Trimethylguanosine nucleoside inhibits cross-linking between Snurportin 1 and m3G-CAPPED U1 snRNA.

Macromolecular nuclear import is an energy-and signal-dependent process. The best characterized type of nuclear import consists of proteins carrying the classical NLS that is mediated by the heterodimeric receptor importin alpha/beta. Spliceosomal snRNPs U1, U2, U4, and U5 nuclear import depend both on the 5' terminal m3G (trimethylguanosine) cap structure of the U snRNA and the Sm core domain. Snurportin 1 recognizes the m3G-cap structure of m3G-capped U snRNPs. In this report, we show how a synthesized trimethylguanosine nucleoside affects the binding of Snurportin 1 to m3G-capped U1 snRNA in a UV-cross-linking assay. The data indicated that TMG nucleoside is an essential component required in the recognition by Snurportin 1, thus suggesting that interaction of Snurportin 1 with U1 snRNA is not strictly dependent on the presence of the whole cap structure, but rather on the presence of the TMG nucleoside structure. These results indicate that the free nucleoside TMG could be a candidate to be an inhibitor of the interaction between Snurportin 1 and U snRNAs. We also show the behavior of free TMG nucleoside in in vitro U snRNPs nuclear import.

Spatiotemporal laser inactivation of inositol 1,4,5-trisphosphate receptors using synthetic small-molecule probes.

A malachite green-conjugated inositol 1,4,5-trisphosphate (MGIP(3)) induces specific inactivation of IP(3) receptor (IP(3)R) in tissue samples upon laser irradiation. To verify potential usefulness of the method for studies of cellular Ca(2+) signaling, we conducted laser inactivation at the single-cell level and show that IP(3)R was inactivated with extremely high spatiotemporal resolution. In the presence of MGIP(3), the Ca(2+) release function of IP(3)R in single B lymphoma cells decayed exponentially with increasing duration of laser irradiation with a time constant of 3.4 s. Moreover, by confining laser irradiation to a spatially distinct region of differentiated PC12 cells, subcellular inactivation of IP(3)R was attained, as revealed by a loss of local Ca(2+) signal. Such real-time inactivation of IP(3)R only within a subcellular region may provide a powerful method for investigating spatiotemporal dynamics of Ca(2+) signaling.

Orphan nuclear receptors, molecular clockwork, and the entrainment of peripheral oscillators.

Here we summarize our work on two aspects of circadian timing: the roles of orphan nuclear receptors in the molecular clockwork, and phase entrainment of peripheral oscillators. With reference to the former, studies on cis-acting regulatory elements within the Bmal1 promoter revealed that REV-ERBalpha, an orphan nuclear receptor provides a link between the positive and negative limbs of the molecular oscillator. Specifically, REV-ERBalpha controls the cyclic transcription of Bmal1 and Clock, the positive limb components. In turn, the circadian expression of Rev-Erbalpha itself is driven directly by the molecular oscillator: it is activated by BMAL1 and CLOCK, and repressed by PERIOD1/2 and CRYPTOCHROME1/2 proteins (the negative limb members). With regard to phase entrainment, it was initially believed that only the suprachiasmatic nucleus (SCN) was capable of generating circadian rhythms. However, circadian oscillators have recently been discovered in many peripheral tissues. In the absence of a functional SCN pacemaker, these peripheral clocks dampen after a few days. Hence, the SCN must periodically synchronize these subsidiary timekeepers. It may accomplish this task mostly through an indirect route: namely, by setting the time of feeding. In addition to feeding cycles, body temperature rhythms and cyclically secreted hormones might also serve as zeitgebers for peripheral clocks.

Small molecule-based laser inactivation of inositol 1,4,5-trisphosphate receptor.

