secondary siRNAs are incapable of recruiting the RdRP, the cc1921 deleted region would be barren. (B) siRNAs from ben-1 (cc1934/cc1921) trans-heterozygote RNAi experiment. siRNAs were analyzed from wild type, ben-1 (cc1921/cc1921), ben-1 (cc1934/cc1934), a mixture of RNA from ben-1 (cc1921/cc1921) and ben-1 (cc1934/cc1934) animals, and from ben-1 (cc1934/cc1921) trans-heterzygotes exposed to dsRNAs corresponding to the R1934, R1934U, and R1934D triggers. In addition, captured RNA datasets from (i) control wild type strains triggered for RNAi in the act-1 gene (RACT1), (ii) the sel-1 gene with the mismatched sel-1 trigger (RMMSEL1), and (iii) no RNAi were included in the analyses (Gent et al., 2010; Maniar and Fire, 2011; Wu et al., 2011). siRNAs were counted as follows: Region 1, all siRNAs that unambiguously overlap the cc1921 deletion; Region 2, all siRNAs that contain sequence spanning the junction of the region deleted in cc1921 with at least 2 nt matches on both sides of the junction; Region 3, all siRNAs that unambiguously overlap the cc1934 deletion; Region 4, all siRNAs that contain sequence spanning the junction of the region deleted in cc1934 with at least 2 nt matches on both sides of the junction. Some variability in overall response is observed in bacterial feeding experiments with C. elegans; the indicated paucity of tertiary siRNAs relative to their neighbors in ben-1 (cc1921/cc1934) heterozygotes has been observed over a variety of different RNAi efficiencies in five independent RNA feeding experiments (bottom panel and data not shown). As the overall efficacy with which RNAi is initiated and amplified can vary between experiments with different triggers (due, in part, to difference in distance between the trigger region and the assayed region), siRNA counts are expressed as a percentage of antisense siRNA reads within a region spanning 50 nt upstream and downstream of the deletion. * Values for cc1934 homozygotes exposed to the R1934 trigger were normalized to equivalent values (as judged by the relative level of sense ben-1 siRNAs) for wild type animals exposed to the R1934 trigger due to a complete lack of an RNAi response in the former. Likewise, counts for control, non-ben-1 RNAi wild type strains were normalized to equivalent values (as judged by the relative level of total antisense siRNAs to all cDNAs) for wild type animals exposed to the R1934 trigger. 'RPKM' is a standard metric of sequence representation calculated as Reads per Kilobase of target per Million sequence instances (in this case antisense cDNA sequences) (Mortazavi et al., 2008). The 5'-P-independent capture method was employed. (See also Figure S2.)

Figure 4

Assays for siRNAs emanating from direct and indirect targets of an actin 3' UTR dsRNA trigger. (A) Experimental design. RNAi against a member of a partially-redundant multigene family is induced using a dsRNA trigger uniquely matching the 3' UTR of one of the gene family. For regions of mismatch between the highly similar coding regions, siRNA populations that derive from the direct target gene (act-1 in this case) can be distinguished by sequence from potential tertiary siRNA populations that derive from the remaining genes in the family. (B–D) Genome-wide gene by gene comparison of antisense siRNA matches. Each point represents one C. elegans mRNA transcript from a duplicate-subtracted reference sequence set (Wormbase). Vertical position on each plot represents antisense siRNA counts for that gene in small RNA captured following act-1 3' UTR RNAi as shown in A. Horizontal positions represent antisense siRNA counts from comparable RNAi experiments with non-actin triggers. (B) Horizontal axis: sel-1 mismatched trigger (see Figure 1), (C, D) Horizontal axis: ben-1 triggers R1934U and R1934D (Figure 3). (E) Distribution of antisense siRNAs matching the act-1 transcript following RNAi feeding with the act-1 3' UTR trigger (blue line). Coverage at each base position is normalized to aggregate antisense siRNA counts for all C. elegans genes, and a smoothing window of 61b is used. Enrichment of antisense counts throughout the act-1 gene is evident in comparison to control samples in which RNAi was triggered to a non-actin target (green, red, orange lines). (F) Equivalent composite coverage plot of antisense siRNAs deriving from act-2, act-3, act-4, or act-5 and not from act-1. Given the high signal of act-1 matching siRNAs in E and the known error frequency in high throughput RNA-seq methods, there is some possibility that a fraction of the signal in F represents mis-sequencing of act-1-matched siRNAs. This value is estimated (magenta line, F) by generating a series of 72 "faux-actin" genes (Supplementary Experimental Procedures). (See also Figure S3.)

Figure 5

Genetic requirements for primary and secondary siRNA generation. (A) Model for RNAi in C. elegans. Exogenous dsRNA is processed by DCR-1 and RDE-4 into primary siRNAs. Short dsRNAs are then delivered to RDE-1, potentially through an additional role of the dsRNA binding factor. RDE-1 then cleaves the passenger strand, allowing subsequent maturation into active RISC. The RDE-1 RISC then recruits the RdRP (RRF-1 or EGO-1) to the target RNA where the latter engages in secondary siRNA synthesis using the target as template. Primary siRNA-complexed RDE-1 may endonucleolytically destroy targ et RNAs, while secondary siRNAs complex with the WAGOs and presumably further downregulate the target RNA via an unknown mechanism. (B) Mutations in RNAi factors affect primary and/or secondary siRNA synthesis. Pie charts indicate fraction of antisense sel-1 siRNAs comprised by trigger-matched (red), target-matched (blue), and ambiguous (black) antisense sel-1 siRNAs. Numbers below pie charts indicate percentage of total small RNAs (sRNAs) comprised of sel-1 siRNAs. (See also Figure S4.)