The piRNA targeting rules and the resistance to piRNA silencing in endogenous genes

Abstract

Piwi-interacting RNAs (piRNAs) silence transposons to safeguard genome integrity in animals. However, the functions of the many piRNAs that do not map to transposons remain unknown. Here, we show that piRNA targeting in Caenorhabditis elegans can tolerate a few mismatches but prefer perfect pairing at the seed region. The broad targeting capacity of piRNAs underlies the germline silencing of transgenes in C. elegans Transgenes engineered to avoid piRNA recognition are stably expressed. Many endogenous germline-expressed genes also contain predicted piRNA targeting sites, and periodic An/Tn clusters (PATCs) are an intrinsic signal that provides resistance to piRNA silencing. Together, our study revealed the piRNA targeting rules and highlights a distinct strategy that C. elegans uses to distinguish endogenous from foreign nucleic acids.

Fig. 1. 22G-RNA loci as a proxy to identify the targets of specific piRNAs

(A) An example of 22G-RNA distributions at one of the RNA targets of the synthetic piRNA (GFP-targeting piRNA#1) in the indicated strains with biological replicates. Each pink bar indicates the 1^st nucleotide position and abundance of 22G-RNAs. The red bar marks the position targeted by the synthetic piRNA. rpm: reads per million. (B) A scatter plot showing the abundance of 22G-RNAs around each potential targeting site (100nt window centered with each target site) of the synthetic piRNA (GFP-targeting #1) in the control strain and in the strain expressing the synthetic piRNA. Note that the potential targeting sites are sites of RNA transcripts that pair to the specific piRNA with six or fewer mismatches. Marked in red are sites at which 22G-RNA levels increased over 4 fold in the strain expressing the synthetic piRNA relative to the control strain. (C) A scatter plot showing the abundance of 22G-RNAs at each potential targeting site of 21U-RNA-X1 in the N2 (wild-type) strain and in the strain containing a deletion of the 21U-RNA-X1 coding loci. Marked in green are sites at which 22G-RNA levels decreased over 4 fold in the strain loss of 21U-RNA-X1 relative to the N2 wild type. (D) The pairing between piRNAs and identified targets. Examples of pairings between the piRNAs and their targets (Top). A bar graph showing the percentage of base pairing at each position within the piRNAs with all 17 identified targets (Bottom). GU wobble pairing is considered as paired here to highlight the near-perfect pairing at the seed region when GU pair is allowed.

Fig. 2. A piRNA reporter assay to investigate the piRNA targeting rules

(A) Fluorescence micrographs showing the expression of transgene GFP^dpiRNA∷CDK-1 in worms carrying an extrachromosomal array to express the gfp-targeting piRNA with perfect pairing or in the control strain that does not express the synthetic piRNA. Arrows mark the germline nuclei with expressed transgene. Circles mark the germline nuclei with silenced transgene. Note that the unmarked green fluorescent signals are auto-fluorescent signals generated from worm intestinal granules. (B) The sequences of the gfp-targeting piRNAs, the positions of the mismatches (red), and their effects on the expression of GFP^dpiRNA∷CDK-1. *: gfp-targeting piRNAs produced by gene-edited animals modified at an endogenous piRNA locus (21U-5499). (C) Percentage of transgenic animals that exhibit the silencing of GFP^dpiRNA∷CDK-1in animals expressing specific gfp-targeting piRNAs. At least 8 independent strains carrying extrachromosomal arrays (roller) are examined for each piRNA.

