GTSF1 accelerates target RNA cleavage by PIWI-clade Argonaute proteins

Abstract

Argonaute proteins use nucleic acid guides to find and bind specific DNA or RNA target sequences. Argonaute proteins have diverse biological functions and many retain their ancestral endoribonuclease activity, cleaving the phosphodiester bond between target nucleotides t10 and t11. In animals, the PIWI proteins-a specialized class of Argonaute proteins-use 21-35 nucleotide PIWI-interacting RNAs (piRNAs) to direct transposon silencing, protect the germline genome, and regulate gene expression during gametogenesis¹. The piRNA pathway is required for fertility in one or both sexes of nearly all animals. Both piRNA production and function require RNA cleavage catalysed by PIWI proteins. Spermatogenesis in mice and other placental mammals requires three distinct, developmentally regulated PIWI proteins: MIWI (PIWIL1), MILI (PIWIL2) and MIWI2^2-4 (PIWIL4). The piRNA-guided endoribonuclease activities of MIWI and MILI are essential for the production of functional sperm^5,6. piRNA-directed silencing in mice and insects also requires GTSF1, a PIWI-associated protein of unknown function^7-12. Here we report that GTSF1 potentiates the weak, intrinsic, piRNA-directed RNA cleavage activities of PIWI proteins, transforming them into efficient endoribonucleases. GTSF1 is thus an example of an auxiliary protein that potentiates the catalytic activity of an Argonaute protein.

Fig. 1. A component of mouse testis lysate potentiates piRNA-directed target RNA cleavage by MIWI.

a, Strategy for programming recombinant MIWI with synthetic piRNA. b, Top, representative denaturing polyacrylamide gel electrophoresis showing target RNA cleavage by MIWI piRISC with and without added testis lysate. Bottom, product formed as a function of time by MIWI piRISC (mean ± s.d., n = 3). Rate constants were determined by fitting the data to the burst-and-steady-state equation (Methods, equation (1)). ${\left[\mathrm{E}\right]}_{\mathrm{active}}^{\mathrm{apparent}}$, the apparent concentration of active MIWI piRISC estimated from data fitting. c, Target RNA cleavage by MIWI piRISC in the presence of lysate from either whole testis or FACS-purified germ cells. DS, diplotene spermatocytes; PS, pachytene spermatocytes; SpII, secondary spermatocytes; Spg, spermatogonia; Sptd, spermatids. For gel source data, see Supplementary Fig. 1.

Fig. 2. The testis protein GTSF1 potentiates target RNA cleavage by MIWI piRISC.

a, Scheme to purify the MIWI-potentiating factor from mouse testis lysate. P20, pellet; S20, supernatant. b, Target-cleavage assay to estimate the apparent molecular mass of the MIWI-potentiating activity by size-exclusion chromatography (SEC). Arrowheads indicate the peak concentration of the molecular mass standards, and their peak elution volumes. V₀, void volume; phenyl (1M), 1 M NaCl eluate from hydrophobic-interaction chromatography; SP 0.5M, 0.5 M NaCl eluate from cation-exchange chromatography. c, Schematic representing known attributes of GTSF1 and the corresponding properties of the MIWI-potentiating activity in testis lysate. IMAC, immobilized metal affinity chromatography. d, Strategy for creating knock-in mouse expressing 3×Flag–HA–GTSF1 (Gtsf1^Flag/Flag). F1, F2, R1 and R2 represent sequencing primers used to validate the insertion. The exon encodes an incomplete isoleucine codon, which is completed upon splicing. e, Western blotting showing immunodepletion of 3×Flag–HA–GTSF1 from secondary spermatocyte lysate using anti-Flag paramagnetic beads. I, input; S, supernatant; E, 3×Flag peptide eluate. f, Product formed by MIWI piRISC in the presence of the indicated components. Numbers below the gel indicate relative amount of product formed in each condition. For gel source data, see Supplementary Fig. 1.

Fig. 3. GTSF1 paralogues can distinguish between MIWI and MILI.

a, Representative denaturing polyacrylamide gel electrophoresis showing that purified GTSF1 recapitulates the effect of testis lysate on MIWI catalysis. b, Amount of product generated as a function of time (mean ± s.d., n = 3). Data were fit to the burst-and-steady-state equation. Data for MIWI piRISC alone are from Fig. 1. c, Product formed as a function of time by MIWI programmed with pi6 and pi9 piRNAs for two independent trials. The mean values of the two trials were fit to the burst-and-steady-state equation. d,e, Representative denaturing polyacrylamide gel images of the assay to test GTSF1 mutants and paralogues in target cleavage by MIWI (d) or MILI (e) piRISC. f, Product formed as a function of time (mean ± s.d., n = 3) fit to the burst-and-steady-state equation. g, Pre-steady-state first-order rate constants of target cleavage by MIWI piRISC in the presence of either wild-type GTSF1 or the PIWI-interacting mutant GTSF1(W98A/W107A/W112A) were plotted as a function of GTSF1 concentration. Data are mean ± s.d., n = 3. k_pot, maximum observable rate. h, Single-turnover rate of target cleavage by EfPiwi piRISC in the presence or absence of EmGtsf1 or yeast tRNA (mean ± s.d., n = 3). For gel source data, see Supplementary Fig. 1.

