Differential regulation of full-length genome and a single-stranded 7S DNA along the cell cycle in human mitochondria.

Mammalian mitochondria contain full-length genome and a single-stranded 7S DNA. Although the copy number of mitochondrial DNA (mtDNA) varies depending on the cell type and also in response to diverse environmental stresses, our understanding of how mtDNA and 7S DNA are maintained and regulated is limited, partly due to lack of reliable in vitro assay systems that reflect the in vivo functionality of mitochondria. Here we report an in vitro assay system to measure synthesis of both mtDNA and 7S DNA under a controllable in vitro condition. With this assay system, we demonstrate that the replication capacity of mitochondria correlates with endogenous copy numbers of mtDNA and 7S DNA. Our study also shows that higher nucleotide concentrations increasingly promote 7S DNA synthesis but not mtDNA synthesis. Consistently, the mitochondrial capacity to synthesize 7S DNA but not mtDNA noticeably varied along the cell cycle, reaching its highest level in S phase. These findings suggest that syntheses of mtDNA and 7S DNA proceed independently and that the mitochondrial capacity to synthesize 7S DNA dynamically changes not only with cell-cycle progression but also in response to varying nucleotide concentrations.

Regulation of the catalytic function of topoisomerase II alpha through association with RNA.

Topoisomerase IIalpha interacts with numerous nuclear factors, through which it is engaged in diverse nuclear events such as DNA replication, transcription and the formation or maintenance of heterochromatin. We previously reported that topoisomerase IIalpha interacts with RNA helicase A (RHA), consistent with a recent view that topoisomerases and helicases function together. Intrigued by our observation that the RHA-topoisomerase IIalpha interaction is sensitive to ribonuclease A, we explored whether the RHA-topoisomerase IIalpha interaction can be recapitulated in vitro using purified proteins and a synthetic RNA. This work led us to an unexpected finding that an RNA-binding activity is intrinsically associated with topoisomerase IIalpha. Topoisomerase IIalpha stably interacted with RNA harboring a 3'-hydroxyl group but not with RNA possessing a 3'-phosphate group. When measured in decatenation and relaxation assays, RNA binding influenced the catalytic function of topoisomerase IIalpha to regulate DNA topology. We discuss a possible interaction of topoisomerase IIalpha with the poly(A) tail and G/U-rich 3'-untranslated region (3'-UTR) of mRNA as a key step in transcription termination.

Transcriptional regulation of multi-drug tolerance and antibiotic-induced responses by the histone-like protein Lsr2 in M. tuberculosis.

Multi-drug tolerance is a key phenotypic property that complicates the sterilization of mammals infected with Mycobacterium tuberculosis. Previous studies have established that iniBAC, an operon that confers multi-drug tolerance to M. bovis BCG through an associated pump-like activity, is induced by the antibiotics isoniazid (INH) and ethambutol (EMB). An improved understanding of the functional role of antibiotic-induced genes and the regulation of drug tolerance may be gained by studying the factors that regulate antibiotic-mediated gene expression. An M. smegmatis strain containing a lacZ gene fused to the promoter of M. tuberculosis iniBAC (PiniBAC) was subjected to transposon mutagenesis. Mutants with constitutive expression and increased EMB-mediated induction of PiniBAC::lacZ mapped to the lsr2 gene (MSMEG6065), a small basic protein of unknown function that is highly conserved among mycobacteria. These mutants had a marked change in colony morphology and generated a new polar lipid. Complementation with multi-copy M. tuberculosis lsr2 (Rv3597c) returned PiniBAC expression to baseline, reversed the observed morphological and lipid changes, and repressed PiniBAC induction by EMB to below that of the control M. smegmatis strain. Microarray analysis of an lsr2 knockout confirmed upregulation of M. smegmatis iniA and demonstrated upregulation of genes involved in cell wall and metabolic functions. Fully 121 of 584 genes induced by EMB treatment in wild-type M. smegmatis were upregulated ("hyperinduced") to even higher levels by EMB in the M. smegmatis lsr2 knockout. The most highly upregulated genes and gene clusters had adenine-thymine (AT)-rich 5-prime untranslated regions. In M. tuberculosis, overexpression of lsr2 repressed INH-mediated induction of all three iniBAC genes, as well as another annotated pump, efpA. The low molecular weight and basic properties of Lsr2 (pI 10.69) suggested that it was a histone-like protein, although it did not exhibit sequence homology with other proteins in this class. Consistent with other histone-like proteins, Lsr2 bound DNA with a preference for circular DNA, forming large oligomers, inhibited DNase I activity, and introduced a modest degree of supercoiling into relaxed plasmids. Lsr2 also inhibited in vitro transcription and topoisomerase I activity. Lsr2 represents a novel class of histone-like proteins that inhibit a wide variety of DNA-interacting enzymes. Lsr2 appears to regulate several important pathways in mycobacteria by preferentially binding to AT-rich sequences, including genes induced by antibiotics and those associated with inducible multi-drug tolerance. An improved understanding of the role of lsr2 may provide important insights into the mechanisms of action of antibiotics and the way that mycobacteria adapt to stresses such as antibiotic treatment.