BACKGROUND: Chromophore-assisted laser inactivation (CALI) is a powerful method for the study of in situ protein function in cellular processes. By using CALI, it is possible to abrogate the function of a target protein with unprecedented spatial and temporal resolution. However, CALI has some limitations, which restrict wider biological application, owing mainly to the use of antibody for target recognition. To circumvent the limitations, we have developed small molecule-based CALI (smCALI). RESULTS: The inositol 1,4,5-trisphosphate receptor (IP3R) was selected as the target protein and a malachite green-conjugated IP3 analog, MGIP3, was used as a small-molecular probe. We examined the effect of MGIP3-based CALI on Ca2+ release via IP3R using permeabilized smooth muscle cells. When the cells were treated with MGIP3 followed by laser irradiation, the IP3-induced Ca2+ release rate was decreased in a concentration- and irradiation time-dependent manner. The effect was specific for IP3R, because the Ca2+ uptake function of the co-localized sarco/endoplasmic reticulum Ca2+-ATPase was not affected. CONCLUSIONS: IP3R was specifically inactivated by smCALI using MGIP3. The efficiency of inactivation was calculated to be substantially greater than that of antibody-based CALI. The efficient and specific inactivation of IP3R would allow us to obtain an insight into spatiotemporal roles of IP3R in various cell functions. Our results may be considered to be a first step for a wider application of smCALI as a useful method to study spatiotemporal protein functions.

Photic regulation of mt(1) melatonin receptors and 2-iodomelatonin binding in the rat and Siberian hamster.

We have investigated the photic regulation of melatonin receptors both at the level of binding capacity and mt(1) mRNA expression in the suprachiasmatic nucleus (SCN) and the pars tuberalis (PT) of the pituitary of two species: a highly photoperiodic one, the Siberian hamster, and a nonphotoperiodic one, the Wistar rat. This study has been performed by looking at the effect of a light pulse applied during the night on the two receptor parameters. The results show that the photic regulations of mt(1) mRNA expression and receptor density are distinct from each other in both the SCN and PT of the two species studied. They also show that, depending on the species and the structure, this regulation may implicate either the circadian clock or melatonin.

Melatonin regulates the mRNA expression of the mt(1) melatonin receptor in the rat Pars tuberalis.

The pars tuberalis (PT) of the pituitary is a major neuroendocrine target site for melatonin as it contains a large number of high-affinity melatonin receptors. We have previously shown that melatonin autoregulates the density of its own receptors in the PT. However, whether melatonin regulation includes mRNA expression in vivo is unclear. In the present study we have used quantitative in situ hybridization to (1) follow the daily profile of mt(1) mRNA expression in the rat PT and (2) investigate whether mt(1) mRNA expression could be regulated in vivo by melatonin. We found clear diurnal variations of mt(1) mRNA expression that persist in constant darkness. We also showed, on pinealectomized animals, that the rhythmic pineal melatonin secretion is necessary for the expression of these daily variations. In a second step, we studied the effect of an acute suppression of endogenous melatonin synthesis on mt(1) melatonin receptors by applying a 1-hour light pulse during the night. We found that light induced a dramatic increase in mt(1) mRNA which was totally prevented by a melatonin injection showing that the acute effect of melatonin on the receptor mRNA is strongly inhibitory. A light pulse applied to animals with a chronic absence of melatonin was ineffective showing that light only affects melatonin receptors via the light-induced plasma melatonin suppression. Altogether our results show that melatonin regulates mt(1) melatonin receptor mRNA expression. However, this regulation seems to be complex: acute changes in plasma melatonin concentration regulate negatively the gene transcription, even if the daily endogenous nocturnal melatonin peak seems a prerequisite for variations in its receptor expression.

Photic regulation of mt1 melatonin receptors in the Siberian hamster pars tuberalis and suprachiasmatic nuclei: involvement of the circadian clock and intergeniculate leaflet.

