A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA.

The 21-nucleotide small temporal RNA (stRNA) let-7 regulates developmental timing in Caenorhabditis elegans and probably in other bilateral animals. We present in vivo and in vitro evidence that in Drosophila melanogaster a developmentally regulated precursor RNA is cleaved by an RNA interference-like mechanism to produce mature let-7 stRNA. Targeted destruction in cultured human cells of the messenger RNA encoding the enzyme Dicer, which acts in the RNA interference pathway, leads to accumulation of the let-7 precursor. Thus, the RNA interference and stRNA pathways intersect. Both pathways require the RNA-processing enzyme Dicer to produce the active small-RNA component that represses gene expression.

Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing.

RNAi is a gene-silencing phenomenon triggered by double-stranded (ds) RNA and involves the generation of 21 to 26 nt RNA segments that guide mRNA destruction. In Caenorhabditis elegans, lin-4 and let-7 encode small temporal RNAs (stRNAs) of 22 nt that regulate stage-specific development. Here we show that inactivation of genes related to RNAi pathway genes, a homolog of Drosophila Dicer (dcr-1), and two homologs of rde-1 (alg-1 and alg-2), cause heterochronic phenotypes similar to lin-4 and let-7 mutations. Further we show that dcr-1, alg-1, and alg-2 are necessary for the maturation and activity of the lin-4 and let-7 stRNAs. Our findings suggest that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation.

Regulation of DAF-2 receptor signaling by human insulin and ins-1, a member of the unusually large and diverse C. elegans insulin gene family.

The activity of the DAF-2 insulin-like receptor is required for Caenorhabditis elegans reproductive growth and normal adult life span. Informatic analysis identified 37 C. elegans genes predicted to encode insulin-like peptides. Many of these genes are divergent insulin superfamily members, and many are clustered, indicating recent diversification of the family. The ins genes are primarily expressed in neurons, including sensory neurons, a subset of which are required for reproductive development. Structural predictions and likely C-peptide cleavage sites typical of mammalian insulins suggest that ins-1 is most closely related to insulin. Overexpression of ins-1, or expression of human insulin under the control of ins-1 regulatory sequences, causes partially penetrant arrest at the dauer stage and enhances dauer arrest in weak daf-2 mutants, suggesting that INS-1 and human insulin antagonize DAF-2 insulin-like signaling. A deletion of the ins-1 coding region does not enhance or suppress dauer arrest, indicating a functional redundancy among the 37 ins genes. Of five other ins genes tested, the only other one bearing a predicted C peptide also antagonizes daf-2 signaling, whereas four ins genes without a C peptide do not, indicating functional diversity within the ins family.

Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA.

Two small RNAs regulate the timing of Caenorhabditis elegans development. Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA, and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA. The lin-4 and let-7 RNA genes are not homologous to each other, but are each complementary to sequences in the 3' untranslated regions of a set of protein-coding target genes that are normally negatively regulated by the RNAs. Here we have detected let-7 RNAs of approximately 21 nucleotides in samples from a wide range of animal species, including vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from several cnidarian and poriferan species, Saccharomyces cerevisiae, Escherichia coli or Arabidopsis. We did not detect lin-4 RNA in these species. We found that let-7 temporal regulation is also conserved: let-7 RNA expression is first detected at late larval stages in C. elegans and Drosophila, at 48 hours after fertilization in zebrafish, and in adult stages of annelids and molluscs. The let-7 regulatory RNA may control late temporal transitions during development across animal phylogeny.

The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans.

The C. elegans heterochronic gene pathway consists of a cascade of regulatory genes that are temporally controlled to specify the timing of developmental events. Mutations in heterochronic genes cause temporal transformations in cell fates in which stage-specific events are omitted or reiterated. Here we show that let-7 is a heterochronic switch gene. Loss of let-7 gene activity causes reiteration of larval cell fates during the adult stage, whereas increased let-7 gene dosage causes precocious expression of adult fates during larval stages. let-7 encodes a temporally regulated 21-nucleotide RNA that is complementary to elements in the 3' untranslated regions of the heterochronic genes lin-14, lin-28, lin-41, lin-42 and daf-12, indicating that expression of these genes may be directly controlled by let-7. A reporter gene bearing the lin-41 3' untranslated region is temporally regulated in a let-7-dependent manner. A second regulatory RNA, lin-4, negatively regulates lin-14 and lin-28 through RNA-RNA interactions with their 3' untranslated regions. We propose that the sequential stage-specific expression of the lin-4 and let-7 regulatory RNAs triggers transitions in the complement of heterochronic regulatory proteins to coordinate developmental timing.

Congenital hypomyelination neuropathy with Ser72Leu substitution in PMP22.

