The miR-17-5p microRNA is a key regulator of the G1/S phase cell cycle transition

Background

MicroRNAs are modifiers of gene expression, acting to reduce translation through either translational repression or mRNA cleavage. Recently, it has been shown that some microRNAs can act to promote or suppress cell transformation, with miR-17-92 described as the first oncogenic microRNA. The association of miR-17-92 encoded microRNAs with a surprisingly broad range of cancers not only underlines the clinical significance of this locus, but also suggests that miR-17-92 may regulate fundamental biological processes, and for these reasons miR-17-92 has been considered as a therapeutic target.

Results

In this study, we show that miR-17-92 is a cell cycle regulated locus, and ectopic expression of a single microRNA (miR-17-5p) is sufficient to drive a proliferative signal in HEK293T cells. For the first time, we reveal the mechanism behind this response - miR-17-5p acts specifically at the G1/S-phase cell cycle boundary, by targeting more than 20 genes involved in the transition between these phases. While both pro- and anti-proliferative genes are targeted by miR-17-5p, pro-proliferative mRNAs are specifically up-regulated by secondary and/or tertiary effects in HEK293T cells.

Conclusion

The miR-17-5p microRNA is able to act as both an oncogene and a tumor suppressor in different cellular contexts; our model of competing positive and negative signals can explain both of these activities. The coordinated suppression of proliferation-inhibitors allows miR-17-5p to efficiently de-couple negative regulators of the MAPK (mitogen activated protein kinase) signaling cascade, promoting growth in HEK293T cells. Additionally, we have demonstrated the utility of a systems biology approach as a unique and rapid approach to uncover microRNA function.     

Automatic discovery of sub-molecular sequence domains in multi-aligned sequences: a dynamic programming algorithm for multiple alignment segmentation

Automatic identification of sub-structures in multi-aligned sequences is of great importance for effective and objective structural/functional domain annotation, phylogenetic treeing and other molecular analyses. We present a segmentation algorithm that optimally partitions a given multi-alignment into a set of potentially biologically significant blocks, or segments. This algorithm applies dynamic programming and progressive optimization to the statistical profile of a multi-alignment in order to optimally demarcate relatively homogenous sub-regions. Using this algorithm, a large multi-alignment of eukaryotic 16S rRNA was analyzed. Three types of sequence patterns were identified automatically and efficiently: shared conserved domain; shared variable motif; and rare signature sequence. Results were consistent with the patterns identified through independent phylogenetic and structural approaches. This algorithm facilitates the automation of sequence-based molecular structural and evolutionary analyses through statistical modeling and high performance computation.     

Purification and characterization of West Nile virus nucleoside triphosphatase (NTPase)/helicase: evidence for dissociation of the NTPase and helicase activities of the enzyme

The nucleoside triphosphatase (NTPase)/helicase associated with nonstructural protein 3 of West Nile (WN) virus was purified from cell culture medium harvested from virus-infected Vero cells. The purification procedure included sequential chromatography on Superdex-200 and Reactive Red 120 columns, followed by a concentration step on an Ultrogel hydroxyapatite column. The nature of the purified protein was confirmed by immunoblot analysis using a WN virus-positive antiserum, determination of its NH(2) terminus by microsequencing, and a binding assay with 5'-[(14)C]fluorosulfonylbenzoyladenosine. Under optimized reaction conditions the enzyme catalyzed the hydrolysis of ATP and the unwinding of the DNA duplex with k(cat) values of 133 and 5.5 x 10(-3) s(-1), respectively. Characterization of the NTPase activity of the WN virus enzyme revealed that optimum conditions with respect to the Mg(2+) requirement and the monovalent salt or polynucleotide response differed from those of other flavivirus NTPases. Initial kinetic studies demonstrated that the inhibition (or activation) of ATPase activity by ribavirin-5'-triphosphate is not directly related to changes in the helicase activity of the enzyme. Further analysis using guanine and O(6)-benzoylguanine derivatives revealed that the ATPase activity of WN virus NTPase/helicase may be modulated, i.e., increased or reduced, with no effect on the helicase activity of the enzyme. On the other hand the helicase activity could be modulated without changing the ATPase activity. Our observations show that the number of ATP hydrolysis events per unwinding cycle is not a constant value.     

