SummaryMammalian genomes are promiscuously transcribed, yielding protein-coding and non-coding products. Many transcripts are short lived due to their nuclear degradation by the ribonucleolytic RNA exosome. Here, we show that abolished nuclear exosome function causes the formation of distinct nuclear foci, containing polyadenylated (pA+) RNA secluded from nucleocytoplasmic export. We asked whether exosome co-factors could serve such nuclear retention. Co-localization studies revealed the enrichment of pA+ RNA foci with “pA-tail exosome targeting (PAXT) connection” components MTR4, ZFC3H1, and PABPN1 but no overlap with known nuclear structures such as Cajal bodies, speckles, paraspeckles, or nucleoli. Interestingly, ZFC3H1 is required for foci formation, and in its absence, selected pA+ RNAs, including coding and non-coding transcripts, are exported to the cytoplasm in a process dependent on the mRNA export factor AlyREF. Our results establish ZFC3H1 as a central nuclear pA+ RNA retention factor, counteracting nuclear export activity.

Brd4 protein has been proposed to act as a cellular receptor for the bovine papillomavirus type 1 (BPV1) E2 protein in the E2-mediated chromosome attachment and mitotic segregation of viral genomes. Here, we provide data that show the involvement of Brd4 in multiple early functions of the BPV1 life cycle, suggest a Brd4-dependent mechanism for E2-dependent transcription activation, and indicate the role of Brd4 in papillomavirus and polyomavirus replication as well as cell-specific utilization of Brd4-linked features in BPV1 DNA replication. Our data also show the potential therapeutic value of the disruption of the E2-Brd4 interaction for the development of antiviral drugs.Papillomavirus (PV) E2 protein is a central regulator of the viral life cycle. In addition to its well-established activity as a transcription modulator and replication initiator protein (6), E2 of bovine papillomavirus type 1 (BPV1) has recently emerged as a trans factor which mediates mitotic segregation of viral genomes by tethering them to host cell chromatin (7,12,19). The first candidate for a receptor of E2 in the latter process, Brd4, is attached to the chromatin through its two bromodomains, which bind to acetylated histones H3 and H4 both in interphase and in mitosis (4, 25). Mutated E2 proteins that are defective in Brd4 binding are unable to bind to mitotic chromosomes (2), and ectopic expression of Brd4 can reconstitute the BPV1 E2-dependent extrachromosomal plasmid maintenance in the yeast Saccharomyces cerevisiae, where such a process normally does not function (3). Ectopic expression of the E2-binding C-terminal domain (CTD) of Brd4 in mammalian cells disrupts the interaction of E2 with cellular Brd4 and relocates E2 from mitotic chromosomes (25,26). Brd4 CTD binds to the N-terminal domain of E2 (25), which is also responsible for interactions critical for transcription activation and replication initiator activities of E2. Therefore, we suspected that Brd4 might have a more complex role in the PV life cycle than initially proposed. We tested this idea in the present study and show that the Brd4 bromodomain protein can indeed participate in the BPV1 E2-dependent transcription activation and DNA replication processes. Brd4 is specifically involved in the E2-activated transcription process; the role of Brd4 in BPV1 DNA replication, however, is either largely or completely independent of its binding to E2. Our data demonstrate the possible involvement of Brd4 also in polyomavirus DNA replication and reveal the varying importance of the Brd4-linked component for BPV1 DNA replication in different cell lines.Cloning of the dominant-negative form of Brd4 (Brd4 CTD). The use of a dominant-negative truncated version of Brd4 is a useful alternative to manipulations with a full-length gene, as overexpression or knockout of Brd4 in mammalian cells has been shown to cause severe alterations in cell growth (5, 15). Overexpression of Brd4 CTD affects neither the growth of several cell lines (C127, C33A, HeLa) (25, 26) nor the cell cycle dist...