The growth factor granulin interacts with cyclin T1 and modulates P-TEFb-dependent transcription.

Cyclin T1, together with the kinase CDK9, is a component of the transcription elongation factor P-TEFb which binds the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. P-TEFb facilitates transcription by phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II. Cyclin T1 is an exceptionally large cyclin and is therefore a candidate for interactions with regulatory proteins. We identified granulin as a cyclin T1-interacting protein that represses expression from the HIV-1 promoter in transfected cells. The granulins, mitogenic growth factors containing repeats of a cysteine-rich motif, were reported previously to interact with Tat. We show that granulin formed stable complexes in vivo and in vitro with cyclin T1 and Tat. Granulin bound to the histidine-rich domain of cyclin T1, which was recently found to bind to the CTD, but not to cyclin T2. Binding of granulin to P-TEFb inhibited the phosphorylation of a CTD peptide. Granulin expression inhibited Tat transactivation, and tethering experiments showed that this effect was due, at least in part, to a direct action on cyclin T1 in the absence of Tat. In addition, granulin was a substrate for CDK9 but not for the other transcription-related kinases CDK7 and CDK8. Thus, granulin is a cellular protein that interacts with cyclin T1 to inhibit transcription.

RNA helicase A interacts with dsDNA and topoisomerase IIalpha.

RNA helicase A (RHA) is a multifunctional protein involved in various nuclear processes such as transcription and RNA export. It is believed that the interacting factors play important roles in determining the functional specificity of RHA. Here we show that RHA directly interacts with double-stranded (ds) nucleic acids (NAs) and assembles complexes with topoisomerase IIalpha. First, electrophoresis mobility shift assays demonstrate that RHA interacts with dsDNAs of different lengths ranging from 15 to 104 bp. Secondly, the binding of RHA to closed circular dsDNA stimulates the relaxation reaction catalyzed by either calf thymus topoisomerase I or HeLa topoisomerase IIalpha. Thirdly, immunoprecipitation, coupled with western blot analysis using anti-RHA and anti-topoisomerase IIalpha antibodies, shows that RHA and topoisomerase IIalpha assemble a complex in the presence of as yet unknown RNA molecules and additional protein factors such as Ubc9. Our observation suggests physical and functional interaction between RHA and topoisomerase IIalpha, which, perhaps, play important roles in regulating chromatin structure. The putative role of RHA-topoisomerase IIalpha complex in RNA polymerase II-mediated transcription is discussed.

A functional interaction between RHA and Ubc9, an E2-like enzyme specific for Sumo-1.