1H NMR conformational study of antiherpetic C5-substituted 2'-deoxyuridines: insight into the nature of structure-activity relationships

1H NMR study and conformational analysis of a broad series of biologically important C5-substituted 2'-deoxyuridines, including alkyl, halogen, vinyl, hydroxymethyl, and hydroxy derivatives as well as nitro, formyl, trifluoromethyl, and dimethylamino substituents, is presented. A thorough analysis of chemical shifts in correlation with C5-substituent electronegativity as well as calculations by SCF semi-empirical method of the formal charge localized on C6 carbon is discussed in terms of charge distribution for electron attracting and electron donating groups. Conformation of the sugar ring is determined from proton-proton coupling constants and described in terms of pseudorotation between two main puckering domains C2'endo (S) and C3'endo (N). Generally, electron donating groups destabilise the N conformation, simultaneously decreasing the mean pseudorotation amplitude. Absolute assignments of the H5' and H5' methylene protons in 1H NMR spectra permitted the unequivocal determination of molar fractions of the three classical exocyclic C4'-C5' rotamers gauche+, trans, and gauche-, and correlation of them with the sugar ring puckering domains. Conformation about the glycosidic bond is described in terms of equilibrium between two conformational regions, anti and syn. Finally, the role of the C5-substituent in the creation of cytotoxic activity is considered on the basis of a simplified model assuming that compound activity is a function of substituent polar surface, its molecular volume, and its molecule polarity defined at the relative partition of the polar atoms.     

Interaction of the 5'-phosphates of the anti-HIV agents, 3'-azido-3'-deoxythymidine and 3'-azido-2',3'-dideoxyuridine, with thymidylate synthase

A study has been made of the interaction of 3'-azido-3'-deoxythymidine 5'-phosphate (AZTMP) and 3'-azido-2',3'-dideoxy-uridine 5'-phosphate (AZdUMP) with thymidylate synthase. With the enzyme from L1210 cells and the tapeworm Hymenolepis diminuta, AZTMP was a weak inhibitor competitive with respect to dUMP (Ki = 6.3 mM and 0.5 mM); hence cytotoxicity of AZT, in cells in which accumulation of AZTMP is not high, is not due to inhibition of cellular thymidylate synthase. AZdUMP, with the L1210 enzyme, was a weak substrate (competition with dUMP described by apparent Ki = 4.7 mM), excluding conversion of AZdUMP to AZTMP as a source of toxicity of 3'-azido-2',3'-dideoxyuridine. An efficient procedure is described for enzymatic phosphorylation on a preparative scale of dideoxynucleosides.     

Theory of spatial position and spatial frequency relations in the receptive fields of simple cells in the visual cortex

Striate cells showing linear spatial summation obey very general mathematical inequalities relating the size of their receptive fields to the corresponding spatial frequency and orientation tuning characteristics. The experimental data show that, in the preferred direction of stimulus motion, the spatial response profiles of cells in the simple family are well described by the mathematical form of Gabor elementary signals. The product of the uncertainties in signalling spatial position (x) and spatial frequency (f) has, therefore, a theoretical minimum value of xf=1/2. We examine the implications that these conclusions have for the relationship between the spatial response profiles of simple cells and the characteristics of their spatial frequency tuning curves. Examples of the spatial frequency tuning curves and their associated spatial response profiles are discussed and illustrated. The advantages for the operation of the visual system of different relationships between the spatial response profiles and the characteristics of the spatial frequency tuning curves are examined. Two examples are discussed in detail, one system having a constant receptive field size and the other a constant bandwidth.     