We describe a patient with congenital hypomyelination neuropathy. The pathological and morphometrical findings in the sural nerve biopsy were consistent with a defect of myelin formation and maintenance. Direct sequence analysis of the genomic regions coding the peripheral myelin proteins P0 and PMP22 disclosed a heterozygous missense point mutation that leads to a Ser72Leu substitution in the second transmembrane of PMP22. Codon 72 mutations of PMP22 are associated with different phenotypes encompassing the Dejerine-Sottas syndrome and including congenital hypomyelination neuropathy.

The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) accesses a cellular mRNA export pathway.

The constitutive transport elements (CTEs) of type D retroviruses are cis-acting elements that promote nuclear export of incompletely spliced mRNAs. Unlike the Rev response element (RRE) of human immunodeficiency virus type 1 (HIV-1), CTEs depend entirely on factors encoded by the host cell genome. We show that an RNA comprised almost entirely of the CTE of Mason-Pfizer monkey virus (CTE RNA) is exported efficiently from Xenopus oocyte nuclei. The CTE RNA and an RNA containing the RRE of HIV-1 (plus Rev) have little effect on export of one another, demonstrating differences in host cell requirements of these two viral mRNA export pathways. Surprisingly, even very low amounts of CTE RNA block export of normal mRNAs, apparently through the sequestration of cellular mRNA export factors. Export of a CTE-containing lariat occurs when wild-type CTE, but not a mutant form, is inserted into the pre-mRNA. The CTE has two symmetric structures, either of which supports export and the titration of mRNA export factors, but both of which are required for maximal inhibition of mRNA export. Two host proteins bind specifically to the CTE but not to non-functional variants, making these proteins candidates for the sequestered mRNA export factors.

Inhibition of mRNA export in vertebrate cells by nuclear export signal conjugates.

Leucine-rich nuclear export signals (NESs) are recognized by the NES receptor exportin 1 and are central to the export of multiple shuttling proteins and RNAs. The export of messenger RNA in vertebrates was, however, thought to occur by a different pathway, because inhibition by injection of a synthetic Rev NES conjugate could not be demonstrated. Here we find that peptide conjugates composed of the NES of either protein kinase A inhibitor protein (PKI) or the HIV-1 Rev protein, when coupled to human serum albumin, are potent inhibitors of mRNA and small nuclear RNA export. These results provide direct evidence that mRNA export in vertebrates depends on interactions between an NES and its cognate NES receptors. PKI NES conjugates are significantly more efficient at inhibiting RNA export than are REV NES conjugates, indicating that different NESs may have different abilities to promote protein and RNA export. Surprisingly, an expected control conjugate containing the mutant Rev NES sequence M10 strongly inhibited the export of intronless dihydrofolate reductase mRNA. Nuclear injection of NES peptide conjugates led to mislocalization to the nucleus of 10-20% of the cytoplasmic Ran GTPase-binding protein (RanBP1) indicating that RanBP1 shuttles between the nucleus and the cytoplasm via an NES pathway. These results demonstrate that in vertebrates the export of mRNA, like that of small nuclear RNA, 5S rRNA, and transport factors such as RanBP1, employs NES-mediated molecular machinery.

Reverse 5' caps in RNAs made in vitro by phage RNA polymerases.

We show that about one-third of the RNAs produced in vitro by viral RNA polymerases in the presence of m7GpppG dinucleotides have unusual 5' caps. In these RNAs, the initiating dinucleotide is incorporated in an orientation opposite to that expected so that the 7-methyl guanine (m7G) nucleotide is adjacent to the body of the RNA, making a "reverse" cap. The doubly methylated dinucleotide, m7GpppGm, containing a 2' O-methylated guanine (Gm) is incorporated only in the reverse orientation. Precursors of U1 snRNAs containing reverse caps are recognized by antibodies specific for the m7G cap structure. When injected into Xenopus laevis oocyte nuclei, reverse-capped pre-U1 RNAs are exported considerably more slowly than normal. Furthermore, U1 RNAs with reverse caps exhibit a striking defect in nuclear import that can be attributed to the failure of reverse caps to be hypermethylated to m2,2,7G caps. Thus, the presence of reverse-capped RNAs in RNA preparations may affect conclusions about the efficiency and extent of certain m7G cap-dependent processes.

Steroid-dependent form of Kinsbourne syndrome: successful treatment with trazodone.

We report the case of a 19 month old girl with myoclonic encephalopathy of infants (MEI) (Kinsbourne syndrome), on long-term therapy with ACTH for the occurrence of frequent relapses (steroid-dependent form). The administration of trazodone per os at low doses as an alternative to the previous treatment ensured complete remission, also on the occasion of a subsequent relapse. No rebound effects were observed after trazodone withdrawal (10 months). At present, 3 years after withdrawal of the therapy, the child is well and free from symptoms. The hypothesis that trazodone may be effective in treating MEI, at least in cases that are steroid-dependent or resistant to ACTH, appears highly interesting. Trazodone is proposed as a possible alternative to treatment with ACTH.

