Global deletion of the RNA decapping enzyme Dcp2 postnatally in male mice results in infertility.

The post-transcriptional regulation of gene expression plays an important role in many essential biological processes. The RNA decapping enzyme Dcp2 is a crucial enzyme involved in RNA degradation. Dcp2 proteins are highly expressed in the testis and brain in adult mice. This study aimed to investigate the in vivo functions of Dcp2. An inducible Dcp2 knockout mouse model was established. No obvious health abnormalities were observed after postnatal global deletion of Dcp2 in male mice. However, Dcp2-deleted male mice were infertile and showed Sertoli cell vacuolization and germ cell degeneration. Dcp2 deletion resulted in testicular atrophy, reduced number of epididymal sperm, and increased apoptosis in seminiferous tubules. However, spermatocyte-specific deletion of Dcp2 did not compromise the fertility. The findings of this study indicated that Dcp2 was important for spermatogenesis and male fertility.

MicroRNA-141-3p/200a-3p target and may be involved in post-transcriptional repression of RNA decapping enzyme Dcp2 during renal development.

The RNA decapping enzyme Dcp2 is a crucial enzyme involved in the process of RNA turnover, which can post-transcriptionally regulate gene expression. Dcp2 has been found to be highly expressed in embryonic, but not adult, kidneys. Here we showed that Dcp2 mRNA was expressed, but Dcp2 proteins were absent, in mouse kidneys after postnatal day 10 (P10). In kidneys of adult Dcp2-IRES-EGFP knock-in mice, Dcp2 was undetectable but EGFP was expressed, indicating that Dcp2 mRNA was not completely silenced in adult kidneys. Using luciferase reporter assays, we found that miR-141-3p/200a-3p directly targeted the 3' UTR of Dcp2 mRNA. Overexpression of miR-141-3p and miR-200a-3p downregulated endogenous Dcp2 protein expression. Furthermore, miR-141-3p and miR-200a-3p expression was low in embryonic kidneys but increased dramatically after P10 and was negatively correlated with Dcp2 protein expression during renal development. These results suggest miR-141-3p/200a-3p may be involved in post-transcriptional repression of Dcp2 expression during renal development. ABBREVIATIONS: IRES: internal ribosome entry site; EGFP: enhanced green fluorescent protein; UTR: untranslated region.

Nudt3 is an mRNA decapping enzyme that modulates cell migration.

Removal of the 5'-end 7-methylguanosine cap structure is a critical step in the highly regulated process of mRNA decay. The Nudix hydrolase, Dcp2, was identified as a first decapping enzyme and subsequently shown to preferentially modulate stability of only a subset of mRNAs. This observation led to the hypothesis that mammalian cells possess multiple decapping enzymes that may function in distinct pathways. Here we report Nudt3 is a Nudix protein that possesses mRNA decapping activity in cells and is a modulator of MCF-7 breast cancer cell migration. Reduction of Nudt3 protein levels in MCF-7 cells promotes increased cell migration and corresponding enhanced filopodia extensions. Importantly, this phenotype was reversed by complementation with wild type, but not catalytically inactive Nudt3 protein indicating Nudt3 decapping activity normally functions to control cell migration. Genome-wide analysis of Nudt3 compromised cells identified elevated levels of transcripts involved in cell motility including integrin ß6, lipocalin-2, and fibronectin. The observed increase in mRNA abundance was dependent on Nudt3 decapping activity where integrin ß6 and lipocalin-2 were modulated directly through mRNA stability, while fibronectin was indirectly controlled. Moreover, increased cell migration observed in Nudt3 knockdown cells was mediated through the extracellular integrin ß6 and fibronectin protein nexus. We conclude that Nudt3 is an mRNA decapping enzyme that orchestrates expression of a subset of mRNAs to modulate cell migration and further substantiates the existence of multiple decapping enzymes functioning in distinct cellular pathways in mammals.

Multiple Nudix family proteins possess mRNA decapping activity.