Exotic sturgeons in the Vistula Lagoon in 2011, their occurrence,diet and parasites,with notes on the fishery background

The Siberian sturgeon Acipenser baerii and the sterlet Acipenser ruthenus were recorded for the first time in the Vistula Lagoon in 2011. Among 66 sturgeons collected between April and December 2011, the Siberian sturgeon was the most numerous species (77%); however, a significant seasonal variability was observed, with sterlet dominating in the catches in late autumn of the same year. The stomach contents of the two species differed widely: Siberian sturgeon (14.9–42.2 cm standard length, SL) fed on crustaceans (Cercopagis pengoi, Oithona sp., Neomysis integer) (10.7% IRI), larvae and pupas of insects (Chaoborus sp., Chironomus sp., Polypedilum sp., Procladius sp., Culex sp.) (88.9% IRI) and fishes (Neogobius melanostomus, Osmerus eperlanus) (0.5% IRI), whereas sterlet (24.0–34.4 cm SL) consumed crustaceans (N. integer) (64.3% IRI), larvae of insects (Chironomus sp., Polypedilum sp.) (20.3% IRI) and fish (N. melanostomus) (15.4% IRI). Single Siberian sturgeon (4.3%) were found to harbour the parasitic nematode (Raphidascaris acus).     

Rapid, sequential changes in surface morphology of PC12 pheochromocytoma cells in response to nerve growth factor

The effect of nerve growth factor (NGF), a substance that promotes the differentiation and maintenance of certain neurons, was studied via scanning electron microscopy utilizing the PC12 clonal NGF-responsive pheochromocytoma cell line. After 2-4 d of exposure to NGF, these cells acquire many of the properties of normal sympathic neurons. However, by phase microscopy, no changes are discernible within the first 12-18 h. Since the primary NGF receptor appears to be a membrane receptor, it seemed likely that some of the initial responses to the factor may be surface related. PC12 cells maintained without NGF are round to ovoid and have numerous microvilli and small blebs. After the addition of NGF, there is a rapidly initiated sequential change in the cell surface. Ruffles appear over the dorsal surface of the cells with 1 min, become prominent by 3 min, and almost disappear by 7 min. Microvilli, conversely, disappear as the dorsal ruffles become prominent. Ruffles are seen at the the periphery of cell at 3 min, are prominent on most of the cells by 7 min and are gone by 15 min. The surface remains smooth from 15 min until 45 min when large blebs appear. The large blebs are present on most cells at 2 h and are gone by 4 h. The surface remains relatively smooth until 6-7 h of NGF treatment, when microvilli reappear as small knobs. These microvilli increase in both number and length to cover the cell surface by 10 h. These changes were not observed with other basic proteins, with α-bungarotoxin (which binds specifically to PC12 membranes), and were not affected by an RNA synthesis inhibitor that blocks initiation of neurite outgrowth. Changes in the cell surface architecture appear to be among the earlist NGF responses yet detected and may represent or reflect primary events in the mechanism of the factor’s action.     

Interactions of 2'-fluoro-substituted dUMP analogues with thymidylate synthase

A series of 2'-fluoro-substituted dUMP/FdUMP analogues were synthesized, their interaction with human recombinant thymidylate synthase investigated, and structural (1)H and (19)F NMR study of the corresponding nucleosides performed. While 2'-F-dUMP (fluorine in the "down" configuration), in striking contrast to 2'-F-ara-UMP (fluorine in the "up" configuration) and 2',2'-diF-dUMP, showed substrate activity, 2'-F-ara-UMP and 2',2'-diF-dUMP were classic inhibitors, and 2',5-diF-ara-UMP behaved as a strong slow-binding inhibitor, suggesting the 2'-F substituent in the "up" position to interfere with the active center cysteine thiol addition to the pyrimidine C(6) and the pyrimidine C(5)-F to prevent this interference. In support, the direct through space heteronuclear coupling J(HF) was observed for the fluorine "up" derivatives, 2'-F-ara-U and 2',5-diF-ara-U, causing the splitting of the H(6) resonance lines. The absence of such splitting in 2',2'-diF-dUrd, indicating an unusual orientation of the base in relation to the furanose, was associated with an exceptionally weak interaction with the enzyme.