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Ribosomes guide pachytene piRNA formation on long intergenic piRNA precursors

Nature Cell Biology volume 22, pages 200–212 (2020)Cite this article

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Abstract

PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs essential for fertility. In adult mouse testes, most piRNAs are derived from long single-stranded RNAs lacking annotated open reading frames (ORFs). The mechanisms underlying how piRNA sequences are defined during the cleavages of piRNA precursors remain elusive. Here, we show that 80S ribosomes translate the 5′-proximal short ORFs (uORFs) of piRNA precursors. The MOV10L1/Armitage RNA helicase then facilitates the translocation of ribosomes into the uORF downstream regions (UDRs). The ribosome-bound UDRs are targeted by piRNA processing machinery, with the processed ribosome-protected regions becoming piRNAs. The dual modes of interaction between ribosomes and piRNA precursors underlie the distinct piRNA biogenesis requirements at uORFs and UDRs. Ribosomes also mediate piRNA processing in roosters and green lizards, implying that this mechanism is evolutionarily conserved in amniotes. Our results uncover a function for ribosomes on non-coding regions of RNAs and reveal the mechanisms underlying how piRNAs are defined.

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Fig. 1: Pachytene piRNA precursors associate with ribosomes.
Fig. 2: Ribosomes bind pervasively to piRNA precursors.
Fig. 3: Ribosomes translate the uORFs of piRNA precursors.
Fig. 4: Ribosomes translate into UDRs of piRNA precursors.
Fig. 5: Endonucleolytic cleavage targets ribosomes on piRNA precursors.
Fig. 6: Two populations of ribosomes at uORFs and UDRs.
Fig. 7: Decrease in piRNA processing intermediates upon temporal ribosome depletion.
Fig. 8: A conserved role for ribosomes in piRNA biogenesis.

Data availability

Deep-sequencing (RNA-Seq, small RNA-Seq, Ribo-Seq and Degradome-Seq) data that support the findings of this study have been deposited in the Gene Expression Omnibus under accession codes GSE65786. All other data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data for Figs. 1 and 3–8 and Extended Data Figs. 2–10 are provided online.

Code availability

All computational codes used in this study can be obtained from the author upon reasonable request. The Ribo-Seq pipeline was developed for this study and is available at https://github.com/LiLabZhaohua/RiboSeqPipeline. 5′ end overlap analysis was performed using our own script, which is available from https://gist.github.com/nimezhu/d8734d2ae6c1619218f1.

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Acknowledgements

We thank P. Zamore, M. Moore, E. Sheppard, K. Kleene, B. Wright, Q. Chen, P. Mu, M. Frohman, P. S. Brookes, G. Xiao, C. Beckham and the Cornell Stem Cell and Transgenic Core Facility for help with the experiments, L. Maquat, E. Phizicky, D. Ermolenko, A. Korostelev, A. Jacobson, J. Lin, C. Roy, P. Yao, D. Mathews, D. Anderson and J. R. Lozada for discussions and N. Chen for the clipart used for the rooster, as well as the University of Rochester Pathology Core, University of Rochester Genomics Research Center, B. Zhang, Z. Guo, G. Zhang, W. Fang and members of the Li Laboratory. This work was supported in part by National Institutes of Health grants K99/R00HD078482 and R35GM128782 and Agriculture and Food Research Initiative Competitive Grant number 2018-67015-27615 from the USDA National Institute of Food and Agriculture (to X.Z.L.). C.C. Is supported by National Institutes of Health grant R01HD084494.

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Author notes

  1. These authors contributed equally: Jiang Zhu, Li Huitong Xie, Ziwei Li.

Authors and Affiliations

  1. Center for RNA Biology: From Genome to Therapeutics, Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, USA

    Yu H. Sun, Jiang Zhu, Li Huitong Xie, Ziwei Li, Rajyalakshmi Meduri & Xin Zhiguo Li

  2. Computational Biology Department, Carnegie Mellon University, Pittsburgh, PA, USA

    Xiaopeng Zhu

  3. College of Public Health, Division of Biostatistics, The Ohio State University, Columbus, OH, USA

    Chi Song

  4. Department of Animal Science, Michigan State University, East Lansing, MI, USA

    Chen Chen

  5. Université de Lyon, ENSL, UCBL, INSERM, CNRS, LBMC, Lyon, France

    Emiliano P. Ricci

  6. Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, MA, USA

    Zhiping Weng

  7. Department of Urology, University of Rochester Medical Center, Rochester, NY, USA

    Xin Zhiguo Li

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Contributions

Y.H.S., X.Z. and C.S. analysed the data with input from E.P.R., Z.W. and X.Z.L. J.Z., Z.L., L.H.X. and R.M. performed the experiments with input from E.P.R., C.C. and X.Z.L. X.Z.L. contributed to the design of the study. All authors contributed to the preparation of the manuscript.