Papillomaviruses establish their productive life cycle in stratified epithelium or mucosa, where the undifferentiated proliferating keratinocytes are the initial targets for the productive viral infection. Papillomaviruses have evolved mechanisms to adapt to the normal cellular growth control pathways and to adjust their DNA replication and maintenance cycle to contend with the cellular differentiation. We provide overview of the papillomavirus DNA replication in the differentiating epithelium and describe the molecular interactions important for viral DNA replication on all steps of the viral life cycle.

Recruitment of the human ribonucleolytic RNA exosome to nuclear polyadenylated (pA+) RNA is facilitated by the Poly(A) Tail eXosome Targeting (PAXT) connection. Besides its core dimer, formed by the exosome co-factor MTR4 and the ZFC3H1 protein, the PAXT connection remains poorly defined. By characterizing nuclear pA+-RNA bound proteomes as well as MTR4-ZFC3H1 containing complexes in conditions favoring PAXT assembly, we here uncover three additional proteins required for PAXT function: ZC3H3, RBM26 and RBM27 along with the known PAXT-associated protein, PABPN1. The zinc-finger protein ZC3H3 interacts directly with MTR4-ZFC3H1 and loss of any of the newly identified PAXT components results in the accumulation of PAXT substrates. Collectively, our results establish new factors involved in the turnover of nuclear pA+ RNA and suggest that these are limiting for PAXT activity.

NEAT1 is one of the most studied lncRNAs, in part because its silencing in mice causes defects in mammary gland development and corpus luteum formation and protects them from skin cancer development. Moreover, depleting NEAT1 in established cancer cell lines reduces growth and sensitizes cells to DNA damaging agents. However, NEAT1 produces two isoforms and because the short isoform, NEAT1_1, completely overlaps the 5 ′ ′ ′ ′ ′ part of the long NEAT1_2 isoform; the respective contributions of each of the isoforms to these phenotypes has remained unclear. Whereas NEAT1_1 is highly expressed in most tissues, NEAT1_2 is the central architectural component of paraspeckles, which are nuclear bodies that assemble in specific tissues and cells exposed to various forms of stress. Using dual RNA-FISH to detect both NEAT1_1 outside of the paraspeckles and NEAT1_2/NEAT1 inside this nuclear body, we report herein that NEAT1_1 levels are dynamically regulated during the cell cycle and targeted for degradation by the nuclear RNA exosome. Unexpectedly, however, cancer cells engineered to lack NEAT1_1, but not NEAT1_2, do not exhibit cell cycle defects. Moreover, Neat1_1-specific knockout mice do not exhibit the phenotypes observed in Neat1-deficient mice. We propose that NEAT1 functions are mainly, if not exclusively, attributable to NEAT1_2 and, by extension, to paraspeckles.

Effective segregation of the bovine papillomavirus type 1 (BPV1), Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated human herpesvirus type 8 (KSHV) genomes into daughter cells is mediated by a single viral protein that tethers viral genomes to host mitotic chromosomes. The linker proteins that mediate BPV1, EBV, and KSHV segregation are E2, LANA1, and EBNA1, respectively. The N-terminal transactivation domain of BPV1 E2 is responsible for chromatin attachment and subsequent viral genome segregation. Because E2 transcriptional activation and chromatin attachment functions are not mutually exclusive, we aimed to determine the requirement of these activities during segregation by analyzing chimeric E2 proteins. This approach allowed us to separate the two activities. Our data showed that attachment of the segregation protein to chromatin is not sufficient for proper segregation. Rather, formation of a segregation-competent complex which carries multiple copies of the segregation protein is required. Complementation studies of E2 functional domains indicated that chromatin attachment and transactivation functions must act in concert to ensure proper plasmid segregation. These data indicate that there are specific interactions between linker molecules and transcription factors/complexes that greatly increase segregation-competent complex formation. We also showed, using hybrid E2 molecules, that restored segregation function does not involve interactions with Brd4.Several animal DNA viruses, including bovine papillomavirus type 1 (BPV1), Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated human herpesvirus type 8 (KSHV), maintain their genomes as nuclear extrachromosomal multicopy plasmids in latently infected proliferating host cells. Each of these viruses carries a sequence-specific DNA binding protein which contains the domain required to bind specific receptor complexes on mitotic chromatin (reviewed in references 15, 27, and 43). Viral genomes are tethered to host mitotic chromosomes by segregation proteins at multimeric binding sites. During cell division, the viral genome is partitioned into daughter cells. Therefore, it is widely accepted that segregation protein tethering and multimeric binding to the viral genome are necessary and sufficient to impart a segregation/partitioning function to viral genomes in proliferating cells. However, the identity of the mitotic receptor and how it interacts with the segregation proteins remain elusive.The E2 protein provides a segregation/partitioning function in BPV1 by binding of E2 multimeric binding sites. E2 is a multifunctional sequence-specific DNA binding protein that is required for viral genome replication (10, 62, 67-69), transcriptional activity (60, 65), and viral genome maintenance (2,23,38,51,59). The E2 protein is comprised of an N-terminal transactivation domain (TAD), a flexible hinge region, and a C-terminal DNA binding/dimerization domain (19). BPV1 E2 associates with mitotic chromosomes throughout mitosis, and this interaction is depende...

