Taiping Chen

Self-association of the single-KH-domain family members Sam68, GRP33, GLD-1, and Qk1: role of the KH domain.

Sam68 is a member of a growing family of proteins that contain a single KH domain embedded in a larger conserved domain of approximately 170 amino acids. Loops 1 and 4 of this KH domain family are longer than the corresponding loops in other KH domains and contain conserved residues. KH domains are protein motifs that are involved in RNA binding and are often present in multiple copies. Here we demonstrate by coimmunoprecipitation studies that Sam68 self-associated and that cellular RNA was required for the association. Deletion studies demonstrated that the Sam68 KH domain loops 1 and 4 were required for self-association. The Sam68 interaction was also observed in Saccharomyces cerevisiae by the two-hybrid system. In situ chemical cross-linking studies in mammalian cells demonstrated that Sam68 oligomerized in vivo. These Sam68 complexes bound homopolymeric RNA and the SH3 domains of p59fyn and phospholipase Cgamma1 in vitro, demonstrating that Sam68 associates with RNA and signaling molecules as a multimer. The formation of the Sam68 complex was inhibited by p59fyn, suggesting that tyrosine phosphorylation regulates Sam68 oligomerization. Other Sam68 family members including Artemia salina GRP33, Caenorhabditis elegans GLD-1, and mouse Qk1 also oligomerized. In addition, Sam68, GRP33, GLD-1, and Qk1 associated with other KH domain proteins such as Bicaudal C. These observations indicate that the single KH domain found in the Sam68 family, in addition to mediating protein-RNA interactions, mediates protein-protein interactions.

Sik (BRK) Phosphorylates Sam68 in the Nucleus and Negatively Regulates Its RNA Binding Ability

ABSTRACT Sik (mouse Src-related intestinal kinase) and its orthologue BRK (human breast tumor kinase) are intracellular tyrosine kinases that are distantly related to the Src family and have a similar structure, but they lack the myristoylation signal. Here we demonstrate that Sik and BRK associate with the RNA binding protein Sam68 (Src associated during mitosis, 68 kDa). We found that Sik interacts with Sam68 through its SH3 and SH2 domains and that the proline-rich P3 region of Sam68 is required for Sik and BRK SH3 binding. In the transformed HT29 adenocarcinoma cell cell line, endogenous BRK and Sam68 colocalize in Sam68-SLM nuclear bodies (SNBs), while transfected Sik and Sam68 are localized diffusely in the nucleoplasm of nontransformed NMuMG mammary epithelial cells. Transfected Sik phosphorylates Sam68 in SNBs in HT29 cells and in the nucleoplasm of NMuMG cells. In functional studies, expression of Sik abolished the ability of Sam68 to bind RNA and act as a cellular Rev homologue. While Sam68 is a substrate for Src family kinases during mitosis, Sik/BRK is the first identified tyrosine kinase that can phosphorylate Sam68 and regulate its activity within the nucleus, where it resides during most of the cell cycle.

Nuclear Retention of MBP mRNAs in the Quaking Viable Mice

Quaking viable (qk(v)) mice fail to properly compact myelin in their central nervous systems. Although the defect in the qk(v) mice involves a mutation affecting the expression of the alternatively spliced qk gene products, their roles in myelination are unknown. We show that the QKI RNA binding proteins regulate the nuclear export of MBP mRNAs. Disruption of the QKI nucleocytoplasmic equilibrium in oligodendrocytes results in nuclear and perikaryal retention of the MBP mRNAs and lack of export to cytoplasmic processes, as it occurs in qk(v) mice. MBP mRNA export defect leads to a reduction in the MBP levels and their improper cellular targeting to the periphery. Our findings suggest that QKI participates in myelination by regulating the mRNA export of key protein components.

A mouse PRMT1 null allele defines an essential role for arginine methylation in genome maintenance and cell proliferation

ABSTRACT Protein arginine methyltransferase 1 (PRMT1) is the major enzyme that generates monomethylarginine and asymmetrical dimethylarginine. We report here a conditional null allele of PRMT1 in mice and that the loss of PRMT1 expression leads to embryonic lethality. Using the Cre/lox-conditional system, we show that the loss of PRMT1 in mouse embryonic fibroblasts (MEFs) leads to the loss of arginine methylation of substrates harboring a glycine-arginine rich motif, including Sam68 and MRE11. The loss of PRMT1 in MEFs leads to spontaneous DNA damage, cell cycle progression delay, checkpoint defects, aneuploidy, and polyploidy. We show using a 4-hydroxytamoxifen-inducible Cre that the loss of PRMT1 in MEFs leads to a higher incidence of chromosome losses, gains, structural rearrangements, and polyploidy, as documented by spectral karyotyping. Using PRMT1 small interfering RNA in U2OS cells, we further show that PRMT1-deficient cells are hypersensitive to the DNA damaging agent etoposide and exhibit a defect in the recruitment of the homologous recombination RAD51 recombinase to DNA damage foci. Taken together, these data show that PRMT1 is required for genome integrity and cell proliferation. Our findings also suggest that arginine methylation by PRMT1 is a key posttranslational modification in the DNA damage response pathway in proliferating mammalian cells.