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Correspondence to Xin Zhiguo Li.

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Extended data

Extended Data Fig. 1 Comparison of the current model (left panel) of pachytene piRNA biogenesis and the revised model (right panel).

The grey bubbles represent the large and small subunits of ribosomes. The blue bubbles represent PIWI proteins. Grey circle in PIWI proteins on the left represents MID domain that recognizes the 5´-phosphate (5´P)70,71, on the right represents PAZ domain, in between represents PIWI domain. In both models, piRNA precursors are synthesized by RNA polymerase II, and contain the 5´-cap, exons, introns, and a poly(A) tail. The transcription of pachytene piRNA genes is controlled by A-MYB. The 5´-end-loaded PIWI proteins specify the endonuclease PLD6 cleavage site, determine the 5´- and 3´-ends of pre-piRNAs, and generate strings of head-to-tail phased piRNAs that will be further trimmed and methylated. In the revised model, ribosomes associate with precursors in a canonical fashion through initiating at the start codon near the 5´-cap. Facilitated by MOV10L1, ribosomes translate into the UDRs (Purple arrows). Ribosome-bound UDRs are targeted by PLD6-mediated cleavage. The cleavage of piRNA precursors upstream of the ribosome E site (at the exit site of the mRNA ribosome channel) does not prevent the ribosome from translating towards the 3´-end of the piRNA precursors. The cleavage machinery, generating 1U bias, appears to pause the ribosomes, leading to the detection of in vivo processed RPFs dwelling at future piRNA sites at the steady-state. As ribosomes translocate, they can release the 5´P of pre-pre-piRNAs for PIWI loading that guides stepwise phased piRNA production. piRNA biogenesis at the UDRs requires TDRD5 protein. Transcripts containing ORFs are translated by polysome, and they are processed to piRNAs in a distinct manner that does not require TDRD5 (Gold arrows).

Extended Data Fig. 2 Ribosomes associate with pachytene piRNA precursors.

a, A254 absorbance profile of testis lysates from adult mice following separation in 10% to 50% sucrose density gradients. The testes were lysed in amended lysis buffer that preserves mitochondria. Top to bottom, relative abundance of 18S rRNA, 28S rRNA, 12S rRNA, Mcm5 mRNA, and two pachytene piRNA precursors: 17-qA3.3-26735 and 7-qD1-16444. For Supplementary Fig. 2a, c, d, experiments have been repeated for at least three times independently with similar results. b, Fraction of RNAs in lysates relative to total RNAs in both lysates and pellets. The long transcripts were quantified using RT-qPCR (three independent biological samples; mean ± standard deviation, n = 3 independent experiments). Quantification of piRNA precursors in pellets during lysate preparation revealed that 63 ± 9% of 7-qD1-16444 and 77 ± 4% of 17-qA3.3-26735 precursors were present in soluble lysates, as were 65 ± 13% of Mcm5 mRNA and 75 ± 1% of 12S rRNA. Each data point was overlaid as dot plots. c, Immunohistochemical staining on 6-μm testis sections from Ddx4-cre:RiboTag mice (left 2 panels) and control littermates (without Ddx4-cre, right 2 panels) using anti-HA (upper 2 panels) and mouse IgG isotype control (bottom 2 panels). HA expression in Ddx4-cre:RiboTag mice (upper left) was most prominent in the cytosol of spermatocytes (marked with arrow), but not in Sertoli cells (marked with arrow). No staining was seen in RiboTag control testis (upper right) or sections probed using mouse IgG isotype control as the primary antibody (bottom two panels). Scale bar, 50 μm. d, Immunostaining of testis cryosections from Neurog3-cre:RiboTag mice (left) and from control littermates (without Neurog3-cre, right) using anti-HA, anti-CRE, DAPI, and merged (top to bottom). Scale bar, 75 μm. e, Schematic of affinity purification procedure for ribosome-associated RNAs. In vitro transcribed Renilla RNA was used as a spike-in for normalization. Statistical Source Data are provided in Source Data Extended Data Fig. 2.

