Circadian peak dopaminergic activity response at the biological clock pacemaker (suprachiasmatic nucleus) area mediates the metabolic responsiveness to a high-fat diet.

Among vertebrate species of the major vertebrate classes in the wild, a seasonal rhythm of whole body fuel metabolism, oscillating from a lean to obese condition, is a common biological phenomenon. This annual cycle is driven in part by annual changes in the circadian dopaminergic signalling at the suprachiasmatic nuclei (SCN), with diminution of circadian peak dopaminergic activity at the SCN facilitating development of the seasonal obese insulin-resistant condition. The present study investigated whether such an ancient circadian dopamine-SCN activity system for expression of the seasonal obese, insulin-resistant phenotype may be operative in animals made obese amd insulin resistant by high-fat feeding and, if so, whether reinstatement of the circadian dopaminergic peak at the SCN would be sufficient to reverse the adverse metabolic impact of the high-fat diet without any alteration of caloric intake. First, we identified the supramammillary nucleus as a novel site providing the majority of dopaminergic neuronal input to the SCN. We further identified dopamine D2 receptors within the peri-SCN region as being functional in mediating SCN responsiveness to local dopamine. In lean, insulin-sensitive rats, the peak in the circadian rhythm of dopamine release at the peri-SCN coincided with the daily peak in SCN electrophysiological responsiveness to local dopamine administration. However, in rats made obese and insulin resistant by high-fat diet (HFD) feeding, these coincident circadian peak activities were both markedly attenuated or abolished. Reinstatement of the circadian peak in dopamine level at the peri-SCN by its appropriate circadian-timed daily microinjection to this area (but not outside this circadian time-interval) abrogated the obese, insulin-resistant condition without altering the consumption of the HFD. These findings suggest that the circadian peak of dopaminergic activity at the peri-SCN/SCN is a key modulator of metabolism and the responsiveness to adverse metabolic consequences of HFD consumption.

Origin recognition complex binding to a metazoan replication origin.

The initiation of DNA replication in eukaryotic cells at the onset of S phase requires the origin recognition complex (ORC) [1]. This six-subunit complex, first isolated in Saccharomyces cerevisiae [2], is evolutionarily conserved [1]. ORC participates in the formation of the prereplicative complex [3], which is necessary to establish replication competence. The ORC-DNA interaction is well established for autonomously replicating sequence (ARS) elements in yeast in which the ARS consensus sequence [4] (ACS) constitutes part of the ORC binding site [2, 5]. Little is known about the ORC-DNA interaction in metazoa. For the Drosophila chorion locus, it has been suggested that ORC binding is dispersed [6]. We have analyzed the amplification origin (ori) II/9A of the fly, Sciara coprophila. We identified a distinct 80-base pair (bp) ORC binding site and mapped the replication start site located adjacent to it. The binding of ORC to this 80-bp core region is ATP dependent and is necessary to establish further interaction with an additional 65-bp of DNA. This is the first time that both the ORC binding site and the replication start site have been identified in a metazoan amplification origin. Thus, our findings extend the paradigm from yeast ARS1 to multicellular eukaryotes, implicating ORC as a determinant of the position of replication initiation.

Box H and box ACA are nucleolar localization elements of U17 small nucleolar RNA.

The nucleolar localization elements (NoLEs) of U17 small nucleolar RNA (snoRNA), which is essential for rRNA processing and belongs to the box H/ACA snoRNA family, were analyzed by fluorescence microscopy. Injection of mutant U17 transcripts into Xenopus laevis oocyte nuclei revealed that deletion of stems 1, 2, and 4 of U17 snoRNA reduced but did not prevent nucleolar localization. The deletion of stem 3 had no adverse effect. Therefore, the hairpins of the hairpin-hinge-hairpin-tail structure formed by these stems are not absolutely critical for nucleolar localization of U17, nor are sequences within stems 1, 3, and 4, which may tether U17 to the rRNA precursor by base pairing. In contrast, box H and box ACA are major NoLEs; their combined substitution or deletion abolished nucleolar localization of U17 snoRNA. Mutation of just box H or just the box ACA region alone did not fully abolish the nucleolar localization of U17. This indicates that the NoLEs of the box H/ACA snoRNA family function differently from the bipartite NoLEs (conserved boxes C and D) of box C/D snoRNAs, where mutation of either box alone prevents nucleolar localization.