Fig. 3. Silencing-prone transgenes can be expressed in the germline by avoiding piRNA targeting

(A) Predicted piRNA sites in GFP mRNA sequence. The numbers of piRNA sites that contain different types of mismatches are shown. The relaxed criteria are used to predict piRNA sites on transgenes: all GU wobble pairing is allowed (considered as paired), and up to 3 non-GU mismatches are allowed when sites have perfect seed pairing, or up to 1 non-GU mismatches are allowed when sites have 1 non-GU mismatches in the seed region. The mismatch at the first nucleotide of a piRNA is not counted/considered. (B) The expression of original GFP∷CDK-1 that contains the predicted piRNA targeting sites, or the modified GFP^dpiRNA∷CDK-1where all predicted piRNA sites have been removed by introducing silent mutations (right). Arrows mark the germline nuclei with expressed GFP^dpiRNA∷CDK-1. Circles mark the germline nuclei with silenced GFP∷CDK-1. (C) Predicted piRNA sites in mCherry mRNA sequence (left). (D) The expression of original mCherry∷ANI-1^681-1159 that contains the predicted piRNA targeting sites, or the modified mCherry^dpiRNA∷ANI-1^681-1159 where the predicted piRNA sites have been removed by introducing silent mutations (right). Arrows mark the expression of mCherry^dpiRNA∷ANI-1^1681-1159 at cleavage furrows of the one-cell embryo. (E) Predicted piRNA sites in Cas9 mRNA sequence. (F) A schematic showing the procedure followed to examine if genome editing occurs in transgenic animals that carry the original or modified Cas9 transgenes. Plasmids containing unc-22 sgRNA and rol-6(su1006) dominant transformation marker plasmid are co-injected into transgenic animals that have been carrying the Cas9 transgene for over 5 generations. F1 transformed roller animals are picked and their F2 progeny are scored for unc-22 gene editing through twitcher phenotype. (G) Sequences of various unc-22 edited alleles obtained in the animals carrying the modified Cas9 transgene injected with plasmid encoding unc-22 sgRNA. Indels are highlighted in red.

Fig. 4. Germline-expressed genes exhibit resistance to piRNA silencing through their intrinsic signals, such as PATCs

(A) Numbers of predicted piRNA sites on germline-expressed RNA transcripts. To predict more confident targeting sites, the stringent piRNA targeting criteria are used here, where up to one GU wobble pair was allowed in the seed region, and overall only up to two mismatches plus an additional GU mismatch were allowed. In addition, the mismatch at the first nucleotide of the piRNA is not counted/considered. The RNA targets of CSR-1 Argonaute (CSR-1 targets) are used to define the germline-expressed genes. (B) qRT-PCR measurements of the abundance of the synthetic piRNAs in comparison to the level of endogenous 21U-5499 (value=1) in the control strain (left) and the expression levels of corresponding mRNA targets in the indicated strains (right). Note that nop-1, cdk-1, and oma-1 targeting piRNAs were produced by gene-edited animals, whereas pie-1-targeting piRNAs were produced by animals carrying extrachromosomal arrays. Error bars represent standard error of the mean from biological duplicated samples. The statistics for synthetic piRNA expression were calculated by comparing the levels of specific piRNAs and 21U-5499. n.s.: not significant, *: p-value<0.05, **: p-value <0.01, ***: p-value <0.001, t-test. (C) A box and whisker blot showing the density of PATC in the germline-specific and somatic specific genes. ***: p-value <0.001, t-test. (D) The density of 22G-RNAs within a 100 nt window around predicted piRNA target sites of germline-specific transcripts with high PATC density (PATC>50) or low PATC density (PATC<10). The plots are centered at sequence targeted by piRNAs (green). The stringent piRNA targeting criteria were used here to predict piRNA target sites. n = number of predicted piRNA sites. (E) The box-and-whisker plots showing the number of predicted piRNA targeted sites on germline-expressed genes that contain the indicated range of PATC density. The stringent piRNA targeting criteria were used here to predict piRNA target sites. n.s.: not significant, ***: p-value <0.001, t-test. (F) Fluorescence micrographs showing the expression of the original mCherry∷ANI-1^681-1159 harboring synthetic introns (no PATC) and mCherry^PATC∷ANI-1^681-1159 harboring PATC-containing introns. (G) 22G-RNA distribution at mCherry coding sequence of the indicated transgenes. Each bar indicates the 1^st nucleotide position and abundance of 22G-RNAs. The red bars mark the location of piRNA targeting sites predicted by using the relaxed piRNA targeting criteria.