Sam68 is a KH-type RNA-binding protein involved in several steps of RNA metabolism with potential implications in cell differentiation and cancer. However, its physiological roles are still poorly understood. Herein, we show that Sam68−/− male mice are infertile and display several defects in spermatogenesis, demonstrating an essential role for Sam68 in male fertility. Sam68−/− mice produce few spermatozoa, which display dramatic motility defects and are unable to fertilize eggs. Expression of a subset of messenger mRNAs (mRNAs) is affected in the testis of knockout mice. Interestingly, Sam68 is associated with polyadenylated mRNAs in the cytoplasm during the meiotic divisions and in round spermatids, when it interacts with the translational machinery. We show that Sam68 is required for polysomal recruitment of specific mRNAs and for accumulation of the corresponding proteins in germ cells and in a heterologous system. These observations demonstrate a novel role for Sam68 in mRNA translation and highlight its essential requirement for the development of a functional male gamete.

We report that mice ablated for the Sam68 RNA-binding protein exhibit a lean phenotype as a result of increased energy expenditure, decreased commitment to early adipocyte progenitors, and defects in adipogenic differentiation. The Sam68(-/-) mice were protected from obesity, insulin resistance, and glucose intolerance induced with a high-fat diet. To identify the alternative splice events regulated by Sam68, genome-wide exon usage profiling in white adipose tissue was performed. Adipocytes from Sam68(-/-) mice retained intron 5 within the mTOR transcript introducing a premature termination codon, leading to an unstable mRNA. Consequently, Sam68-depleted cells had reduced mTOR levels resulting in lower levels of insulin-stimulated S6 and Akt phosphorylation leading to defects in adipogenesis, and this defect was rescued by the exogenous expression of full-length mTOR. Sam68 bound intronic splice elements within mTOR intron 5 required for the usage of the 5' splice site. We propose that Sam68 regulates alternative splicing during adipogenesis.

The Src substrate associated in mitosis of 68 kDa (Sam68) is a KH-type RNA binding protein that has been shown to regulate several aspects of RNA metabolism; however, its physiologic role has remained elusive. Herein we report the generation of Sam68-null mice by homologous recombination. Aged Sam68−/− mice preserved their bone mass, in sharp contrast with 12-month-old wild-type littermates in which bone mass was decreased up to approximately 75%. In fact, the bone volume of the 12-month-old Sam68−/− mice was virtually indistinguishable from that of 4-month-old wild-type or Sam68−/− mice. Sam68−/− bone marrow stromal cells had a differentiation advantage for the osteogenic pathway. Moreover, the knockdown of Sam68 using short hairpin RNA in the embryonic mesenchymal multipotential progenitor C3H10T1/2 cells resulted in more pronounced expression of the mature osteoblast marker osteocalcin when differentiation was induced with bone morphogenetic protein-2. Cultures of mouse embryo fibroblasts generated from Sam68+/+ and Sam68−/− littermates were induced to differentiate into adipocytes with culture medium containing pioglitazone and the Sam68−/− mouse embryo fibroblasts shown to have impaired adipocyte differentiation. Furthermore, in vivo it was shown that sections of bone from 12-month-old Sam68−/− mice had few marrow adipocytes compared with their age-matched wild-type littermate controls, which exhibited fatty bone marrow. Our findings identify endogenous Sam68 as a positive regulator of adipocyte differentiation and a negative regulator of osteoblast differentiation, which is consistent with Sam68 being a modulator of bone marrow mesenchymal cell differentiation, and hence bone metabolism, in aged mice.

Regeneration of skeletal muscle requires the continued presence of quiescent muscle stem cells (satellite cells), which become activated in response to injury. Here, we report that whole-body protein arginine methyltransferase PRMT7(-/-) adult mice and mice conditionally lacking PRMT7 in satellite cells using Pax7-CreERT2 both display a significant reduction in satellite cell function, leading to defects in regenerative capacity upon muscle injury. We show that PRMT7 is preferentially expressed in activated satellite cells and, interestingly, PRMT7-deficient satellite cells undergo cell-cycle arrest and premature cellular senescence. These defects underlie poor satellite cell stem cell capacity to regenerate muscle and self-renew after injury. PRMT7-deficient satellite cells express elevated levels of the CDK inhibitor p21CIP1 and low levels of its repressor, DNMT3b. Restoration of DNMT3b in PRMT7-deficient cells rescues PRMT7-mediated senescence. Our findings define PRMT7 as a regulator of the DNMT3b/p21 axis required to maintain muscle stem cell regenerative capacity.

