MicroRNAs (miRNAs) are endogenous B22-nucleotide RNAs, which suppress gene expression by selectively binding to the 3 0 -noncoding region of specific messenger RNAs through base-pairing. Given the diversity and abundance of miRNA targets, miRNAs appear to functionally interact with various components of many cellular networks. By analyzing the interactions between miRNAs and a human cellular signaling network, we found that miRNAs predominantly target positive regulatory motifs, highly connected scaffolds and most downstream network components such as signaling transcription factors, but less frequently target negative regulatory motifs, common components of basic cellular machines and most upstream network components such as ligands. In addition, when an adaptor has potential to recruit more downstream components, these components are more frequently targeted by miRNAs. This work uncovers the principles of miRNA regulation of signal transduction networks and implies a potential function of miRNAs for facilitating robust transitions of cellular response to extracellular signals and maintaining cellular homeostasis.

MicroRNAs (miRNAs) are a class of noncoding small RNAs that regulate gene expression by base pairing with target mRNAs at the 3′-terminal untranslated regions (3′-UTRs), leading to mRNA cleavage or translational repression. Single-nucleotide polymorphisms (SNPs) located at miRNA-binding sites (miRNA-binding SNPs) are likely to affect the expression of the miRNA target and may contribute to the susceptibility of humans to common diseases. We herein performed a genome-wide analysis of SNPs located in the miRNA-binding sites of the 3′-UTR of various human genes. We found that miRNA-binding SNPs are negatively selected in respect to SNP distribution between the miRNA-binding ‘seed’ sequence and the entire 3′-UTR sequence. Furthermore, we comprehensively defined the expression of each miRNA-binding SNP in cancers versus normal tissues through mining EST databases. Interestingly, we found that some miRNA-binding SNPs exhibit significant different allele frequencies between the human cancer EST libraries and the dbSNP database. More importantly, using human cancer specimens against the dbSNP database for case-control association studies, we found that twelve miRNA-binding SNPs indeed display an aberrant allele frequency in human cancers. Hence, SNPs located in miRNA-binding sites affect miRNA target expression and function, and are potentially associated with cancers.

Mutations in FUS/TLS (fused in sarcoma/translated in liposarcoma) cause an inheritable form of amyotrophic lateral sclerosis (ALS6). In contrast to FUS(WT), which is concentrated in the nucleus, these mutants are abnormally distributed in the cytoplasm where they form inclusions and associate with stress granules. The data reported herein demonstrate the importance of protein arginine methylation in nuclear-cytoplasmic shuttling of FUS and abnormalities of ALS-causing mutants. Depletion of protein arginine methyltransferase 1 (PRMT1; the enzyme that methylates FUS) in mouse embryonic fibroblasts by gene knockout, or in human HEK293 cells by siRNA knockdown, diminished the ability of ALS-linked FUS mutants to localize to the cytoplasm and form inclusions. To examine properties of FUS mutants in the context of neurons vulnerable to the disease, FUS(WT) and ALS-linked FUS mutants were expressed in motor neurons of dissociated murine spinal cord cultures. In motor neurons, shRNA-mediated PRMT1 knockdown concomitant with the expression of FUS actually accentuated the shift in distribution of ALS-linked FUS mutants from the nucleus to the cytoplasm. However, when PRMT1 was inhibited prior to expression of ALS-linked FUS mutants, by pretreatment with a global methyltransferase inhibitor, ALS-linked FUS mutants were sequestered in the nucleus and cytoplasmic inclusions were reduced, as in the cell lines. Mitochondria were significantly shorter in neurons with cytoplasmic ALS-linked FUS mutants, a factor that could contribute to toxicity. We propose that arginine methylation by PRMT1 participates in the nuclear-cytoplasmic shuttling of FUS, particularly of ALS6-associated mutants, and thus contributes to the toxic gain of function conferred by these disease-causing mutations.

