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Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals

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Science  22 Jan 2016:
Vol. 351, Issue 6271, pp. 391-396
DOI: 10.1126/science.aad6780
  • Fig. 1 Dietary effects on sRNAs in sperm.

    (A) Size distribution of sequencing reads for cauda sperm sRNAs. (B to D) 5′ fragments of tRNA are shown schematically, with arrowheads indicating dominant 3′ ends. (E) Dietary effects on sperm sRNA content. The scatter plot shows RNA abundance (in parts per million) for sperm isolated from control animals (x axis, log10) versus low-protein sperm (y axis), with various RNA classes indicated. Multiple points for tRFs result from sequence differences between genes encoding a given tRNA isoacceptor. (F) Heat map showing RNAs responding to diet across eight paired sperm samples.

  • Fig. 2 Cleavage of tRNA occurs predominantly in the epididymis.

    (A) Sperm RNA payload diverges dramatically from testicular RNA. The scatter plot shows sRNAs in the testis versus sperm, as in Fig. 1E. (B) Schematic of the epididymis. Sperm exiting the testis enter the proximal (caput) epididymis, then proceed distally to the corpus and cauda epididymis, and exit via the vas deferens. (C) tRFs are primarily generated in the epididymis. Northern blots are shown for 5′ ends of tRNA-Gly-GCC or tRNA-Val-CAC on RNA extracted from the testis, caput epididymis, and cauda epididymis. Arrows indicate ~30- to 34-nt 5′ tRFs. 5S RNA served as a loading control. (D) Pie charts showing the percentage of sRNAs mapping to the indicated features. (E) Scatter plot of sRNA abundance for sperm versus epididymosomes. Sperm-enriched RNAs include piRNAs and fragments of mRNAs involved in spermatogenesis (e.g., Prm1) and represent RNAs synthesized during testicular spermatogenesis.

  • Fig. 3 Changes in sperm tRF payload during epididymal transit.

    (A) Proximal-distal biases observed for RNAs in the epididymis are recapitulated in sperm samples. (B) Proximal-distal biases for tRFs, aggregated by anticodon, in the epididymis and sperm. (C) TaqMan assay of the indicated tRFs in caput sperm and reconstituted sperm, showing gain of tRFs relative to let-7 (t test, P = 0.05 for Gly-GCC and 0.004 for Val-CAC). (D) Deep sequencing of reconstituted sperm. tRFs are aggregated by codon and normalized to levels of tRF-Glu-CTC. Caput versus cauda differences were broadly recapitulated in reconstitutions, with tRFs such as tRF-Val-CAC being delivered to caput sperm via fusion with the cauda epididymosomes.

  • Fig. 4 Regulation of MERVL-driven transcripts by tRF-Gly-GCC.

    (A) Scatter plot shows mRNA abundance in ES cells transfected with green fluorescent protein (GFP) small interfering RNA (siRNA), as compared with LNA antisense oligonucleotides to the 5′ end of tRF-Gly-GCC. (B) Effect of tRF-Gly-GCC inhibition on MERVL-regulated genes is isoacceptor-specific. Affymetrix data for ES cells transfected with LNA antisense oligos, relative to matched GFP controls, showing genes changing twofold in two or more samples. (C) RNA-seq data for ESCs transfected with GFP siRNA or anti-tRF-Gly-GCC. (D) Genomic context of tRF-Gly-GCC target genes, showing nearby MERVL LTRs. (E) Inhibition of tRF-Gly-GCC affects MERVL targets in embryos. Averaged single-embryo RNA-seq data for control (n = 28) or tRF-inhibited (n = 27) four-cell-stage (4C) embryos. Among twofold up-regulated genes, known MERVL targets (17) are indicated. (F) Single-embryo data for two MERVL targets. TPM, transcripts per million.

  • Fig. 5 Paternal dietary effects on preimplantation development.