RNA decapping is an important contributor to gene expression and is a critical determinant of mRNA decay. The recent demonstration that mammalian cells harbor at least two distinct decapping enzymes that preferentially modulate a subset of mRNAs raises the intriguing possibility of whether additional decapping enzymes exist. Because both known decapping proteins, Dcp2 and Nudt16, are members of the Nudix hydrolase family, we set out to determine whether other members of this family of proteins also contain intrinsic RNA decapping activity. Here we demonstrate that six additional mouse Nudix proteins--Nudt2, Nudt3, Nudt12, Nudt15, Nudt17, and Nudt19--have varying degrees of decapping activity in vitro on both monomethylated and unmethylated capped RNAs. The decapping products from Nudt17 and Nudt19 were analogous to Dcp2 and predominantly generated m7GDP, while cleavage by Nudt2, Nudt3, Nudt12, and Nudt15 was more pleiotropic and generated both m7GMP and m7GDP. Interestingly, all six Nudix proteins as well as both Dcp2 and Nudt16 could hydrolyze the cap of an unmethylated capped RNA, indicating that decapping enzymes may be less constrained for the presence of the methyl moiety. Investigation of Saccharomyces cerevisiae Nudix proteins revealed that the yeast homolog of Nudt3, Ddp1p, also possesses decapping activity in vitro. Moreover, the bacterial Nudix pyrophosphohydrolase RppH displayed RNA decapping activity and released m7GDP product comparable to Dcp2, indicating that decapping is an evolutionarily conserved activity that preceded mammalian cap formation. These findings demonstrate that multiple Nudix family hydrolases may function in mRNA decapping and mRNA stability.

Multiple mRNA decapping enzymes in mammalian cells.

Regulation of RNA degradation plays an important role in the control of gene expression. One mechanism of eukaryotic mRNA decay proceeds through an initial deadenylation followed by 5' end decapping and exonucleolytic decay. Dcp2 is currently believed to be the only cytoplasmic decapping enzyme responsible for decapping of all mRNAs. Here we report that Dcp2 protein modestly contributes to bulk mRNA decay and surprisingly is not detectable in a subset of mouse and human tissues. Consistent with these findings, a hypomorphic knockout of Dcp2 had no adverse consequences in mice. In contrast, the previously reported Xenopus nucleolar decapping enzyme, Nudt16, is an ubiquitous cytoplasmic decapping enzyme in mammalian cells. Like Dcp2, Nudt16 also regulates the stability of a subset of mRNAs including a member of the motin family of proteins involved in angiogenesis, Angiomotin-like 2. These data demonstrate mammalian cells possess multiple mRNA decapping enzymes, including Nudt16 to regulate mRNA turnover.

Transcript-specific decapping and regulated stability by the human Dcp2 decapping protein.

mRNA decapping is a critical step in the control of mRNA stability and gene expression and is carried out by the Dcp2 decapping enzyme. Dcp2 is an RNA binding protein that must bind RNA in order to recognize the cap for hydrolysis. We demonstrate that human Dcp2 (hDcp2) preferentially binds to a subset of mRNAs and identify sequences at the 5' terminus of the mRNA encoding Rrp41, a core subunit component of the RNA exosome, as a specific hDcp2 substrate. A 60-nucleotide element at the 5' end of Rrp41 mRNA was identified and shown to confer more efficient decapping on a heterologous RNA both in vitro and upon transfection into cells. Moreover, reduction of hDcp2 protein levels in cells resulted in a selective stabilization of the Rrp41 mRNA, confirming it as a downstream target of hDcp2 regulation. These findings demonstrate that hDcp2 can specifically bind to and regulate the stability of a subset of mRNAs, and its intriguing regulation of the 3'-to-5' exonuclease exosome subunit suggests a potential interplay between 5'-end mRNA decapping and 3'-end mRNA decay.

3' Terminal oligo U-tract-mediated stimulation of decapping.

Decapping is a critical step in the control of gene expression and is regulated by both positive and negative trans factors. Less is known about cis elements that promote decapping. In plants, following microRNA (miRNA)-directed cleavage of an mRNA, a uridine tract can be added onto the exposed 3' end of the resulting 5' fragment, which can promote 5' end decay. We now demonstrate that in mammalian cell extract, addition of five uridine residues to the 3' end of an RNA (U5) promotes decapping relative to an RNA lacking the uridines (U0). Although the decapping stimulation observed in extract required hDcp2, recombinant hDcp2 was unable to support differential decapping of the U0 and U5 RNAs, indicating that the stimulation was likely due to an indirect recruitment of hDcp2 to the RNA. Consistent with the promotion of 5' end decapping by the uridine tract, affinity purification with the U5 RNA revealed the presence of a decapping subcomplex at least consisting of hDcp2, Dcp1a, Edc4, LSm1, and LSm4 that were specifically bound to the U5 RNA but not the U0 RNA. In addition to promoting decapping, the U-tract stabilized the 3' end of the RNA by preventing 3' to 5' exonucleolytic decay to ensure 5' end directional degradation. These data suggest that following post-transcriptional oligo uridylation of an mRNA or mRNA fragment, the U-tract has the capacity to specifically stimulate 5' end decapping to expedite mRNA decay.

