RNA 2'-O-methylation promotes persistent R-loop formation and AID-mediated IgH class switch recombination.

BACKGROUND: RNA-DNA hybrids or R-loops are associated with deleterious genomic instability and protective immunoglobulin class switch recombination (CSR). However, the underlying phenomenon regulating the two contrasting functions of R-loops is unknown. Notably, the underlying mechanism that protects R-loops from classic RNase H-mediated digestion thereby promoting persistence of CSR-associated R-loops during CSR remains elusive. RESULTS: Here, we report that during CSR, R-loops formed at the immunoglobulin heavy (IgH) chain are modified by ribose 2'-O-methylation (2'-OMe). Moreover, we find that 2'-O-methyltransferase fibrillarin (FBL) interacts with activation-induced cytidine deaminase (AID) associated snoRNA aSNORD1C to facilitate the 2'-OMe. Moreover, deleting AID C-terminal tail impairs its association with aSNORD1C and FBL. Disrupting FBL, AID or aSNORD1C expression severely impairs 2'-OMe, R-loop stability and CSR. Surprisingly, FBL, AID's interaction partner and aSNORD1C promoted AID targeting to the IgH locus. CONCLUSION: Taken together, our results suggest that 2'-OMe stabilizes IgH-associated R-loops to enable productive CSR. These results would shed light on AID-mediated CSR and explain the mechanism of R-loop-associated genomic instability.

Biogenesis of intronic miRNAs located in clusters by independent transcription and alternative splicing.

miRNAs are generally classified as "intergenic" or "intronic" based upon their genomic location. Intergenic miRNAs are known to be transcribed as independent transcription units, while intronic miRNAs are believed to be processed from the introns of their hosting transcription units and hence share common regulatory mechanisms and expression patterns with its host gene. Recent reports in the literature suggest that some intronic miRNAs, which do not show concordance in expression with their respective host genes, might be transcribed and regulated as independent transcription units. However, there is no direct evidence for the existence of independently transcribed intronic miRNA in humans to date. We have characterized the full-length primary transcripts (pri-miRNAs) of three human intronic miRNAs-miR 106b, miR 93, and miR 24-1-by RNA ligase-mediated RACE and show that human intronic miRNA can indeed be transcribed as independent transcription units. Also, clustered miRNAs are generally believed to arise from a common primary transcript and are expected to have similar expression profiles. However, we have identified several novel alternatively spliced transcripts by RT-PCR, each of which harbors a single pre-miRNA from a cluster of closely located intronic miRNAs. We show that these transcripts represent unique pri-miRNAs for each of these clustered miRNAs. We also report the identification of conserved splice acceptor signals which are responsible for maturation of these novel splice variants. Our results suggest that alternative splicing might play a role in uncoupling the expression of clustered miRNAs from each other, which otherwise are generally believed to be co-transcribed and co-expressed.

Targeting dePARylation for cancer therapy.

Poly(ADP-ribosyl)ation (PARylation) mediated by poly ADP-ribose polymerases (PARPs) plays a key role in DNA damage repair. Suppression of PARylation by PARP inhibitors impairs DNA damage repair and induces apoptosis of tumor cells with repair defects. Thus, PARP inhibitors have been approved by the US FDA for various types of cancer treatment. However, recent studies suggest that dePARylation also plays a key role in DNA damage repair. Instead of antagonizing PARylation, dePARylation acts as a downstream step of PARylation in DNA damage repair. Moreover, several types of dePARylation inhibitors have been developed and examined in the preclinical studies for cancer treatment. In this review, we will discuss the recent progress on the role of dePARylation in DNA damage repair and cancer suppression. We expect that targeting dePARylation could be a promising approach for cancer chemotherapy in the future.

The role of dePARylation in DNA damage repair and cancer suppression.

Poly(ADP-ribosyl)ation (PARylation) is a reversible post-translational modification regulating various biological pathways including DNA damage repair (DDR). Rapid turnover of PARylation is critically important for an optimal DNA damage response and maintaining genomic stability. Recent studies show that PARylation is tightly regulated by a group of enzymes that can erase the ADP-ribose (ADPR) groups from target proteins. The aim of this review is to present a comprehensive understanding of dePARylation enzymes, their substrates and roles in DDR. Special attention will be laid on the role of these proteins in the development of cancer and their feasibility in anticancer therapeutics.

Correction: LGR5 regulates gastric adenocarcinoma cell proliferation and invasion via activating Wnt signaling pathway.

The second corresponding author Dr. Xiaochun Yu is only affiliated with [3] Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope, 1500 E. Duarte Rd, Duarte, CA 91010, USA. He is not affiliated with [1] College of Life Sciences, Hebei University, Baoding 071002 Hebei, China.

NADP+ is an endogenous PARP inhibitor in DNA damage response and tumor suppression.