In the Siberian hamster suprachiasmatic nuclei and pars tuberalis of the pituitary, high affinity mt1 melatonin receptors are present. We have previously shown that night applied light pulse induced an increase in mt1 mRNA expression in the suprachiasmatic nuclei of this species, independently of the endogenous melatonin. Here, we report the photic regulation of melatonin receptor density and mRNA expression in the suprachiasmatic nuclei and pars tuberalis of pinealectomized Siberian hamsters and the implication in this control of either the circadian clock or the intergeniculate leaflet. The results show that: (1) A 1-h light pulse, delivered during the night, induces a transitory increase in mt1 mRNA expression in the suprachiasmatic nuclei and pars tuberalis. After 3 h this increase has totally disappeared (suprachiasmatic nuclei) or is greatly reduced (pars tuberalis). (2) The melatonin receptor density, in the suprachiasmatic nuclei, is not affected by 1 or 3 h of light, while it is strongly increased in the pars tuberalis. (3) In hamsters kept in constant darkness, the mt1 mRNA rise is gated to the subjective night in the suprachiasmatic nuclei and pars tuberalis. In contrast, the light-induced increase in melatonin binding is also observed in the subjective day in the pars tuberalis. (4) intergeniculate leaflet lesion totally inhibits the mt1 mRNA expression rise in the suprachiasmatic nuclei, while it has no effect on the light-induced increase in mt1 mRNA in the pars tuberalis. However, the light-induced increase in melatonin receptor density is totally prevented by the intergeniculate leaflet lesion in the pars tuberalis. These results show that: (1) the photic regulations of mt1 mRNA expression and receptor density are independent of each other in both the suprachiasmatic nuclei and pars tuberalis; and (2) the circadian clock and the intergeniculate leaflet are implicated in the photic regulation of melatonin receptors but their level of action differs totally between the suprachiasmatic nuclei and pars tuberalis.

Serotonergic modulation of photically induced increase in melatonin receptor density and Fos immunoreactivity in the suprachiasmatic nuclei of the rat.

The mammalian suprachiasmatic nuclei (SCN) contain a circadian clock which is regulated by neuronal photic and non-photic afferences. Among these, the serotonergic input originating from the dorsal raphe nucleus (DRN) is extremely important. In rats, a light pulse administered during the dark period is known to induce the expression of the immediate early gene c-fos and to increase melatonin receptor density in the SCN. The aim of this study was to assess whether, in rats, these two phenomena were regulated by serotonin, acting via 5-HT1A receptors. Three days after pinealectomy, 4 groups of rats were injected i.p. 90 min before sacrifice with respectively: (1) vehicle, (2) the 5-HT1A-agonist 8-OH-DPAT (5 mg/kg), (3) the 5-HT1A-antagonist NAN-190 (10 mg/kg) or (4) NAN-190 and the 8-OH-DPAT. Half of the animals from each group were exposed to light for 60 min before sacrifice and the other half remained in darkness. Sacrifice took place 5 to 6 h after lights off. Our results show that the antagonist NAN-190: (1) completely blocked the photically-induced increase of melatonin receptor density in the SCN, with an IC50 = 0.352 +/- 0.103 mg/kg, and (2) partially blocked (30%) the photic induction of Fos (the protein product of c-fos) in the ventrolateral subdivision of the SCN. The agonist 8-OH-DPAT enhanced the photically-induced increase of melatonin receptors by 10% and decreased the photically-induced increase in Fos by 18%. Both drugs were devoid of any effect in non-light exposed animals. From these results we may suggest that, in rats, there is a serotonergic control of the neuronal path driving photic information to the SCN. This regulation seems to occur through 5-HT1A or 5-HT1a-like receptors.

Circadian photic regulation of melatonin receptor density in rat suprachiasmatic nuclei: comparison with light induction of fos-related protein.

High-affinity melatonin receptors are present in rat suprachiasmatic nuclei (SCN), and their density exhibits a daily rhythm regulated by the light/dark cycle. In this study we demonstrate that the light regulation of these receptors depends on a circadian mechanism. Pinealectomized rats kept in constant darkness were subjected to 1-hr light pulses delivered across the circadian cycle. The density of melatonin receptors was significantly increased when photic exposure was performed during subjective night, and not different from control animals kept in darkness when the light pulse was applied during subjective day. The protein product (Fos) of the immediate early gene c-fos studied in the same paradigm showed globally the same circadian sensitivity phase. These results clearly show that, although the rhythmic appearance of melatonin receptor density in SCN follows and is directly regulated by the standard light/dark cycle, this light regulation is not passive. As is the case with Fos-like protein, it is only during a precise phase of the circadian cycle that light is able to regulate the density of melatonin receptors in SCN.