RNA helicase A (RHA) is a member of the DEAH helicase family of proteins. Recent studies imply the role of RHA in the regulation of the topology of chromatin DNA, which could influence diverse nuclear processes such as transcription activity of the chromatin DNA and chromosome condensation. We previously reported that Ubc9, an E2-like enzyme specific for small ubiquitin-like modifier 1 (Sumo-1), is required for the interaction between RHA and topoisomerase IIalpha. Here, we describe that Ubc9 is a novel factor that functionally interacts with RHA and activates the transcription activity of RHA, measured in the CREB-mediated pathway. We demonstrate that the N-terminal domain of RHA, encompassing amino acid residues 1-137, is sufficient for its interaction with Ubc9. Our data also show that interaction with Ubc9 leads to the Sumo-1 conjugation of RHA both in vitro and in vivo. However, the catalytic activity of Ubc9 seems to be dispensable for the transcription activation activity of RHA. Our observation suggests multiple roles for Ubc9 in the regulation of the RHA function.

Selective regulation of gene expression by nuclear factor 110, a member of the NF90 family of double-stranded RNA-binding proteins.

Members of the nuclear factor 90 (NF90) family of double-stranded RNA (dsRNA)-binding proteins have been implicated in several biological processes including the regulation of gene expression. cDNA sequences predict that the proteins have a functional nuclear localization signal and two dsRNA-binding motifs (dsRBMs), and are identical at their N termini. Isoforms are predicted to diverge at their C termini as well as by the insertion of four amino acid residues (NVKQ) between the two dsRBMs. In this study, we verified the expression of four of the isoforms by cDNA cloning and mass spectrometric analysis of proteins isolated from human cells. Cell fractionation studies showed that NF90 and its heteromeric partner, NF45, are predominantly nuclear and largely chromatin-associated. The C-terminally extended NF90 species, NF110, are almost exclusively chromatin-bound. Both NF110 isoforms are more active than NF90 isoforms in stimulating transcription from the proliferating cell nuclear antigen reporter in a transient expression system. NF110b, which carries the NVKQ insert, was identified as the strongest activator. It stimulated transcription of some, but not all, promoters in a fashion that suggested that it functions in concert with other transcription factors. Finally, we demonstrate that NF110b associates with the dsRBM-containing transcriptional co-activator, RNA helicase A, independently of RNA binding.

Identification of toposome, a novel multisubunit complex containing topoisomerase IIalpha.

Topoisomerase IIalpha plays essential roles in chromosome segregation. However, it is not well understood how topoisomerase IIalpha exerts its function during mitosis. In this report, we find that topoisomerase IIalpha forms a multisubunit complex, named toposome, containing two ATPase/helicase proteins (RNA helicase A and RHII/Gu), one serine/threonine protein kinase (SRPK1), one HMG protein (SSRP1), and two pre-mRNA splicing factors (PRP8 and hnRNP C). Toposome separates entangled circular chromatin DNA about fourfold more efficiently than topoisomerase IIalpha. Interestingly, this decatenation reaction yields knotted circles, which are not seen in reactions provided with monomeric circular DNA. Our results also show that interaction among toposome-associated proteins is highest in G2/M phase but drastically diminishes in G1/S phase. These results suggest that toposome is a dynamic complex whose assembly or activation is subject to cell cycle regulation.

Gene expression profile of HIV-1 Tat expressing cells: a close interplay between proliferative and differentiation signals.

BACKGROUND: Expression profiling holds great promise for rapid host genome functional analysis. It is plausible that host expression profiling in an infection could serve as a universal phenotype in virally infected cells. Here, we describe the effect of one of the most critical viral activators, Tat, in HIV-1 infected and Tat expressing cells. We utilized microarray analysis from uninfected, latently HIV-1 infected cells, as well as cells that express Tat, to decipher some of the cellular changes associated with this viral activator. RESULTS: Utilizing uninfected, HIV-1 latently infected cells, and Tat expressing cells, we observed that most of the cellular host genes in Tat expressing cells were down-regulated. The down-regulation in Tat expressing cells is most apparent on cellular receptors that have intrinsic receptor tyrosine kinase (RTK) activity and signal transduction members that mediate RTK function, including Ras-Raf-MEK pathway. Co-activators of transcription, such as p300/CBP and SRC-1, which mediate gene expression related to hormone receptor genes, were also found to be down-regulated. Down-regulation of receptors may allow latent HIV-1 infected cells to either hide from the immune system or avoid extracellular differentiation signals. Some of the genes that were up-regulated included co-receptors for HIV-1 entry, translation machinery, and cell cycle regulatory proteins. CONCLUSIONS: We have demonstrated, through a microarray approach, that HIV-1 Tat is able to regulate many cellular genes that are involved in cell signaling, translation and ultimately control the host proliferative and differentiation signals.

