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, 10 (1), 828

Decoding the 5' Nucleotide Bias of PIWI-interacting RNAs

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Decoding the 5' Nucleotide Bias of PIWI-interacting RNAs

Chad B Stein et al. Nat Commun.

Abstract

PIWI-interacting RNAs (piRNAs) are at the center of a small RNA-based immune system that defends genomes against the deleterious action of mobile genetic elements (transposons). PiRNAs are highly variable in sequence with extensive targeting potential. Their diversity is restricted by their preference to start with a Uridine (U) at the 5' most position (1U-bias), a bias that remains poorly understood. Here we uncover that the 1U-bias of Piwi-piRNAs is established by consecutive discrimination against all nucleotides but U, first during piRNA biogenesis and then upon interaction with Piwi's specificity loop. Sequence preferences during piRNA processing also restrict U across the piRNA body with the potential to directly impact target recognition. Overall, the uncovered signatures could modulate specificity and efficacy of piRNA-mediated transposon restriction, and provide a substrate for purifying selection in the ongoing arms race between genomes and their mobile parasites.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
The Specificity Loop (SL) contributes to, but does not solely determine the 1U bias of Piwi-piRNAs in ovarian somatic sheath cells (OSC). a Structural model of Piwi’s SL and of SL mutants in complex with monophosphorylated Uridine (U) based on the crystal structure of SIWI (PDB 5GUH). The SL is highlighted in purple for wild type (wt), blue for the substitute SL from Dm Ago3, yellow for the synthetic 3× (GSS) loop, and orange for the GGG linker in the loop deficient (LD) mutant. The recognition interface between the SL and the Uracil nucleobase is indicated in red for wt. b Flag-HA (FH) tagged Piwi constructs harboring wild type (wt) or mutant specificity loops (SL) (FH-Piwi-SL). The first nucleotide preference of small RNAs that are physiologically associated with the original Argonaute protein is indicated (1st N). c FH-Piwi-SL proteins were expressed in OSC, immunoprecipitated (IP) using anti-FLAG antibody, and detected by western blotting (WB) by anti-HA antibody. d Nucleotide (22-30nt) length distribution of FH-Piwi-SL-piRNAs that associate with wt (gray) and the three categories of SL mutants. e Annotation of FH-Piwi-SL-piRNAs by their antisense targeting-potential for different transposon families. f Genomic origin of FH-Piwi-SL-piRNAs with respect to annotation categories in sense (s) and antisense (as) orientation. g Nucleotide frequencies at the first position of FH-Piwi-SL-piRNAs. [M ≤ 100: Unique mapping reads and primary alignments of multimapping reads are considered for the analyses represented in dg; read length was restricted to 24–29nt for analyses presented in eg]. (See also Supplementary Fig. 1 and Supplementary Data 3.)
Fig. 2
Fig. 2
The identity of Piwi’s SL is not required for Piwi’s function in vivo. a Flies expressing Gal4 instead of Piwi from the piwi-promoter ([piwi]>Gal4) are heterozygous fertile, but homozygous sterile. A Gal4-inducible Piwi transgene (UASp_FH-Piwi(wt)) and select SL mutants rescue fertility and ovarian morphology. b Localization of FH-Piwi-SL transgenes (IF: anti-HA) and all Piwi (IF: anti-Piwi) in piwi heterozygous (*) ovaries. c Nucleotide (nt) size distribution of piRNAs that associate with FH-Piwi wt (gray) and SL mutants. [PiRNAs associated with FH-Piwi-SL(wt), FH-Piwi-SL(Ago3), and FH-Piwi-SL(GSS) were purified from rescued piwi null ovaries. FH-Piwi-SL(LD)-piRNAs were purified from piwi heterozygous flies (*)]. Scale bars indicate 20 µm. d Annotation of piRNAs by their potential to target different transposon families by antisense complementarity. e Genomic origin of piRNAs with respect to annotation categories in sense (s) and antisense (as) orientation. f Nucleotide frequencies at the first position of FH-Piwi-SL piRNAs. [M ≤ 100: Unique mapping reads and primary alignments of multimapping reads are considered for the analyses represented in df; read length was restricted to 24–29 nt for analyses presented in df.] (See also Supplementary Fig. 2 and Supplementary Data 4.)
Fig. 3
Fig. 3
Preferences during piRNA biogenesis establish sequence restrictions. a Patterns of nucleotide frequencies within a 101 nt window surrounding the 5′ end of Piwi-piRNAs (position 0) in OSC. Data represent the mean of three biological replicates (n = 3) and standard deviations are indicated. Nucleotide 1–26 of the observed piRNAs are indicated by a gray box. The insert illustrates the background model with all possible 1U, 1C, 1G, or 1A starting piRNAs from Piwi-piRNA clusters. b Patterns of nucleotide frequencies for all four nucleotides of Piwi-piRNAs from OSC, fly ovaries, and c mouse Piwil1-associated piRNAs. Patterns of nucleotide frequencies of piRNAs associated with Piwi-SL mutants in OSC (n = 3) (d), and fly ovaries (n = 1) (e). Correlation coefficient (Pearson) compared to Piwi-SL-wt are indicated (d, e). [M = 1: Only unique mapping reads are considered to ensure unambiguous genomic coordinates. (See also Supplementary Fig. 3 and Supplementary Data 5.)
Fig. 4
Fig. 4
The 1U-bias of Piwi-piRNAs is established by a general mechanism that restricts all nucleotides but U in the first position. Piwi-piRNAs from OSC are grouped by piRNA generating clusters. The observed a 1U, b 1C, c 1A, and d 1G piRNAs (y-axis: piRNA f(1×)) are correlated with the expected genomic nucleotide frequencies (x-axis: genomic f(x)) of their generating cluster. Inserts represent the corresponding nucleotide bias (observed/expected nucleotide frequencies) as box plots with the median indicated as center line with the upper and lower quartile represented by the box. PiRNAs that are associated with FH-Piwi(wt) (gray) and two FH-Piwi-SL mutants, (Ago3) (blue) and (GSS) (yellow), are shown as median of three biological replicates (n = 3). [M = 1: unique mapping reads are considered]. (See also Supplementary Fig. 4 and Supplementary Data 6.)
Fig. 5
Fig. 5
Model for the establishment of a 1U-bias. Our data support that the 1U-bias is established in two steps by differential gating against all nucleotides but Uridine. First, purines (A/G) are reduced during piRNA biogenesis in an SL-independent manner. Consecutively, 1C is discriminated against upon piRISC formation by interactions with Piwi’s SL. (See also Supplementary Fig. 5.)

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