MicroRNAs (miRNAs) are small noncoding RNA molecules of 20-24 nucleotides that regulate gene expression. In animals, miRNAs form imperfect interactions with sequences in the 39 Untranslated region (39UTR) of mRNAs, causing translational inhibition and mRNA decay. In contrast, plant miRNAs mostly associate with protein coding regions. Here we show that human miR-148 represses DNA methyltransferase 3b (Dnmt3b) gene expression through a region in its coding sequence. This region is evolutionary conserved and present in the Dnmt3b splice variants Dnmt3b1, Dnmt3b2, and Dnmt3b4, but not in the abundantly expressed Dnmt3b3. Whereas overexpression of miR-148 results in decreased DNMT3b1 expression, short-hairpin RNAmediated miR-148 repression leads to an increase in DNMT3b1 expression. Interestingly, mutating the putative miR-148 target site in Dnmt3b1 abolishes regulation by miR-148. Moreover, endogenous Dnmt3b3 mRNA, which lacks the putative miR-148 target site, is resistant to miR-148-mediated regulation. Thus, our results demonstrate that the coding sequence of Dnmt3b mediates regulation by the miR-148 family. More generally, we provide evidence that coding regions of human genes can be targeted by miRNAs, and that such a mechanism might play a role in determining the relative abundance of different splice variants.

Summary The MRN (MRE11-RAD50-NBS1) complex has been implicated in many aspects of the DNA damage response. It has key roles in sensing and processing DNA double-strand breaks, as well as in activation of ATM (ataxia-telangiectasia mutated). We reveal a new function for MRN in ATR (ATM and RAD3-related) activation by using defined ATR-activating DNA structures in Xenopus egg extracts. Strikingly, we demonstrate that MRN is required to recruit TOPBP1 to an ATR-activating structure that contains a single-stranded DNA (ssDNA) and a double-stranded DNA (dsDNA) junction, and that this recruitment is necessary for phosphorylation of Chk1. We also show that the 911 (RAD9-RAD1-HUS1) complex is not required for TOPBP1 recruitment, but is essential for TOPBP1 function. Thus, whereas MRN is required for TOPBP1 recruitment at a ss/dsDNA junction, 911 is required for TOPBP1 “activation”. These findings provide new molecular insight into how ATR is activated.

The human telomerase RNA (hTR), together with the telomerase reverse transcriptase, hTERT, constitute the core components of telomerase that is essential for telomere maintenance. While hTR is ubiquitously expressed, hTERT is normally restricted to germ cells and certain stem cells, but both are often deregulated during tumorigenesis. Here, we investigated the effects of changes in hTR cellular levels. Surprisingly, while inhibition of hTR expression triggers a rapid, telomerase-independent, growth arrest associated with p53 and CHK1 activation, its increased expression neutralizes activation of these pathways in response to genotoxic stress. These hTR effects are mediated through ATR and are sufficiently strong to impair ATR-mediated DNA-damage checkpoint responses. Furthermore, in response to low UV radiation, which activates ATR, endogenous hTR levels increase irrespective of telomerase status. Thus, we uncovered a novel, telomerase-independent, function of hTR that restrains ATR activity and participates in the recovery of cells from UV radiation.

Activation of tumor suppressor p53 in response to genotoxic stress imposes cellular growth arrest or apoptosis. We identified Cdc6, a licensing factor of the prereplication complex, as a novel target of the p53 pathway. We show that activation of p53 by DNA damage results in enhanced Cdc6 destruction by the anaphase-promoting complex. This destruction is triggered by inhibition of CDK2-mediated CDC6 phosphorylation at serine 54. Conversely, suppression of p53 expression results in stabilization of Cdc6. We demonstrate that loss of p53 results in more replicating cells, an effect that can be reversed by reducing Cdc6 protein levels. Collectively, our data suggest that initiation of DNA replication is regulated by p53 through Cdc6 protein stability.

Experimental and clinical observations have highlighted the role of cytotoxic T cells in human tumor control. However, the parameters that control tumor cell sensitivity to T cell attack remain incompletely understood. To identify modulators of tumor cell sensitivity to T cell effector mechanisms, we performed a whole genome haploid screen in HAP1 cells. Selection of tumor cells by exposure to tumor-specific T cells identified components of the interferon-γ (IFN-γ) receptor (IFNGR) signaling pathway, and tumor cell killing by cytotoxic T cells was shown to be in large part mediated by the pro-apoptotic effects of IFN-γ. Notably, we identified schlafen 11 (SLFN11), a known modulator of DNA damage toxicity, as a regulator of tumor cell sensitivity to T cell-secreted IFN-γ. SLFN11 does not influence IFNGR signaling, but couples IFNGR signaling to the induction of the DNA damage response (DDR) in a context dependent fashion. In line with this role of SLFN11, loss of SLFN11 can reduce IFN-γ mediated toxicity. Collectively, our data indicate that SLFN11 can couple IFN-γ exposure of tumor cells to DDR and cellular apoptosis. Future work should reveal the mechanistic basis for the link between IFNGR signaling and DNA damage response, and identify tumor cell types in which SLFN11 contributes to the anti-tumor activity of T cells.