Nucleolar localization elements of Xenopus laevis U3 small nucleolar RNA.

The Nucleolar Localization Elements (NoLEs) of Xenopus laevis U3 small nucleolar RNA (snoRNA) have been defined. Fluorescein-labeled wild-type U3 snoRNA injected into Xenopus oocyte nuclei localized specifically to nucleoli as shown by fluorescence microscopy. Injection of mutated U3 snoRNA revealed that the 5' region containing Boxes A and A', known to be important for rRNA processing, is not essential for nucleolar localization. Nucleolar localization of U3 snoRNA was independent of the presence and nature of the 5' cap and the terminal stem. In contrast, Boxes C and D, common to the Box C/D snoRNA family, are critical elements for U3 localization. Mutation of the hinge region, Box B, or Box C' led to reduced U3 nucleolar localization. Results of competition experiments suggested that Boxes C and D act in a cooperative manner. It is proposed that Box B facilitates U3 snoRNA nucleolar localization by the primary NoLEs (Boxes C and D), with the hinge region of U3 subsequently base pairing to the external transcribed spacer of pre-rRNA, thus positioning U3 snoRNA for its roles in rRNA processing.

[Internal initiation of translation in eukaryotes. Chemical probing of the encephalomyocarditis virus RNA IRES-element in the 48S preinitiation complex]. / Vnutrenniaia initsiatsiia transliatsii u éukariot. Khimicheskoe zondirovanie IRES-élementa RNK virusa éntsefalomiokardita v sostave 48S preynitsiatornogo kompleksa.

Using in vitro T7 polymerase system, the transcript containing the IRES-element (nts 315-833), and the initial part of the coding sequence of encephalomyocarditis virus (EMCV) RNA (nts 834-1155) was prepared. Its complex with the 40S ribosomal subunit (48S preinitiation complex) was then isolated by sucrose gradient sedimentation from ascites carcinoma Krebs2 cell extracts after preincubation with the transcript. The complex was treated with dimethylsulphate (DMS), a common reagent for chemical probing of A and C residues in single-stranded RNA regions. The modified nucleotides were identified by primer extension inhibition analysis in reverse transcription reaction. The pattern of modification of the 48S complex was compared with that for the corresponding free mRNP. Multiple protection of A residues against DMS modification was found in the domains of the IRES-element proximal to the initiation AUG codon (nt 834-836). The mechanism of internal translational initiation of EMCV RNA and other picornaviral RNAs is discussed.

RNA--protein interactions within the internal translation initiation region of encephalomyocarditis virus RNA.

Various derivatives of the internal ribosomal entry site (IRES) of encephalomyocarditis virus (EMCV) RNA have been used to analyze by UV-cross-linking its interaction with mRNA binding proteins from ascites carcinoma Krebs-2 cells. A doublet of proteins with Mr 58 and 60 kD bound to two regions of the IRES. One site is centered at nt 420-421 of EMCV RNA whereas the other is located between nt 315-377. Both sites form hairpin structures, the loops of which contain UCUUU motif, conserved among cardio- and aphthoviruses. The interaction of p58 and p60 with IRES is affected by the integrity of the stem-loop structure proximal to the start AUG codon (nts 680-787), although, under similar conditions, cross-linking of these proteins to this region was not detected. Deletions in the main recognition site of p58 strongly reduce the initiation activity of the IRES in vitro. However, elimination of p58 (p60) binding by these mutations does not completely abolish the ability of the IRES to direct polypeptide synthesis starting from the authentic AUG codon. The IRES can be assembled in vitro from two covalently unlinked transcripts, one containing the target site for p58 and the other encompassing the remaining part of the IRES fused to a reporter gene, resulting in considerable restoration of its activity. Implications of these findings for the mechanism of initiation resulting from internal entry of ribosomes are discussed.