Arginine methylation of histones is a well-known regulator of gene expression. Protein arginine methyltransferase 6 (PRMT6) has been shown to function as a transcriptional repressor by methylating the histone H3 arginine 2 [H3R2(me2a)] repressive mark; however, few targets are known. To define the physiological role of PRMT6 and to identify its targets, we generated PRMT6−/− mouse embryo fibroblasts (MEFs). We observed that early passage PRMT6−/− MEFs had growth defects and exhibited the hallmarks of cellular senescence. PRMT6−/− MEFs displayed high transcriptional levels of p53 and its targets, p21 and PML. Generation of PRMT6−/−; p53−/− MEFs prevented the premature senescence, suggesting that the induction of senescence is p53-dependent. Using chromatin immunoprecipitation assays, we observed an enrichment of PRMT6 and H3R2(me2a) within the upstream region of Trp53. The PRMT6 association and the H3R2(me2a) mark were lost in PRMT6−/− MEFs and an increase in the H3K4(me3) activator mark was observed. Our findings define a new regulator of p53 transcriptional regulation and define a role for PRMT6 and arginine methylation in cellular senescence.

The quaking (qkI) gene encodes 3 major alternatively spliced isoforms that contain unique sequences at their C termini dictating their cellular localization. QKI-5 is predominantly nuclear, whereas QKI-6 is distributed throughout the cell and QKI-7 is cytoplasmic. The QKI isoforms are sequence-specific RNA binding proteins expressed mainly in glial cells modulating RNA splicing, export, and stability. Herein, we identify a new role for the QKI proteins in the regulation of microRNA (miRNA) processing. We observed that small interfering RNA (siRNA)-mediated QKI depletion of U343 glioblastoma cells leads to a robust increase in miR-7 expression. The processing from primary to mature miR-7 was inhibited in the presence QKI-5 and QKI-6 but not QKI-7, suggesting that the nuclear localization plays an important role in the regulation of miR-7 expression. The primary miR-7-1 was bound by the QKI isoforms in a QKI response element (QRE)-specific manner. We observed that the pri-miR-7-1 RNA was tightly bound to Drosha in the presence of the QKI isoforms, and this association was not observed in siRNA-mediated QKI or Drosha-depleted U343 glioblastoma cells. Moreover, the presence of the QKI isoforms led to an increase presence of pri-miR-7 in nuclear foci, suggesting that pri-miR-7-1 is retained in the nucleus by the QKI isoforms. miR-7 is known to target the epidermal growth factor (EGF) receptor (EGFR) 3= untranslated region (3=-UTR), and indeed, QKI-deficient U343 cells had reduced EGFR expression and decreased ERK activation in response to EGF. Elevated levels of miR-7 are associated with cell cycle arrest, and it was observed that QKI-deficient U343 that harbor elevated levels of miR-7 exhibited defects in cell proliferation that were partially rescued by the addition of a miR-7 inhibitor. These findings suggest that the QKI isoforms regulate glial cell function and proliferation by regulating the processing of certain miRNAs.

The Src-associated substrate in mitosis Sam68 is a KH type RNA-binding protein known to be a substrate of numerous tyrosine kinases, and often referred to as a STAR (signal transduction activator of RNA) protein. Herein, we observed that Sam68-null mice display mammary gland and the uterine development defects. Moreover, we report that Sam68 haploinsufficiency impedes mammary tumor onset in vivo driven by the potent mammary-targeted polyoma middle T-antigen (MMTV-PyMT) oncogene. The effect was cell autonomous as the Sam68 knockdown in PyMT-transformed cell lines also delayed tumorigenesis and metastasis formation in nude mice. Interestingly, tumor extracts isolated from PyMT/Sam68(+/-) mice compared with PyMT/Sam68(+/+) mice contained activated Src and FAK kinases. These findings suggest that Sam68 may be a modulator of tyrosine kinase activity in vivo and a signaling requirement for mammary tumorigenesis and metastasis.

The MRE11/RAD50/NBS1 complex is the primary sensor rapidly recruited to DNA double-strand breaks (DSBs). MRE11 is known to be arginine methylated by PRMT1 within its glycine-arginine-rich (GAR) motif. In this study, we report a mouse knock-in allele of Mre11 that substitutes the arginines with lysines in the GAR motif and generates the MRE11 RK protein devoid of methylated arginines. The Mre11 RK/RK mice were hypersensitive to γ-irradiation (IR) and the cells from these mice displayed cell cycle checkpoint defects and chromosome instability. Moreover, the Mre11 RK/RK MEFs exhibited ATR/CHK1 signaling defects and impairment in the recruitment of RPA and RAD51 to the damaged sites. The M RK RN complex formed and localized to the sites of DNA damage and normally activated the ATM pathway in response to IR. The M RK RN complex exhibited exonuclease and DNA-binding defects in vitro responsible for the impaired DNA end resection and ATR activation observed in vivo in response to IR. Our findings provide genetic evidence for the critical role of the MRE11 GAR motif in DSB repair, and demonstrate a mechanistic link between post-translational modifications at the MRE11 GAR motif and DSB processing, as well as the ATR/ CHK1 checkpoint signaling.