Fig. 4. Cleavage by MIWI piRISC is sensitive to the complementarity between the piRNA and target.

a, MIWI piRISC target cleavage in the presence of GTSF1 for targets with varying complementarity to synthetic piRNA guide. Top, multiple-turnover conditions; bottom, single-turnover conditions. All reactions contained saturating amounts of GTSF1. b, Target-cleavage assay using targets complementary to piRNA guide nucleotides g2–g16 or g2–g30 and MIWI or MILI loaded with piRNAs of the indicated lengths, with or without GTSF1 (mean ± s.d., n = 3). c, Absolute and relative pre-steady-state and steady-state rates of cleavage of the g2–g30 target by MIWI loaded with piRNAs of the indicated lengths in the presence of GTSF1 (mean ± s.d., n = 3), E_apparent, apparent active enzyme concentration estimated from fitting data to equation (1). For gel source data, see Supplementary Fig. 1.

Fig. 5. A model for the function of guide length and GTSF1 in target cleavage by PIWI proteins.

a, The energy of base pairing, estimated by standard nearest-neighbour methods versus pre-and steady-state rates of GTSF1-potentiated target cleavage (mean ± s.d., n = 3), directed by piRNAs of different lengths loaded into MIWI. The same target, which was fully complementary to each piRNA, was used in all experiments. b, piRNA-directed, MIWI-catalysed target cleavage is envisioned to require two sequential conformational changes: (1) a target-dependent conformational change in piRISC (E_PAZ) in which the piRNA 3′ end leaves the PAZ domain, allowing extensive base pairing of the piRNA with the target substrate (S); and (2) GTSF1-dependent conversion of this piRISC pre-catalytic state (E_C) to the fully competent catalytic state (E_C′). P, cleaved target products. K_d, dissociation constant; k_c and k_−c, rate constants for pre-catalytic state; k_on and k_off, rate constants for GTSF1 binding; k_chem, rate constant for the endoribonucleolytic chemistry step; k′_−c, rate constant for piRISC (EPAZ) regeneration. c, Proposed effects of different piRNA lengths, extent of guide–target complementarity and GTSF1 on the forward and reverse rates of the two conformational rearrangements. The wide, central cleft, as observed in the cryo-electron microscopy structure of E. fluviatilis Piwi-A, is envisioned to allow the central region of a 30 nt piRNA to be mobile and exposed to solvent when its 3′ end is secured to the PAZ domain (top right).

Extended Data Fig. 1. Recombinant MIWI programmed with guide piRNA cleaves complementary target RNA.

a, Purification of apo-MIWI and of MIWI loaded with a synthetic piRNA guide (piRISC). b, Coomassie-stained SDS-PAGE of purified apo-MIWI and MIWI piRISC. E1, E2: first and second eluates from sequential incubation of the paramagnetic beads with 3XFLAG peptide. E1 was used for the cleavage assay. c, Representative denaturing polyacrylamide gel of target cleavage assay using apo-MIWI or MIWI piRISC. A digital overexposure is shown below the gel. d, MIWI piRISC loaded with synthetic guides differing at their 5′ or 3′ termini was assayed for the ability to cleave a complementary target RNA. e, Northern blot to estimate the yield of purified MIWI piRISC. f, Silver-stained gel showing purified MIWI piRISC with and without an amino-terminal epitope tag. g, Target cleavage assay comparing the activity of piRISC with and without an amino-terminal epitope tag. Data were fit to Equation 1 (see Methods); error of fit (SD) is reported. h, Mass spectrometry was used to determine arginine methylation status for recombinant, affinity-purified MIWI (n = 3); arginine methylation status of endogenous MIWI from mouse testis is provided for comparison. Arginine position reports the amino acid number in endogenous mouse MIWI and in parentheses, the corresponding residue in the epitope-tagged recombinant protein. Only modified arginine residues detected in all three independent preparations are reported. N.D., not detected. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 2. The target-cleavage potentiating component in testis lysate component transiently interacts with MIWI.

a, Immobilized, unloaded apo-MIWI was preincubated with testis lysate, purified as depicted, and then assayed for target cleavage activity. b, Immobilized MIWI piRISC was preincubated with testis lysate. purified as depicted, and then assayed for target cleavage activity. A digital overexposure is shown below the gel. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 3. Biochemical properties of the MIWI potentiating component in testis lysate.

