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.