    (A) Embryos generated by IVF were cultured for varying times and then subjected to single-embryo RNA-seq. (B) Single-embryo data for preimplantation embryos represented via principal components analysis: The first two principal components explain 74% of the data set variance. (C) Abundance of mRNA in two-cell embryos generated via IVF using control versus low-protein sperm (n = 41 C and 39 LP embryos). Cumulative distribution plots for tRF-Gly-GCC targets (P = 4.5 × 10−7, Kolmogorov-Smirnov test), other MERVL targets (17) (P = 2.5 × 10−13), and all remaining genes, showing the percentage of genes with the average log2(LP/C) indicated on the x axis. Low-protein embryos exhibit a significant shift to lower expression of MERVL targets. Bottom panels show individual embryo data for two targets. (D) Small RNAs isolated from control or low-protein cauda sperm were microinjected into control zygotes. RNA-seq (n = 42 C and 46 LP embryos) reveals down-regulation of tRF-Gly-GCC targets (P = 4.8 × 10−14) driven by low-protein RNAs. (E) Effects of synthetic tRF-Gly-GCC on two-cell gene regulation, showing significant (P = 0.0001) down-regulation of target genes in embryos injected with tRF-Gly-GCC (n = 26) versus GFP controls (n = 11). The inset shows effects of tRF-Glu-CTC (n = 6). (F) Effects of epididymal passage on embryonic gene regulation. Intact sperm isolated from the rete testis (n = 12) or cauda epididymis (n = 9) were injected into control oocytes, and mRNA abundance was analyzed as described above.

Supplementary Materials

  • Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals

    Upasna Sharma, Colin C. Conine, Jeremy M. Shea, Ana Boskovic, Alan G. Derr, Xin Y. Bing, Clemence Belleannee, Alper Kucukural, Ryan W. Serra, Fengyun Sun, Lina Song, Benjamin R. Carone, Emiliano P. Ricci, Xin Z. Li, Lucas Fauquier, Melissa J. Moore, Robert Sullivan, Craig C. Mello, Manuel Garber, Oliver J. Rando*

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S13
    • Captions for Tables S1 to S8
    • Full Reference List
    Table S1
    Additional Data table S1 (separate file) Complete small RNA dataset. All murine small RNA-Seq datasets. Rows give read counts mapping to rRNAs, tRNAs, repeatmasker consensus sequences, unique piRNA clusters, and microRNAs are all provided here. Reads mapping to Refseq are excluded in the interest of space, but are provided for tissue averages in Table S2.
    Table S2
    Additional Data table S2 (separate file) Small RNA abundance for all tissues. Small RNA data for all tissues are shown here normalized to ppm (excluding rRNAs). For each tissue, total reads were obtained by summing reads from each relevant library.
    Table S3
    Additional Data table S3 (separate file) Small RNA-Seq of B. taurus caput sperm reconstitutions. Data for four replicate sperm reconstitutions are shown, with counts of RNAs mapping to rRNAs, tRNAs, or microRNAs shown as indicated. Samples include purified caput sperm, mock-treated caput sperm (incubated at 37 °C), caput sperm incubated with cauda epididymosomes, cauda epididymosomes alone, and a single sample of cauda sperm.
    Table S4
    Additional Data table S4 (separate file) ES cell mRNA abundance. Affymetrix microarray data are shown for ES cells transfected with GFP knockdown siRNAs, or with an LNA antisense targeting the 5’ end of tRNA-Gly-GCC.
    Table S5
    Additional Data table S5 (separate file) tRF-Gly-GCC effects on translation in ES cells. RNA-Seq data and ribosome footprinting for ES cells subject to mock tranfection, GFP knockdown, or tRF-Gly-GCC inhibition (4 replicates each).
    Table S6
    Additional Data table S6 (separate file) tRF-Gly-GCC effects on 4-cell stage gene expression. Single embryo RNA-Seq data for 4-cell stage embryos. Zygotes generated via IVF were microinjected with H3.3-GFP mRNA, with or without an antisense oligo targeting tRF-Gly-GCC, then allowed to develop to the 4-cell stage and subject to single embryo RNA-Seq.
    Table S7
    Additional Data table S7 (separate file) Dietary effects on preimplantation gene regulation. Single-embryo RNA-Seq data for embryos at varying stages of development, generated using IVF with sperm obtained from male mice consuming the indicated diets.
    Table S8
    Additional Data table S8 (separate file) RNA effects on preimplantation gene regulation. Single-embryo RNA-Seq data for ICSI experiments, sperm small RNA injections, and tRF-Gly-GCC injections. All data are for late 2-cell stage embryos, and data are only shown for transcripts expressed at greater than 5 parts per million in a given experiment.

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