Source data

Extended Data Fig. 3 Ribosomes directly bind to pachytene piRNA precursors.

a, Schematic of Ribo-seq library construction. PNK: T4 Polynucleotide Kinase b, Northern hybridization of a piRNA from a pachytene piRNA precursor, 7-qD1-16444. Total RNAs from equal amounts of testis lysates treated with or without RNaseA&T1 were resolved by electrophoresis and detected by northern hybridization. The intensity of probe signals has been confirmed to be linear with respect to the amount of RNA (r > 0.99). c, Length histogram of RPFs from mRNA protein coding regions. d, Fragment length analysis plot of total RPF reads per transcript and FLOSS relative to the nuclear coding sequence average from wild-type testes. Fragment Length Organization Similarity Score (FLOSS) was computed as previously described23. For different raw read counts, to detect extremely large FLOSS values, outlier cutoffs were calculated using Tukey’s fences method and smoothed using LOESS regression. The purple curve shows the smoothed cutoff line. piRNAs were downsampled to match the read number of RPFs from piRNA precursors before calculating FLOSS. Statistical Source Data are provided in Source Data Extended Data Fig. 3. Unprocessed blots are provided in Source Data Extended Data Figure 3.

Source data

Extended Data Fig. 4 RPF signals on piRNA precursors respond to translation inhibitors.

a, Examples of pachytene piRNA genes with single uORF (two on the left), and pachytene piRNA genes with multiple uORF (one on the right). Browser views of piRNA precursor loci with normalized reads of RPFs and piRNAs. Grey shades highlight the uORF regions. Ppm, parts per million. b, A254 absorbance profile of testis lysates from adult mice with (blue) and without (black) harringtonine treatment following separation in 15% to 60% sucrose density gradients. Harr, harringtonine. c, Aggregated data for RPF abundance on pachytene expressed mRNAs from untreated testis (top), and from harringtonine treated testis (bottom) across 5´UTRs, ORFs, and 3´-UTRs of pachytene-expressed mRNAs. The x-axis shows the median length of these regions, and the y-axis represents the 10% trimmed mean of relative abundance. d, Cumulative distribution of Z scores comparing lengths of the longest ORFs from mRNAs (black line), lncRNAs (grey line), and pachytene piRNA precursors (pre-piRNAs) (purple line) with the length of the predicted longest ORF from the random shuffled background. Given a sequence X, its nucleotides were shuffled to generate a sequence Y. The length N of the longest ORF in Y was counted. After repeating this procedure 10,000 times, the Empirical Null Distribution of ORF lengths conditioned on the nucleotide distribution of X was obtained. The Z score of the ORF length of X was calculated based on this empirical null distribution. The dashed line denotes a Z-score of 2.58, which corresponds to p = 0.01 according to a two-side Z-test. The 29 ORFs from piRNA precursor transcripts with a Z score > 2.58 were from transposons embedded in the transcripts. e, Length histograms of total testis small RNAs from Control (Mov10l1CKO/∆, left) and Mov10l1 CKO (Mov10l1CKO/∆ Neurog3-cre, right). Statistical Source Data are provided in Source Data Extended Data Fig. 4.

Source data

Extended Data Fig. 5 uORFs on the piRNA precursors recruit ribosomes through canonical translation initiation.

a, Boxplots of RPF abundance at each start codon relative to the median density across the gene with and without harringtonine (Harr) treatment. The occupancy at a codon 20, 40, and 60 positions downstream of the start codon is depicted as a control. Sample size n = 100 pachytene piRNA precursors. Experiments have been repeated from two independent biological samples and mean is used for plotting. p value was determined by two-side paired Wilcoxon signed-rank test. b, Histogram of the distance from the 5´-cap to the start codon. AUG start sites typically fall in the first few hundred nt of transcripts. c, Sequence logos depicting the KOZAK consensus sequences. The start codons of single-ORF transcripts displayed weaker KOZAK consensus sequence with the critical A at the -3 position and the critical G at the +4 position. The A at the AUG codon being the +1. The start codons of the first ORFs for multi-ORF transcripts lack the critical A at the -3 position and the critical G at the +4 position. d, Boxplots of the mean PhastCons score72 (probability that each nucleotide belongs to a conserved element) of the genomic regions of putative 5´-UTR, uORF, and UDR of pachytene piRNA precursors, and 5´-UTR, ORF, and 3´-UTR of pachytene expressed mRNAs among placental mammals. Sample size n = 100 pachytene piRNA precursors; Sample size n =115 pachytene expressed mRNAs. e, Scatterplots of RPF abundance in ORFs relative to the overall abundance on the transcript from adult wild-type testes. f, A254 absorbance profile of testis lysates from Flag-knockin mice following separation in 15% to 60% sucrose density gradients. Western blot analysis of the FLAG-tagged micropeptide from testes of flag-knockin mice with anti-FLAG antibodies. Scheme of the flag-knockin mice was listed on the right. Experiments have been repeated for two times independently with similar results. g, Immunolabeling of squashed pachytene spermatocytes from flag-knockin mice and from control mice. Three examples of each genotype were shown using anti-FLAG, DAPI, and merged. Statistical Source Data are provided in Source Data Extended Data Fig. 5. Unprocessed blots are provided in Source Data Extended Data Figure 5.