Regulation of inter- and intramolecular interaction of RNA, DNA, and proteins by MLE.

Drosophila maleless (MLE) is a member of helicase superfamily 2 and functions as a dosage compensation factor essential for the development of male flies. This function provides a good opportunity to investigate diverse biochemical activities associated with MLE in the context of a defined in vivo pathway, i.e., the transcriptional activation of X-linked genes. We have shown for the first time that MLE catalyzes the unwinding of both DNA and RNA and that MLE helicase activity is essential for its in vivo function. Also, we have provided evidence that MLE stimulates the transcriptional activity of roX2 on the X chromosome. We have also found that MLE interacts with dsDNA, topoisomerase II, and nucleosome. This observation supports a current model of dosage compensation: transcriptional activation of X-linked genes is causally associated with conformational change in the male X chromosome, subsequent to the targeted association of the dosage compensation complex (DCC).

Nuclear factor 45 (NF45) is a regulatory subunit of complexes with NF90/110 involved in mitotic control.

Nuclear factor 90 (NF90) and its C-terminally extended isoform, NF110, have been isolated as DNA- and RNA-binding proteins together with the less-studied protein NF45. These complexes have been implicated in gene regulation, but little is known about their cellular roles and whether they are redundant or functionally distinct. We show that heterodimeric core complexes, NF90-NF45 and NF110-NF45, exist within larger complexes that are more labile and contain multiple NF90/110 isoforms and additional proteins. Depletion of the NF45 subunit by RNA interference is accompanied by a dramatic decrease in the levels of NF90 and NF110. Reciprocally, depletion of NF90 but not of NF110 greatly reduces the level of NF45. Coregulation of NF90 and NF45 is a posttranscriptional phenomenon, resulting from protein destabilization in the absence of partners. Depletion of NF90-NF45 complexes retards cell growth by inhibition of DNA synthesis. Giant multinucleated cells containing nuclei attached by constrictions accumulate when either NF45 or NF90, but not NF110, is depleted. This study identified NF45 as an unstable regulatory subunit of NF90-NF45 complexes and uncovered their critical role in normal cell division. Furthermore, the study revealed that NF90 is functionally distinct from NF110 and is more important for cell growth.

RH70, a bidirectional RNA helicase, co-purifies with U1snRNP.

In this report, I describe the co-purification of a novel 70-kDa RNA helicase (RH70) and U1snRNP through six column steps. Peptide sequence analysis by mass spectrometry and Edman degradation revealed that RH70 is the previously reported DDX17. Biochemical characterization of RH70, obtained by partial separation from U1snRNP, yielded the following results. (a) RH70 mediates the unwinding of duplex RNA but not DNA in an ATP-dependent manner. (b) Both the RNA-dependent ATPase and RNA helicase activities of RH70 are highly specific for ATP, exhibiting an apparent K(m) of 0.5 mm. (c) RH70 catalyzes the unwinding of duplex RNA containing single-stranded regions at either the 5'- or 3'-end. Its association with U1snRNP and ATP specificity suggest a role for RH70 in pre-mRNA splicing, in particular, at the early stages of the splicing reaction involving U1snRNP.

MLE functions as a transcriptional regulator of the roX2 gene.