[Utilization of the stable ectopic expression cell line as a model for the investigation of RIG-G gene].

OBJECTIVE: To investigate the biological function of RIG-G gene by establishing a cell line stably expressing RIG-G protein. METHODS: Ectopic RIG-G gene was transfected into U937 cells by using Tet-off expression system. Changes before and after RIG-G expression were detected for cell growth, cell morphology, cell surface antigen and cell cycle regulating proteins. RESULTS: RIG-G protein arrested the cells at G0/G1 phase and inhibited cell growth by increasing the cell cycle inhibitors P21 and P27. As compared to that in control group, the proportion of cells at G0/G1 phase in RIG-G-expressing cell group increased from (43.9 +/- 5.6)% to (63.9 +/- 2.3)% (P < 0.01). The rate of growth inhibition was (68.7 +/- 0.2)%. In addition, an increase in CD11C expression [(61.3 +/- 1.1)% vs. (18.0 +/- 0.4)% (P < 0.01)] and in cells with morphologic features of partial differentiation (smaller cell size, reduced nucleus-cytoplasm ratio, notched nucleus and coarse chromatin) was also observed in RIG-G-expressing cells. CONCLUSIONS: RIG-G has potential abilities to inhibit cell proliferation and promote cell differentiation.

RIG-G as a key mediator of the antiproliferative activity of interferon-related pathways through enhancing p21 and p27 proteins.

The RIG-G gene, originally isolated from an acute promyelocytic leukemia cell line NB4, codes for a 60-kDa cytoplasmic protein that is induced by all-trans retinoic acid (ATRA) treatment along with the induction of morphological differentiation of NB4 cells. Here, we provide evidence that ectopic expression of Rig-G in U937 cells can lead to a significant accumulation of cells at G(1)/S transition. Growth arrest seems to occur by modulating several major cell cycle regulatory players. Interestingly, Rig-G alters JAB1 cellular distribution through interacting with this protein and increases the intracellular level of p27 by preventing it from the JAB-1-dependent and ubiquitin/proteasome-mediated degradation. Furthermore, we demonstrate a role of Rig-G for c-myc down-regulation that results in an up-regulation of p21, tightly associated with cell cycle arrest. In addition, our studies reveal that Rig-G is a direct target of STAT1, a key transcription factor in regulating IFN responses, and may be one of the first experimentally proven molecular mediators for the antiproliferative effect of IFN-alpha. Considering that IFN-alpha and ATRA synergistically inhibit growth along the intracellular pathways triggered by the two compounds in many cell types, we suggest that Rig-G may also represent one of the key molecular nodes of signaling cross-talk between ATRA and IFN-alpha.

Nanomolar concentration of NSC606985, a camptothecin analog, induces leukemic-cell apoptosis through protein kinase Cdelta-dependent mechanisms.

As a promising new class of anticancer drugs, camptothecins have advanced to the forefront of several areas of therapeutic and developmental chemotherapy. In the present study, we report that NSC606985, a rarely studied camptothecin analog, induces apoptosis in acute myeloid leukemia (AML) cells NB4 and U937 and inhibits the proliferation without cell death in breakpoint cluster region-Abelson murine leukemia (bcr-abl) kinase-carrying leukemic K562 cells. For apoptosis induction or growth arrest, nanomolar concentrations of NSC606985 are sufficient. At such low concentrations, this agent also significantly inhibits the clonogenic activity of hematopoietic progenitors from patients with AML. For apoptosis induction, NSC606985 rapidly induces the proteolytic activation of protein kinase Cdelta (PKCdelta) with loss of mitochondrial transmembrane potential (DeltaPsim) and caspase-3 activation. Cotreatment with rottlerin, a PKCdelta-specific inhibitor, completely blocks NSC606985-induced mitochondrial DeltaPsim loss and caspase-3 activation, while the inhibition of caspase-3 by z-DEVD-fluoromethyl ketone (Z-DEVD-fmk) only partially attenuates PKCdelta activation and apoptosis. These data indicate that NSC606985-induced PKCdelta activation is an early event upstream to mitochondrial DeltaPsim loss and caspase-3 activation, while activated caspase-3 has an amplifying effect on PKCdelta proteolysis. In addition, NSC606985-induced apoptosis by PKCdelta also involves caspase-3-independent mechanisms. Taken together, our results suggest that NSC606985 is a potential agent for the treatment of AML.