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Extended Data Fig. 6 UDR RPFs are bona fide ribosome footprints.

a, Boxplots of the change in ribosome density (upper) or piRNA abundance (lower) per gene in Mov10l1 mutants (Mov10l1CKO/∆ Neurog3-cre, lower) compared to littermate controls (Mov10l1CKO/∆, upper) in testes. Experiments in a and f have been repeated for three biological replicates and mean is used for plotting. Sample sizes in a and f, n = 100 pachytene piRNA precursors; n =115 pachytene expressed mRNAs. b, Aggregated data for RPF abundance on mRNAs (10% trimmed mean). c, Browser views of piRNA precursor loci with normalized degradome reads. No degradome peak around stop codon was observed. d, Aggregated data for RNA-seq abundance on pachytene piRNA precursors (10% trimmed mean). e, Western blot analyses of RPL5 (Ribosomal large subunit component), RPS6 (Ribosomal small subunit component), and MOV10L1. Experiments have been repeated for two times independently with similar results. f, Boxplots of the change in ribosome density (upper) or piRNA abundance (lower) per gene in Tdrd5 mutants compared to littermate controls in testes. The piRNAs were analyzed using publicly available datasets32. g, Metagene analysis of piRNA abundance from 50 nt upstream stop codon to 1,000 nt downstream stop codon. h, Schematic of affinity purification of germ-line specific RPFs. i, Length histograms of anti-HA immunoprecipitated RPFs from uORF (top) and UDR (bottom) pachytene piRNA precursors in testis. j, A254 absorbance profile of 15% to 60% sucrose density gradients of adult mouse testis lysed in conventional lysis conditions in which mitochondria were disrupted. Normal salt (blue) and high salt (grey). The substantial decrease in the monosome peak with high salt treatment confirms that the high salt buffer can dissociate translation-inactive ribosomes73. k, Aggregated data for RPF abundance on mRNAs (10% trimmed mean) (normal salt, upper, and high salt, lower). The high salt treatment releases the ribosomes at the stop codon of mRNAs as these terminated ribosomes have released their nascent polypeptides. Statistical Source Data are provided in Source Data Extended Data Fig. 6. Unprocessed blots are provided in Source Data Extended Data Figure 6.

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Extended Data Fig. 7 Ribosomes bind piRNA precursors at the future piRNA sites.

a, Boxplots of distance spectra of 5´-ends of anti-HA immunoprecipitated RPFs from adult testis that overlap simulated sequences. The 5´-end overlap analyses between RPFs and simulated sequences were computed 10,000 times (sample size n = 10,000 5´-end overlap analyses). b, Boxplots of distance spectra of 5´-ends of sheared RNA fragments that overlap piRNAs of adults. The sheared RNA fragments were randomly downsampled 10,000 times to match the read numbers of RPFs immunoprecipitated from adult testis (sample size n = 10,000 5´-end overlap analyses). c, Distance spectrum of 5´-ends of RPFs from a publicly available dataset42 that overlap piRNAs from our dataset. We believe the higher accessibility of RNase I (used in42) compared to RNaseT1&A (used in this study) sometimes clipped one nucleotide from the 5´-end and resulted in a peak at the second position. Experiments have been repeated from two independent biological samples and mean is used for plotting. Higher accessibility of RNase I compared to Xrn1 has been reported66. d, Boxplots of the fraction of RPF with a 5´ upstream A in total RPFs per transcript. Mean of three biological replicates of anti-HA immunoprecipitated RPFs from adult testis were used. e, Distance spectra of 5´-ends of anti-HA immunoprecipitated degradome reads that overlap piRNAs in adults. f, Scatterplots of piRNA abundance versus 5´P RNA abundance (anti-HA immunoprecipitated degradome reads) from adults per pachytene piRNA precursors (sample size n = 100 pachytene piRNA precursors). Rpkm, reads per kilobase pair per million reads mapped to the genome. The partial Spearman’s correlation efficient and the corresponding p-value based on two-sided Spearman’s correlation test were listed, controlling for the piRNA precursor level measured by RNA-seq. Statistical Source Data are provided in Source Data Extended Data Fig. 7.