Dosage compensation is a process that equalizes transcription activity between the sexes. In Drosophila, two non-coding RNA, roX1 and roX2, and at least six protein regulators, MSL-1, MSL-2, MSL-3, MLE, MOF, and JIL-1, have been identified as essential for dosage compensation. Although there is accumulating evidence of the intricate functional and physical interactions between protein and RNA regulators, little is known about how roX RNA expression and function are modulated in coordination with protein regulators. In this report, we have found that a relatively short (about 350 bp) upstream genomic region of the roX2 gene, Prox2, harbors an activity that drives transcription of the downstream gene. Our study has shown that MLE can stimulate the transcription activity of Prox2 and that MLE associates with Prox2 through direct interaction with a newly identified 54-bp repeat, Prox. Our observations suggest a novel mechanism by which roX2 RNA is regulated at the transcriptional level.

Effect of an exogenous trigger on the pathogenesis of lupus in (NZB x NZW)F1 mice.

OBJECTIVE: This study examined the interactions between exogenous and endogenous factors shaping the phenotype of lupus in autoimmune (NZB x NZW)F(1) mice exposed to pristane, a model environmental trigger. METHODS: Frequencies of various autoantibodies in untreated NZB/NZW mice were determined by various means (immunoprecipitation, enzyme-linked immunosorbent assay [ELISA], Crithidia luciliae kinetoplast staining). Pristane or saline was administered intraperitoneally to 9-12-week-old NZB/NZW mice, followed by serial studies of autoantibodies, total Ig levels (ELISA), and proteinuria (dipstick). RESULTS: Besides antichromatin/DNA responses, NZB/NZW mice spontaneously produced novel autoantibodies against the double-stranded RNA binding protein RNA helicase A (RHA). In contrast, NZB/NZW mice (n = 70) did not produce autoantibodies against the nuclear RNP (nRNP), Sm, Ro, or La antigens. Pristane exposure synergistically activated the production of antichromatin/DNA antibodies and dramatically accelerated renal disease. Production of anti-nRNP/Sm and Su autoantibodies also was induced, indicating that the unresponsiveness of NZB/NZW mice to these antigens can be overcome. Curiously, pristane treatment did not enhance the production of anti-RHA, suggesting that these autoantibodies are regulated differently than anti-DNA/chromatin and Sm. In contrast to previous reports that suggest a critical role of deficient interleukin-12 (IL-12) production in the pathogenesis of lupus, there was overproduction of IL-12 in the peritoneal cavity of pristane-treated NZB/NZW mice, and their spleen cells also produced large amounts of IL-12. CONCLUSION: These data lead us to propose that environmental influences exacerbate autoimmune manifestations in genetically lupus-susceptible mice through their stimulatory effects on proinflammatory cytokines, such as IL-12.

DEN1 is a dual function protease capable of processing the C terminus of Nedd8 and deconjugating hyper-neddylated CUL1.

Nedd8 activates ubiquitination by increasing the efficiency of polyubiquitin chain assembly through its covalent conjugation to cullin molecules. Here we report the isolation, cloning, and characterization of a novel human Nedd8-specific protease called DEN1. Human DEN1 is encoded by AAH31411.1, a previously uncharacterized protein of 212 amino acids that shares homology with the Ulp1 cysteinyl SUMO deconjugating enzyme family. Recombinant human DEN1, purified from bacteria, selectively binds to Nedd8 and hydrolyzes C-terminal derivatives of Nedd8. Interestingly, DEN1 deconjugates cullin 1 (CUL1)-Nedd8 in a concentration-dependent manner. At a low concentration, DEN1 processes hyper-neddylated CUL1 to yield a mononeddylated form, which presumably contains the Lys-720CUL1-Nedd8 linkage. At elevated concentrations, DEN1 is able to complete the removal of Nedd8 from CUL1. These activities distinguish DEN1 from the COP9 signalosome, which is capable of efficiently cleaving the Lys-720CUL1-Nedd8 conjugate, but lacks Nedd8 C-terminal hydrolytic activity and poorly processes hyperneddylated CUL1. These results suggest a unique role for DEN1 in regulating the modification of cullins by Nedd8.