PurposeThe National Comprehensive Cancer Network (NCCN) has proposed guidelines for the genetic testing of the BRCA1 and BRCA2 genes, based on studies in western populations. This current study assessed potential predictive factors for BRCA mutation probability, in an Asian population.MethodsA total of 359 breast cancer patients, who presented with either a family history (FH) of breast and/or ovarian cancer or early onset breast cancer, were accrued at the National Cancer Center Singapore (NCCS). The relationships between clinico-pathological features and mutational status were calculated using the Chi-squared test and binary logistic regression analysis.ResultsOf 359 patients, 45 (12.5%) had deleterious or damaging missense mutations in BRCA1 and/or BRCA2. BRCA1 mutations were more likely to be found in ER-negative than ER-positive breast cancer patients (P=0.01). Moreover, ER-negative patients with BRCA mutations were diagnosed at an earlier age (40 vs. 48 years, P=0.008). Similarly, triple-negative breast cancer (TNBC) patients were more likely to have BRCA1 mutations (P=0.001) and that these patients were diagnosed at a relatively younger age than non-TNBC patients (38 vs. 46 years, P=0.028). Our analysis has confirmed that ER-negative status, TNBC status and a FH of hereditary breast and ovarian cancer (HBOC) are strong factors predicting the likelihood of having BRCA mutations.ConclusionsOur study provides evidence that TNBC or ER-negative patients may benefit from BRCA genetic testing, particularly younger patients (<40 years) or those with a strong FH of HBOC, in Asian patients.

Bovine papillomavirus type 1 (BPV1), Epstein-Barr virus (EBV), and human herpesvirus 8 genomes are stably maintained as episomes in dividing host cells during latent infection. The mitotic segregation/partitioning function of these episomes is dependent on single viral protein with specific DNA-binding activity and its multimeric binding sites in the viral genome. In this study we show that, in the presence of all essential viral trans factors, the segregation/partitioning elements from both BPV1 and EBV can provide the stable maintenance function to the mouse polyomavirus (PyV) core origin plasmids but fail to do so in the case of complete PyV origin. Our study is the first which follows BPV1 E2-and minichromosome maintenance element (MME)-dependent stable maintenance function with heterologous replication origins. In mouse fibroblast cell lines expressing PyV large T antigen (LT) and either BPV1 E2 or EBV EBNA1, the long-term episomal replication of plasmids carrying the PyV minimal origin together with the MME or family of repeats (FR) element can be monitored easily for 1 month under nonselective conditions. Our data demonstrate clearly that the PyV LT-dependent replication function and the segregation/partitioning function of the BPV1 or EBV are compatible in certain, but not all, configurations. The quantitative analysis indicates a loss rate of 6% per cell, doubling in the case of MME-dependent plasmids, and 13% in the case of FR-dependent plasmids in nonselective conditions. Our data clearly indicate that maintenance functions from different viruses are principally interexchangeable and can provide a segregation/partitioning function to different heterologous origins in a variety of cells.