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Extended Data Fig. 8 Phased cleavages occur on ribosome-bound piRNA precursors.

a, Distance spectrum of 5´-ends of anti-HA immunoprecipitated RPFs from uORFs (left) and from UDRs (right) that overlap 3´-ends of MILI-bound pre-piRNAs in Pnldc1 mutant testis (three biological replicates). Data are mean ± standard deviation. b, Distance spectrum of 5´-ends of anti-HA immunoprecipitated RPFs from uORFs (left) and from UDRs (right) that overlap 3´-ends of MIWI-bound pre-piRNAs in Pnldc1 mutant testis (three biological replicates)6. Data are mean ± standard deviation. c, Distance spectrum of 5´-ends of piRNAs from uORFs (left) and from UDRs (right) that overlap 3´-ends of pre-piRNAs in Pnldc1 mutant testis6. Experiments have been repeated for two biological replicates and mean is used for plotting. d, Sequence logos depicting the nucleotide bias at 3´-ends and 1 nt downstream of the 3´-ends of total pre-piRNA species (top), MILI-bound pre-piRNA species (middle), and MIWI-bound pre-piRNA species (bottom) from Pnldc1 mutants. Statistical Source Data are provided in Source Data Extended Data Fig. 8.

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Extended Data Fig. 9 Dual mode interactions between ribosomes and piRNA precursors at uORFs and UDRs.

a, Schematic of affinity purification procedure for ribosome-associated RNAs. Renilla RNA was used as a negative control. Firefly RNA was used as a spike-in for normalization. b, RNA enrichment in affinity-purified ribosomes versus control monosome IP from littermates (without Cre). The abundance of transcripts was quantified using qRT-PCR with three independent biological samples (mean ± standard deviation; n = 3 independent experiments). *p < 0.05 by one-side Student’s t-test comparing the RNAs in vivo with the in vitro-added negative control. Each data point was overlaid as dot plots. c, Histology of testis sections with (right) and without (left) puromycin treatment harvested at different time points. Experiments have been repeated for two times independently with similar results. Puromycin did not disrupt seminiferous tubule structures or lead to visible morphological changes up to 8 hours after intra-testicular injection. Scale bar, 50 μm. d, Scatterplot of mRNA abundance between untreated and puromycin treated adult mouse testis. Each data point represents an mRNA. Sample size n = 8,623 mRNAs. Tpm, transcripts per million. Pearson’s correlation and the corresponding p-value based on two-sided Pearson’s correlation test is listed. Statistical Source Data are provided in Source Data Extended Data Fig. 9.

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Extended Data Fig. 10 Ribosome-mediated piRNA biogenesis is conserved.

a, Length histograms of RPFs from piRNA producing loci (upper) and of piRNAs (bottom) in the testis of roosters (left) and green lizards (right). Ppm, parts per million. b, Fragment length analysis plot of total RPF reads per transcript and FLOSS relative to the nuclear coding sequence average from testes of roosters (upper) and green lizards (bottom). For different raw read counts, to detect extremely large FLOSS values, outlier cutoffs were calculated using Tukey’s fences method and smoothed using LOESS regression. piRNAs were downsampled to match the read number of RPFs from piRNA precursors before calculating FLOSS. c, Sequence logos depicting the nucleotide bias at 5´-ends and 1 nt upstream of the 5´-ends of total RPF species (top) and RPF species with a 5´-upstream A (bottom) from roosters and green lizards. d, Boxplots of the fraction of RPF with a 5´-upstream A in total RPFs per transcript in roosters (upper) and green lizards (lower). Sample size n = 9,461 rooster mRNAs; sample size n = 95 rooster piRNA clusters. Sample size n = 14,680 lizard mRNAs; sample size n = 94 lizard piRNA clusters. e, Boxplots of distance spectra of 5´-ends of anti-HA immunoprecipitated RPFs from adult testis that overlap simulated sequences. The 5´-end overlap analyses between RPFs and simulated sequences were computed 10,000 times for roosters (upper) and green lizards (lower) (sample size n =10,000 5´-end overlap analyses). Statistical Source Data are provided in Source Data Extended Data Fig. 10.

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Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1: PCR primers. Supplementary Table 2: Pachytene piRNA precursors. Supplementary Table 3: Ribo-Seq data statistics. Supplementary Table 4: Small RNA-Seq data statistics. Supplementary Table 5: RNA-Seq data statistics. Supplementary Table 6: Degradome-Seq data statistics.

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Sun, Y.H., Zhu, J., Xie, L.H. et al. Ribosomes guide pachytene piRNA formation on long intergenic piRNA precursors. Nat Cell Biol 22, 200–212 (2020). https://doi.org/10.1038/s41556-019-0457-4

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