Poly(ADP-Ribose) polymerase 1 (PARP-1) regulates ribosomal biogenesis in Drosophila nucleoli.

Poly(ADP-ribose) polymerase 1 (PARP1), a nuclear protein, utilizes NAD to synthesize poly(AD-Pribose) (pADPr), resulting in both automodification and the modification of acceptor proteins. Substantial amounts of PARP1 and pADPr (up to 50%) are localized to the nucleolus, a subnuclear organelle known as a region for ribosome biogenesis and maturation. At present, the functional significance of PARP1 protein inside the nucleolus remains unclear. Using PARP1 mutants, we investigated the function of PARP1, pADPr, and PARP1-interacting proteins in the maintenance of nucleolus structure and functions. Our analysis shows that disruption of PARP1 enzymatic activity caused nucleolar disintegration and aberrant localization of nucleolar-specific proteins. Additionally, PARP1 mutants have increased accumulation of rRNA intermediates and a decrease in ribosome levels. Together, our data suggests that PARP1 enzymatic activity is required for targeting nucleolar proteins to the proximity of precursor rRNA; hence, PARP1 controls precursor rRNA processing, post-transcriptional modification, and pre-ribosome assembly. Based on these findings, we propose a model that explains how PARP1 activity impacts nucleolar functions and, consequently, ribosomal biogenesis.

Poly (ADP-ribose) polymerase 1 is required for protein localization to Cajal body.

Recently, the nuclear protein known as Poly (ADP-ribose) Polymerase1 (PARP1) was shown to play a key role in regulating transcription of a number of genes and controlling the nuclear sub-organelle nucleolus. PARP1 enzyme is known to catalyze the transfer of ADP-ribose to a variety of nuclear proteins. At present, however, while we do know that the main acceptor for pADPr in vivo is PARP1 protein itself, by PARP1 automodification, the significance of PARP1 automodification for in vivo processes is not clear. Therefore, we investigated the roles of PARP1 auto ADP-ribosylation in dynamic nuclear processes during development. Specifically, we discovered that PARP1 automodification is required for shuttling key proteins into Cajal body (CB) by protein non-covalent interaction with pADPr in vivo. We hypothesize that PARP1 protein shuttling follows a chain of events whereby, first, most unmodified PARP1 protein molecules bind to chromatin and accumulate in nucleoli, but then, second, upon automodification with poly(ADP-ribose), PARP1 interacts non-covalently with a number of nuclear proteins such that the resulting protein-pADPr complex dissociates from chromatin into CB.

Deregulation of HEF1 impairs M-phase progression by disrupting the RhoA activation cycle.

The focal adhesion-associated signaling protein HEF1 undergoes a striking relocalization to the spindle at mitosis, but a function for HEF1 in mitotic signaling has not been demonstrated. We here report that overexpression of HEF1 leads to failure of cells to progress through cytokinesis, whereas depletion of HEF1 by small interfering RNA (siRNA) leads to defects earlier in M phase before cleavage furrow formation. These defects can be explained mechanistically by our determination that HEF1 regulates the activation cycle of RhoA. Inactivation of RhoA has long been known to be required for cytokinesis, whereas it has recently been determined that activation of RhoA at the entry to M phase is required for cellular rounding. We find that increased HEF1 sustains RhoA activation, whereas depleted HEF1 by siRNA reduces RhoA activation. Furthermore, we demonstrate that chemical inhibition of RhoA is sufficient to reverse HEF1-dependent cellular arrest at cytokinesis. Finally, we demonstrate that HEF1 associates with the RhoA-GTP exchange factor ECT2, an orthologue of the Drosophila cytokinetic regulator Pebble, providing a direct means for HEF1 control of RhoA. We conclude that HEF1 is a novel component of the cell division control machinery and that HEF1 activity impacts division as well as cell attachment signaling events.

Human papillomavirus type 11 alters the transcription and expression of loricrin, the major cell envelope protein.

Human papillomavirus (HPV) does not induce lysis of infected keratinocytes, and the exact mechanisms of viral escape are not known. As keratinocytes differentiate, the cornified cell envelope (CCE) develops, providing a protective barrier to the host. We previously showed that the normally durable CCE in HPV 11-infected epithelium is fragile compared to CCEs in healthy epithelium. In this study, we examined uninfected and HPV 11-infected human genital epithelium for expression of loricrin, the major CCE protein in healthy epidermis. In HPV 11-infected human genital epithelium, detection of loricrin was reduced in immunoelectron microscopic and immunoblot assays, suggesting that loricrin incorporation into the CCE was reduced or that loricrin synthesis was reduced. Loricrin detection was reduced in immunohistochemical assays in areas of high viral replication. Mathematical modeling by least squares was performed using the amino acid composition of highly purified CCE fragments, confirming that loricrin was markedly reduced and that the small proline-rich proteins and cytokeratins were increased in those derived from HPV 11-infected epithelium compared to healthy genital epithelium. In RNase protection and RT-PCR assays, loricrin transcripts were markedly reduced in HPV 11-infected epithelium compared to uninfected epithelium. Loricrin transcripts were detectable by RNA in situ analysis in isolated cells of HPV 11-infected epithelium, but were absent in large regions of epithelium. We conclude that HPV 11 infection reduces transcription of the loricrin gene and that this leads to a reduction in loricrin incorporation into the CCE. Further studies will examine the effects of specific HPV gene products on transcription of loricrin and other CCE components, as it is likely that viral egress from the infected keratinocyte depends on these effects.