The mouse cytosine-5 RNA methyltransferase NSun2 is a component of the chromatoid body and required for testis differentiation

Abstract

Posttranscriptional regulatory mechanisms are crucial for protein synthesis during spermatogenesis and are often organized by the chromatoid body. Here, we identify the RNA methyltransferase NSun2 as a novel component of the chromatoid body and, further, show that NSun2 is essential for germ cell differentiation in the mouse testis. In NSun2-depleted testes, genes encoding Ddx4, Miwi, and Tudor domain-containing (Tdr) proteins are repressed, indicating that RNA-processing and posttranscriptional pathways are impaired. Loss of NSun2 specifically blocked meiotic progression of germ cells into the pachytene stage, as spermatogonial and Sertoli cells were unaffected in knockout mice. We observed the same phenotype when we simultaneously deleted NSun2 and Dnmt2, the only other cytosine-5 RNA methyltransferase characterized to date, indicating that Dnmt2 was not functionally redundant with NSun2 in spermatogonial stem cells or Sertoli cells. Specific NSun2- and Dnmt2-methylated tRNAs decreased in abundance when both methyltransferases were deleted, suggesting that RNA methylation pathways play an essential role in male germ cell differentiation.

Fig 1

Testis size is reduced and sperm differentiation is blocked in NSun2^−/− mice [Nsun2^{tm1a(EUCOMM)Wtsi}]. (A) A testis from an NSun2^−/− mouse (−/−) is smaller than one from a wild-type littermate (wt). (B) Quantification of data from panel A. Error bars, standard deviations (SD). (C and D) Hematoxylin and eosin staining of testis sections from wild-type and NSun2^−/− mice. The arrows indicate the absence of elongated sperm in the NSun2^−/− testis. ps, primary spermatocytes; rs, round spermatids; es, elongated spermatids. (E) qPCR confirming repression of NSun2, Tnp2, and Prm1 RNAs in the NSun2^−/− testis. (F to I) Hematoxylin and eosin staining of sections from wt and NSun2^−/− testes at the indicated time points.

Fig 2

Germ cells arrest at the leptotene and zygotene stages of prophase I at 6 weeks of age. (A to E) Surface-spread germ cells isolated from wild-type (A, C, and E) and NSun2^−/− (B and D) testes labeled for Sycp3 (green) and γH2X (red). DAPI (4′,6-diamidino-2-phenylindole) (blue) served as a counterstain. (F) Quantification of germ cells at the indicated stages of early prophase. The error bars indicate SD.

Fig 3

Factors of the RNA-processing machinery are repressed in the NSun2^−/− testis. (A) Overview of murine spermatogenesis in days (d). The blue lines indicate the appearance of the indicated germ cells, and the dotted lines mark germ cells missing in the NSun2^−/− testis. The red dotted lines with asterisks mark the time points when samples for RNA expression arrays were taken. (B) Heat map showing significantly changed genes (probes) at an FDR of <0.01 in wild-type versus NSun2^−/− testes at P15 in six biological replicates (A to F). (C) Fold change in expression of RNAs (log₂FC) in NSun2^−/− versus wild-type (wt) testes at postnatal day 15 (15d). (D) Fold change in expression of Miwi, Trd5, and Trd6 RNAs in NSun^−/− versus wt testes at postnatal day 15 (−/− 15d), NSun2^−/− versus wt testes at postnatal day 49 (−/− 49d), and testes of wt mice at 49 days versus 15 days (Wt 49d). (E and F) qRT-PCR determining RNA expression of NSun2 and Miwi RNAs (E) and selected transposons (F) in NSun2^−/− and wt testes. The error bars indicate SD. (G) Venn diagram showing the overlap of significantly repressed genes in NSun2^−/− versus wt testes at 49 days (blue) and significantly upregulated genes in wt testes at 49 versus 15 days (orange). (H) Heat map of the 602 genes (probes) shown in panel G in wild-type and NSun2^−/− testes at P15 and P49 in six biological replicates (A to F). (I) Fold change of the 27 most repressed RNAs in the NSun2^−/− testis at 49 compared to 15 days.

Fig 4

Localization of NSun2 in adult testis. (A and B) Immunofluorescence labeling of NSun2 (NSun2p; green) in wild-type (wt) (A) and NSun2^−/− (−/−) (B) testes. (C and D) Higher magnification of wt (C) and NSun2^−/− (D) testes showing that NSun2 protein is lost in round spermatids. (E and F) Colocalization of NSun2 (green) and Ddx25 (red) in wt (E) and NSun2^−/− (F) testes. Nuclei were counterstained in blue (DAPI) (A to F). The arrows (A, C, and E) point to NSun2 staining in round spermatids.

Fig 5

NSun2 localizes to the chromatoid body. (A and B) NSun2 (green) colocalizes with Ddx4 (red) in round spermatids using two different antibodies for NSun2 (Meth2) (A) and 20854-AP (NSun2p) (B). (C) Coimmunoprecipitation of NSun2 with Ddx4 (top) and Maelstrom (bottom) in wild-type and NSun2^−/− testes. Preimmune serum served as the negative control. (D and E) NSun2 (green) does not colocalize with Ddx4 (red) at the intermitochondrial cement in spermatocytes (D) or sp56 (red) at the acrosomal matrix in spermatids (E). (i to iv) Higher magnifications of the respective boxed areas in panels A, B, D, and E). (A, B, D, and E) Nuclei were counterstained in blue (DAPI).

Fig 6

Deletion of NSun2 and Dnmt2 reduced tRNA stability in testes at P15 and 3 months of age. (A) RNA expression levels of NSun2 (left) and Dnmt2 (right) at the indicated postnatal days. The error bars indicate SD. (B) Protein expression levels of NSun2 (top) and Dnmt2 (middle) at the indicated postnatal days (P) in wild-type and NSun2-Dnmt2 double knockouts (DKO). GAPDH served as a loading control (bottom). (C) Northern blot analyses of NSun2- and Dnmt2-methylated tRNAs at P15 and 3 months of age. 5S RNA served as a loading control.

Fig 7

Cytosine-5 methylation is dispensable in spermatogonial and Sertoli cells. (A to D) Immunofluorescence labeling of Mili (green) (A), Miwi (green) (B), Ki67 (green) (C), and Gata4 (sc-1237) (red) and Dmrt1 (green) to mark Sertoli and spermatogonial stem cells, respectively (D), in wild-type testis or upon deletion of Dnmt2 (Dnmt2^−/−), NSun2 (NSun2^−/−), or both Dnmt2 and NSun2 (DKO). DAPI served as a nuclear counterstain.