We have shown that Dart5-mediated methylation of Sm proteins is not essential for snRNP biogenesis. The results uncover a novel role for dart5 in specification of the germline and in spermatocyte maturation. Because disruption of both dart5 and valois causes the specific loss of sDMA-modified Sm proteins and studies in C. elegans show that Sm proteins are required for germ-granule localization, we propose that Sm protein methylation is a pivotal event in germ-cell development.

Small nuclear ribonucleoproteins (snRNPs) are core components of the spliceosome. The U1, U2, U4, and U5 snRNPs each contain a common set of seven Sm proteins. Three of these Sm proteins are posttranslationally modified to contain symmetric dimethylarginine (sDMA) residues within their C-terminal tails. However, the precise function of this modification in the snRNP biogenesis pathway is unclear. Several lines of evidence suggest that the methyltransferase protein arginine methyltransferase 5 (PRMT5) is responsible for sDMA modification of Sm proteins. We found that in human cells, PRMT5 and a newly discovered type II methyltransferase, PRMT7, are each required for Sm protein sDMA modification. Furthermore, we show that the two enzymes function nonredundantly in Sm protein methylation. Lastly, we provide in vivo evidence demonstrating that Sm protein sDMA modification is required for snRNP biogenesis in human cells.

BackgroundCoilin is the signature protein of the Cajal body, a conserved nuclear organelle involved in multiple aspects of small ribonucleoprotein (RNP) biogenesis. Coilin is required for Cajal body homeostasis in both plants and animals. Mice lacking coilin are viable when the mutation is crossed to an outbred strain but only partially viable when crossed to inbred lines.Methodology/Principal FindingsIn order to clarify this issue, we backcrossed the coilin deletion onto the C57BL6/J background for ten generations and then investigated the consequences of coilin removal on overall viability and reproductive success. We conclude that semi-lethal phenotype observed in mixed-background crosses is due to loss of the Coilin gene (or a very tightly-linked locus). Interestingly, coilin knockout embryos die relatively late in gestation, between E13.5 and birth. We show that the maternal contribution of coilin is not important for organismal viability. Importantly, coilin knockout mice display significant fertility and fecundity defects. Mutant males that escape the embryonic lethality display reduced testis size, however, both males and females contribute to the observed reduction in reproductive fitness.Conclusions/SignificanceThe evolutionary conservation of coilin from plants to animals suggests that the protein plays an important role, perhaps coordinating the activities of various RNA-processing machineries. Our observations are consistent with the idea that coilin functions to ensure robust organismal development, especially during periods of rapid growth.

Notch signaling is a highly conserved cell-cell communication pathway regulating normal development and tissue homeostasis. Aberrant Notch signaling represents an important oncogenic mechanism for T cell acute lymphoblastic leukemia (T-ALL), an aggressive subset of the most common malignant childhood cancer ALL. Therefore, understanding the molecular regulation of Notch signaling is critical to identifying new approaches to block aberrant Notch oncogenic activity. The family of three MAML transcriptional co-activators is crucial for Notch signaling activation. The prototypic member MAML1 is the major co-activator that regulates Notch oncogenic activities in leukemic cells. However, the molecular basis underlying MAML1 co-activator function that contributes to Notch signaling remains unclear. In this study, we performed proteomic studies and identified DDX5, an ATP-dependent DEAD-box RNA helicase, as a component of the MAML1 protein complex. DDX5 interacts with MAML1 in vitro and in vivo, and is associated with the endogenous NOTCH1 transcription activation complex in human T-ALL leukemic cells. Lentivirus-mediated shRNA knockdown of DDX5 resulted in decreased expression of Notch target genes, reduced cell proliferation, and increased apoptosis in cultured human leukemic cells with constitutive activation of Notch signaling. Also, DDX5 depletion inhibited the growth of human leukemia xenograft in nude mice. Moreover, DDX5 is highly expressed in primary human T-ALL leukemic cells based on the analyses of Oncomine and GEO databases and Immunohistochemical staining. Our overall findings revealed a critical role of DDX5 in promoting efficient Notch-mediated transcription in leukemic cells, suggesting that DDX5 might be critical for NOTCH1-mediated T-ALL pathogenesis and thus is a potential new target for modulating the Notch signaling in leukemia.