The Mouse Cytosine-5 RNA Methyltransferase NSun2 Is a Component of the Chromatoid Body and Required for Testis Differentiation

Abstract

dPosttranscriptional 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.

Novel RNA Modifications in the Nervous System: Form and Function

“…Gain-of-function experiments demonstrate that CDR1as acts as a natural miR-7 antagonist in neuronal tissues. Expression of CDR1as in zebrafish results in severe impairment of midbrain development, similar to miR-7 depletion, which indicates the ability of CDR1as to regulate miRNA-7 Hussain et al, 2013b; Gene silencing (Hussain et al, 2013b) Protein translation Stress response Differentiation and development (Blanco et al, 2011;Hussain et al, 2013c) Inosine ADARs (Nishikura, 2010) RNA editing (Slotkin and Nishikura, 2013) Generation of nongenomically encoded proteins (Rueter et al, 1995;Wahlstedt et al, 2009) circRNAs Unknown MicroRNA sponge (Hansen et al, 2013;Memczak et al, 2013) Modulation of gene expression levels (Memczak et al, 2013). Interestingly, miR-7 targets have been previously linked to Parkinson's disease etiology, and circCDR1as was shown to be strongly downregulated in the hippocampi of patients with Alzheimer's disease (Junn et al, 2009;Lukiw, 2013).…”

“…Functionally, m 5 C has been shown not only to affect degradation and stress-induced ribonuclease cleavage of tRNAs but also to change global protein translation (Alexandrov et al, 2006;Chow et al, 2007;Metodiev et al, 2014). In addition, hypomethylation of vRNAs alters their processing into microRNA-like RNAs (Hussain et al, 2013b). In rRNA, m 5 C is also thought to play a role in translation (Chow et al, 2007;Metodiev et al, 2014).…”

“…Although no gross phenotype has been observed in DNMT2-deficient mice or plants (Goll et al, 2006;, DNMT2 loss-offunction mutant flies show increased sensitivity to oxidative stress, and DNMT2 loss in zebrafish affects liver, retina, and brain development (Rai et al, 2007;Schaefer et al, 2010). Studies performed in NSUN2-deficient mice, flies, and cell lines, suggest roles for m 5 C RNA modification in cellular signaling, stem cell biology, tissue development, differentiation, and cancer Sakita-Suto et al, 2007;Hussain et al, 2009;Frye et al, 2010;Blanco et al, 2011;Abbasi-Moheb et al, 2012;Hussain et al, 2013c). NSUN2 is highly expressed during mouse embryogenesis and is specifically enriched in the brain (Blanco et al, 2011).…”

“…But how loss of this methylation causes the disease symptoms described above is not yet fully understood. Loss of tRNA methylation could be the main defect leading to these complex intellectual disorders because the vast majority of NSUN2 targets are tRNAs (Squires et al, 2012;Hussain et al, 2013b;Khoddami and Cairns, 2013). It is known that loss of DNMT2-mediated m 5 C methylation increases tRNA stress-induced cleavage in flies, and cleavage of tRNAs and repression of protein translation is a conserved response to several stress stimuli in eukaryotes (Thompson et al, 2008;Yamasaki et al, 2009;Emara et al, 2010;Schaefer et al, 2010;Spriggs et al, 2010;Ivanov et al, 2011;Gebetsberger et al, 2012;Sobala and Hutvagner, 2013).…”

Sperm tsRNAs and acquired metabolic disorders

“…The modified nucleoside, 5-methylcytidine, has been reported to protect tRNA Asp GTC , Val AAC and Gly GCC from endonucleolytic cleavage in Drosophila to maintain tRNA secondary structure (Schaefer et al 2010). RNA methyltransferases, DNA methyltransferase-2 and NSun2 can methylate cytosine to form m 5 C in tRNAs (Goll et al 2006, Hussain et al 2013. DNA methyltransferase-2 is required for the establishment and hereditary maintenance of Kit and Sox9 paramutation phenotypes (Kiani et al 2013).…”