Structure-Function Analysis of Qk1: a Lethal Point Mutation in Mouse quaking Prevents Homodimerization

ABSTRACT Qk1 is a member of the KH domain family of proteins that includes Sam68, GRP33, GLD-1, SF1, and Who/How. These family members are RNA binding proteins that contain an extended KH domain embedded in a larger domain called the GSG (for GRP33–Sam68–GLD-1) domain. An ethylnitrosourea-induced point mutation in the Qk1 GSG domain alters glutamic acid 48 to a glycine and is known to be embryonically lethal in mice. The function of Qk1 and the GSG domain as well as the reason for the lethality are unknown. Here we demonstrate that the Qk1 GSG domain mediates RNA binding and Qk1 self-association. By using in situ chemical cross-linking studies, we showed that the Qk1 proteins exist as homodimers in vivo. The Qk1 self-association region was mapped to amino acids 18 to 57, a region predicted to form coiled coils. Alteration of glutamic acid 48 to glycine (E➤G) in the Qk1 GSG domain (producing protein Qk1:E➤G) abolishes self-association but has no effect on the RNA binding activity. The expression of Qk1 or Qk1:E➤G in NIH 3T3 cells induces cell death by apoptosis. Approximately 90% of the remaining transfected cells are apoptotic 48 h after transfection. Qk1:E➤G was consistently more potent at inducing apoptosis than was wild-type Qk1. These results suggest that the mousequaking lethality (E➤G) occurs due to the absence of Qk1 self-association mediated by the GSG domain.

Ablation of the Sam68 RNA Binding Protein Protects Mice from Age-Related Bone Loss

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.

Parathyroid Hormone-related Protein Interacts with RNA

Parathyroid hormone-related protein (PTHrP) is a secreted protein that acts as an autocrine and paracrine mediator of cell proliferation and differentiation. In addition to its biological activity that is mediated through signal transduction cascades, there is evidence for an intracellular role for PTHrP in cell cycle progression and apoptosis. These effects are mediated through a mid-region nuclear targeting sequence (NTS) that localizes PTHrP to the region of the nucleolus where ribonucleoprotein complexes form in vivo. In this work, we show that endogenous, transfected, andin vitro translated PTHrP proteins bind homopolymeric and total cellular RNAs at salt concentrations up to 1 m. A peptide representing the PTHrP NTS was effective in competing with the wild-type protein for RNA binding, whereas a similar peptide representing the nucleolin NTS was not. Site-directed mutagenesis revealed that the binding of PTHrP to RNA was direct and was dependent on preservation of a core GXKKXXK motif, embedded in the PTHrP NTS, which is shared with other RNA-binding proteins. The current observations are the first to document RNA binding by a secreted cellular protein and predict a role for PTHrP in regulating RNA metabolism that may be related to its localization in the nucleolus of cells in vivo.

The physiological and pathophysiological role of PRMT1-mediated protein arginine methylation

Post-translational modifications are well-known effectors in DNA damage signaling and epigenetic gene expression. Protein arginine methylation is a covalent modification that results in the addition of methyl groups to the nitrogen atoms of the arginine side chains and is catalyzed by a family of protein arginine methyltransferases (PRMTs). In the past, arginine methylation was mainly observed on abundant proteins such as RNA-binding proteins and histones, but recent advances have revealed a plethora of arginine-methylated proteins implicated in a variety of cellular processes including signal transduction, epigenetic regulation and DNA repair pathways. Herein, we discuss these recent advances, focusing on the role of PRMT1, the major asymmetric arginine methyltransferase, in cellular processes and its link to human diseases.

The Identification of Two Drosophila K Homology Domain Proteins KEP1 AND SAM ARE MEMBERS OF THE Sam68 FAMILY OF GSG DOMAIN PROTEINS

Sam68 is a member of a growing family of RNA-binding proteins that contains an extended K homology (KH) domain embedded in a larger domain called the GSG (GRP33,Sam68, GLD1) domain. To identify GSG domain family members, we searched data bases for expressed sequence tags encoding related portions of the Sam68 KH domain. Here we report the identification of two novel Drosophila KH domain proteins, which we termed KEP1 (KH encompassingprotein) and SAM. SAM bears sequence identity with mammalian Sam68 and may be the Drosophila Sam68 homolog. We demonstrate that SAM, KEP1, and the recently identifiedDrosophila Who/How are RNA-binding proteins that are able to self-associate into homomultimers. The GSG domain of KEP1 and SAM was necessary to mediate the RNA binding and self-association. To elucidate the cellular roles of these proteins, SAM, KEP1, and Who/How were expressed in mammalian and Drosophila S2 cells. KEP1 and Who/How were nuclear and SAM was cytoplasmic. The expression of KEP1 and SAM, but not Who/How, activated apoptotic pathways inDrosophila S2 cells. The identification of KEP1 and SAM implies that a large GSG domain protein family exists and helps redefine the boundaries of the GSG domain. Taken together, our data suggest that KEP1 and SAM may play a role in the activation or regulation of apoptosis and further implicate the GSG domain in RNA binding and oligomerization.

Allele-Specific H3K79 Di- versus Trimethylation Distinguishes Opposite Parental Alleles at Imprinted Regions

ABSTRACT Imprinted gene expression corresponds to parental allele-specific DNA CpG methylation and chromatin composition. Histone tail covalent modifications have been extensively studied, but it is not known whether modifications in the histone globular domains can also discriminate between the parental alleles. Using multiplex chromatin immunoprecipitation-single nucleotide primer extension (ChIP-SNuPE) assays, we measured the allele-specific enrichment of H3K79 methylation and H4K91 acetylation along the H19/Igf2 imprinted domain. Whereas H3K79me1, H3K79me2, and H4K91ac displayed a paternal-specific enrichment at the paternally expressed Igf2 locus, H3K79me3 was paternally biased at the maternally expressed H19 locus, including the paternally methylated imprinting control region (ICR). We found that these allele-specific differences depended on CTCF binding in the maternal ICR allele. We analyzed an additional 11 differentially methylated regions (DMRs) and found that, in general, H3K79me3 was associated with the CpG-methylated alleles, whereas H3K79me1, H3K79me2, and H4K91ac enrichment was specific to the unmethylated alleles. Our data suggest that allele-specific differences in the globular histone domains may constitute a layer of the “histone code” at imprinted genes.