a, The MIWI-potentiating factor is specific to testis lysate. b, Testis lysate does not enhance target cleavage by purified mouse AGO2. c, The MIWI-potentiating factor is sensitive to alkylation by N-ethylmaleimide. d, Effect of divalent cation chelators on MIWI-potentiating activity. For 0 nM chelator, water was added for EDTA and EGTA and ethanol for 1,10-phenanthroline. e, Strategy to test metal rescue after incubation of testis lysate with divalent cation chelators. f, g, Metal rescue experiments were performed as in (e) using EDTA (f) or 1,10-phenanthroline (g). h, Fractionation of testis lysate using Zn²⁺ and Ni²⁺ immobilized metal affinity chromatography (IMAC). In c and f–h, a digital overexposure is shown below each gel. Numbers below the gel indicate relative product produced in each lane. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 4. Gtsf1, Gtsf1l, and Gtsf2 mRNA abundance and recombinant protein expression.

a, For each germ cell type, mRNA abundance (from publicly available data; see Methods) is reported as mean ± SD (n = 3) in molecules/cell. Spg: spermatogonia; SpI: primary spermatocytes; SpII: secondary spermatocytes; RS: round spermatids. b, Right: Coomassie-stained SDS-PAGE of purified, recombinant wild-type GTSF1, GTSF1 mutants, and wild-type GTSF1Like and GTSF2. Left: Oriole protein stain detection of purified, recombinant, C-terminal V5SBP-tagged GTSF1 orthologs. Mm: Mus musculus (mouse); Bm: Bombyx mori (silkmoth). Coomassie protein stain detection of purified, recombinant Ephydatia muelleri Gtsf1. Bottom: Clustal Omega amino acid sequence alignment of the three mouse GTSF1 paralogs. c, Pre-steady-state rates (k_burst) of target cleavage by MIWI and MILI determined by fitting the data in Fig. 3d–f (n = 3) to the burst-and-steady-state equation. LQ: limit of quantification. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 5. Efficient target cleavage by MIWI guided by endogenous piRNAs requires GTSF1.

Representative target cleavage assay using MIWI piRISC programmed with an abundant mouse pachytene piRNA antisense to the L1MC transposon (left) or targeting the Scpep1 mRNA (right). piRNA sequences are in orange; below each piRNA sequence, the target in the RNA sequence is in black. Scissors indicates the cleavage site of the target RNA. The last lane in each gel shows the extent of cleavage using the reciprocal, non-cognate target, incubated with piRISC for 240 min as a negative control. Where data points from the two trials overlap, jitter has been introduced for clarity. Curves show the fit of the burst-and-steady equation to the mean of the two trials. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 6. GTSF proteins are PIWI selective.

a, MIWI piRISC target cleavage assay in the presence of lysates from different animals. T.ni: Trichoplusia ni. Macaque: rhesus macaque. A digital overexposure is shown below the gel. b, Representative target cleavage assay using the indicated PIWI protein in the presence of different GTSF1 orthologs (100 nM). Numbers below the gel lanes report fraction target cleaved (mean ± SD; n = 3). c, Left: Percent identity of different GTSF1 orthologs Right: Unrooted tree of GTSF1 orthologs. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 7. Expression of GTSF1 paralogs in mouse, rat, rhesus macaque, and human whole testes.

Gtsf genes are syntenic in mice, rats, macaques, and humans. Primates, including humans, do not express GTSF2. RNA-seq from total testis RNA of the indicated animals sourced from previously published datasets (see Methods).

Extended Data Fig. 8. Complementarity and GTSF1 requirements for target cleavage by AGO2, MIWI, and MILI.

a, MIWI target cleavage ± GTSF1 using synthetic piRNA guide 1. b, Target cleavage by AGO2 siRISC compared with MIWI or MILI piRISC loaded with synthetic piRNA guide 1 or 2, for targets with complete (g2–g30) or partial (g2–g16) complementarity to the piRNA. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 9. Model of the free-energy landscape of GTSF1-induced potentiation of target cleavage by PIWI.

Two conformational changes in piRISC (E) are proposed to be required for efficient target cleavage catalyzed by PIWI proteins. E_PAZ: piRISC with the 3′ end of the piRNA bound to the PAZ domain; E_C: piRISC with the piRNA fully paired to the target RNA, i.e., the pre-catalytic conformation; E_C′: E_C in the catalytically competent conformation; S^‡: transition-state. For piRNAs of biologically relevant length, extensive complementarity is proposed to promote the first conformational change; GTSF1 is proposed to promote the second. Blue: energy barrier to catalysis without GTSF1, i.e., the spontaneous conversion of E_C to E_C′.