ADP-ribosylation is a unique posttranslational modification catalyzed by poly(ADP-ribose) polymerases (PARPs) using NAD+ as ADP-ribose donor. PARPs play an indispensable role in DNA damage repair and small molecule PARP inhibitors have emerged as potent anticancer drugs. However, to date, PARP inhibitor treatment has been restricted to patients with BRCA1/2 mutation-associated breast and ovarian cancer. One of the major challenges to extend the therapeutic potential of PARP inhibitors to other cancer types is the absence of predictive biomarkers. Here, we show that ovarian cancer cells with higher level of NADP+, an NAD+ derivative, are more sensitive to PARP inhibitors. We demonstrate that NADP+ acts as a negative regulator and suppresses ADP-ribosylation both in vitro and in vivo. NADP+ impairs ADP-ribosylation-dependent DNA damage repair and sensitizes tumor cell to chemically synthesized PARP inhibitors. Taken together, our study identifies NADP+ as an endogenous PARP inhibitor that may have implications in cancer treatment.

PARP2 mediates branched poly ADP-ribosylation in response to DNA damage.

Poly(ADP-ribosyl)ation (PARylation) is a posttranslational modification involved in multiple biological processes, including DNA damage repair. This modification is catalyzed by poly(ADP-ribose) polymerase (PARP) family of enzymes. PARylation is composed of both linear and branched polymers of poly(ADP-ribose) (PAR). However, the biochemical mechanism of polymerization and biological functions of branched PAR chains are elusive. Here we show that PARP2 is preferentially activated by PAR and subsequently catalyzes branched PAR chain synthesis. Notably, the direct binding to PAR by the N-terminus of PARP2 promotes the enzymatic activity of PARP2 toward the branched PAR chain synthesis. Moreover, the PBZ domain of APLF recognizes the branched PAR chain and regulates chromatin remodeling to DNA damage response. This unique feature of PAR-dependent PARP2 activation and subsequent PARylation mediates the participation of PARP2 in DNA damage repair. Thus, our results reveal an important molecular mechanism of branched PAR synthesis and a key biological function of branched PARylation.

LGR5 regulates gastric adenocarcinoma cell proliferation and invasion via activating Wnt signaling pathway.

LGR5 plays a critical role in tissue development and the maintenance of adult stem cells in gastrointestinal tract. However, the oncogenic role of LGR5 in the development of gastric adenocarcinoma remains elusive. Here, we show that LGR5 promotes gastric adenocarcinoma cell proliferation and metastasis. We find that knock down of LGR5 or suppression of Wnt signaling pathway by inhibitor C59 arrests gastric adenocarcinoma cell proliferation and invasion. Moreover, treatment of Wnt3a, the activator of Wnt signaling pathway, partially recovers the proliferation defect observed in LGR5 knockdown gastric adenocarcinoma cells. Moreover, LGR5 facilitates ß-catenin nuclear accumulation, a surrogate marker of the activation of Wnt signaling pathway. In addition, C59 treatment suppresses transcription of Axin2 and TCF1, both of which are the target genes of ß-catenin in gastric adenocarcinoma cells. Gastric adenocarcinoma cells with overexpressed LGR5 form a large quantity of visible actin filaments and pseudopods, suggesting that LGR5 significantly enhances the ability of cell movement, which might capacitate gastric adenocarcinoma cells with enhanced LGR5 expression to gain invasive and migratory properties. Taken together, our results show that LGR5 contributes to cell proliferation and invasion through the activation of Wnt/ß-catenin-signaling pathway in gastric adenocarcinoma cells.

Gene Silencing and Activation of Human Papillomavirus 18 Is Modulated by Sense Promoter Associated RNA in Bidirectionally Transcribed Long Control Region.

BACKGROUND: Recently various studies have demonstrated the role of promoter associated non-coding RNAs (pRNA) in dsRNA induced transcriptional gene silencing and activation. However the exact mechanistic details of these processes with respect to the orientation of pRNAs are poorly defined. METHODOLOGY/PRINCIPAL FINDINGS: We have identified novel sense and antisense long control region (LCR) associated RNAs (pRNAs) in HPV18 positive cervical cancer cell lines HeLa, C-4 I and C-4 II. Using dsRNAs against these pRNAs, we were able to achieve upregulation or downregulation of the sense and antisense pRNAs and the downstream E6 and E7 oncogenes. We present evidence that knockdown of the sense pRNA is associated with reduction in E6 and E7 oncogenes and an upregulation of antisense pRNA. Conversely upregulation of sense pRNA is accompanied by an induction of the oncogenes and a concomitant reduction in antisense pRNA. Moreover, the exact role of sense and antisense pRNAs in dsRNA mediated gene modulation was confirmed by their selective degradation using antisense phosphorothioate oligodeoxynucleotides (ODN). Degradation of sense pRNA with antisense ODN led to loss of dsRNA induced silencing and activation, suggesting that dsRNA mediated gene modulation requires sense pRNA. Both processes were accompanied with congruent changes in the methylation pattern of activating and repressive histones. CONCLUSION/SIGNIFICANCE: Thus this data identifies and demonstrates the role of previously unknown important regulatory transcripts in HPV18 gene expression which can prove valuable targets in cervical cancer therapeutics. This mode of gene regulation by bidirectional transcription could be operational in other promoters as well and serve as a mechanism of regulating gene expression.