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.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the posttranscriptional level to lead to mRNA degradation or repressed protein production. The expression of miRNA is deregulated in many types of cancers. To determine whether genetic alterations in miRNA genes are associated with cancers, we have systematically screened sequence variations in several hundred human miRNAs from >100 human tumor tissues and 20 cancer cell lines. We identified 8 new single-nucleotide polymorphisms (SNPs) and 14 novel mutations (or very rare SNPs) that specifically present in human cancers. These mutations/SNPs are distributed in the regions of pri-, pre- and even mature miRNAs, respectively. Importantly, whereas most of the mutations did not exert detectable effects on miRNA function, a G --> A mutation at 19 nt downstream of miRNA let-7e led to a significant reduction of its expression in vivo, indicating that miRNA mutation could contribute to tumorigenesis. These data suggest that further screening for genetic variations in miRNA genes from a wide variety of human cancers should increase the discovery and identification of molecular diagnostic and therapeutic targets and complement the mutation analysis of consensus coding sequences in human cancers.

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PTEN is a tumor suppressor that primarily dephosphorylates phosphatidylinositol 3,4,5-trisphosphate to down-regulate the phosphoinositide 3-kinase/Akt signaling pathway. Although the cellular functions of PTEN as a tumor suppressor have been well characterized, the mechanism by which PTEN activity is modulated by other signal molecules in vivo remains poorly understood. In searching for potential PTEN modulators through protein-protein interaction, we identified the major vault protein (MVP) as a dominant PTENbinding protein in a yeast two-hybrid screen. MVP is the major structural component of vault, the largest intracellular ribonucleoprotein particle. PTEN was originally identified as a tumor suppressor gene based on its high frequency of mutation in a variety of tumors (1-3). Germ-line mutations of PTEN are the cause of Cowden disease, an autosomal-dominant hamartoma syndrome that results in an increased risk for development of tumors in a variety of tissues (4 -7). The genetic evidence that PTEN is an important tumor suppressor is supported by the fact that heterozygous disruption of the PTEN gene in knockout mice results in the spontaneous development of tumors (8 -10). Although PTEN as a protein phosphatase is capable of dephosphorylating tyrosine and threonine/serine residues (11, 12), the primary substrates of PTEN are 3Ј-phosphoinositides, PtdIns-3,4-P 2 and PtdIns-3,4,5-P 3 (13). Genetic and biochemical studies have demonstrated that the tumor suppressor functions of PTEN are linked primarily with the lipid phosphatase activity and its association with the well defined phosphoinositide 3-kinase pathway (reviewed in Refs. 14 -20). Substantial progress has been made in the characterization of PTEN as a tumor suppressor as well as in the regulation of many cellular processes including growth, adhesion, migration, invasion, and apoptosis. Nevertheless, the mechanism by which PTEN activity is modulated in various cellular signaling complexes remains elusive. It is assumed that the activity and the cellular function of PTEN may be regulated through in vivo proteinprotein interactions. PTEN contains a number of putative regulatory modules, including the N-terminal phosphoinositide binding motif, a C2 domain, a PDZ-binding site, and two proline-, glutamic acid-, serine-, and threonine-rich segments (21). The C2 domain of PTEN has been implicated in mediating membrane association (22). The C-terminal tail of PTEN interacts with several PDZ domain-containing proteins such as hDLG, hMAST205, MAGI-2, and MAGI-3 (23-25). The interaction of PTEN with these proteins may be important for its biological function, as it has been reported that MAGI-2 and MAGI-3 can enhance the activity of PTEN (23, 24). In contrast, several groups found that the PDZ-binding site of PTEN is not required for tumor suppression or other biological activities (21,(25)(26)(27)(28). Therefore, the complete spectrum of PTEN-interacting proteins and the effects of the interactions on PTEN function are not yet defined.To date, the vault complex w...

MicroRNAs (miRNAs) are non-coding small RNAs of ∼22 nt that regulate the gene expression by base pairing with target mRNAs, leading to mRNA cleavage or translational repression. It is currently estimated that miRNAs account for ∼1% of predicted genes in higher eukaryotic genomes and that up to 30% of genes might be regulated by miRNAs. However, only very few miRNAs have been functionally characterized and the general functions of miRNAs are not globally studied. In this study, we systematically analyzed the expression patterns of miRNA targets using several public microarray profiles. We found that the expression levels of miRNA targets are lower in all mouse and Drosophila tissues than in the embryos. We also found miRNAs more preferentially target ubiquitously expressed genes than tissue-specifically expressed genes. These results support the current suggestion that miRNAs are likely to be largely involved in embryo development and maintaining of tissue identity.