Alertness and incidence of seizures in patients with Lennox-Gastaut syndrome.

Sixteen subjects affected by Lennox-Gastaut syndrome (11 males and five females, ranging from 5 to 15 years of age [mean age, 9 years 11 months]) were followed for periods of 9 months to 5 years 9 months, and were studied during three to five prolonged hospitalizations for total periods of 2-9 months with a veiw to examining the distribution of epileptic seizures during four states of vigilance, evaluated from a behavioral point of view: sleep, drowsiness, inactive wakefulness, and active wakefulness. it was ascertained that the overall average of 406 seizures daily, directly observed, was distributed as follows: 26 (6.40%) during sleep; 128 (31.52%) during drowsiness; 219 (53.94%) during inactive wakefulness; and 33 (8.12%) during active wakefulness. The comparison between the incidence of seizures observed during active wakefulness and those observed during both drowsiness and inactive wakefulness was significant (p less than 0.001). The latter two states represent, in our study, the shortest period of the day (8 h as compared with the 16 h of sleep and active wakefulness), thus making the results of the comparison even more significant. The results of this study suggest the importance of a stimulating environment for children affected by Lennox-Gastaut syndrome, and they point out that an overdose of antiepileptic drugs, not uncommon in the treatment of this syndrome, may make seizures more frequent.

[Studies of the expectancy wave (CNV) in patients with interruption of the thalamo-prefrontal interconnnection pathways caused by psychosurgical prefrontal lobotomy operations]. / Rilievi sull'onda d'aspettativa (CNV) in pazienti con interruzione delle vie di interconnessione talamo-prefrontali da interventi psicochirurgici di lobotomia prefrontale.

Frontal and vertex CNVs were studied in 8 selected nonschizophrenic patients subjected to unilateral or bilateral extensive prefrontal lobotomy. The dorsomedial thalamo-frontal pathways had been severed and their regeneration must be considered impossible. Standard CNV task (S1-S2-R) was followed in order to elicit CNVs from the frontal areas anterior to the line of sections and at Cz. In 7 out of 8 patients it was quite easy to evoke CNV with almost normal features and equal latencies in each case from all the cortical areas explored. These results show that CNV formation is not grossly altered in the prefrontal areas which have been irreversibly deprived of normal bi-directional mediothalamic-frontocortical connnections. This suggests that the role of the dorsomedial thalamo-frontal pathways are not essential in the genesis of the frontal CNV in humans. These findings would suggest that the CNV is a diffuse electrical event essentially related to a unitary cerebral process mediated fundamentally by nonspecific ascending meso-diencephalic reticular systems. The differences in morphology and polarity of the CNVs detectable in various brain structures are presumably related to their intrinsic anatomo-functional characteristics and to the method commonly utilized in recording the CNV.

[Event-related slow potentials (ERSPs) of the brain in cases of temporal psychomotor and petit mal status]. / Gli ERSPs in casi di stato epilettico psicomotorio temporale e di piccolo male.

Two cases have been studied by means of the usual method for eliciting CNV (S-1.5 OR 1 SEC-S-operant response) during and after the end of an episode of prolonged epileptic twilight state with almost continuous strictly unilateral temporal lobe discharge. From the clinical viewpoint in both cases the twilight state, lasting respectively about 12 and 48 hours, was characterized by a slightly clouded consciousness and moderate impairment of awareness and of psychic performances, at times associated with simple and complex psychomotor automatism and hallucinations. The EEG recorded an almost continous left temporal discharge of pseudorhythmic mixed slow waves and sharps. The third case had typical prolonged petit mal states with continuous spike-and-slow-wave activity, impaired intellectual and motor performances (very long reaction time etc.). In this patient for eliciting ERSPs, besides the standard method, we have used a paradigm in which S consisted of a colored slide, with various semantic contents, remaining visible for 5 seconds on a screen. At the trials of the standard paradigm during the epiliptic twilight state, all patients showed they had understood the signal to interrupt (S loud repetitive tone) in the shortest time possible and could clearly distinguish them from the S. The operant response was almost always made with sufficient precision and sometimes with fairly short reaction time, especially by the patients with temporal psychomotor status. During the episodes of prolonged clouded consciosness in all series of trials administered to the patients, no negative slow potential shifts were observed in the averaged EEG recordings obtained from F-T, F-T or F, FCZ and referred to to linked mastoids. On repetition of the examinations some time after the end of the epileptic twilight state, fairly normal ERSPs were obtained in all cases. Taking also into account the results of previous researches, these studies show that the temporal lobe and "centrencephalic" epileptic discharges, under certain conditions, may influence negatively the neurophysiological mechanisms which contribute to the information of complex contingent connections and which also underly the particular attentional, cognitive and sensorimotor functions involved in the inhibiting the appearance of ERSPs probably related to more specific perceptual and integrative functions. Some AA. maintain that negative slow potential shifts express the functional activity of the brain structures, particularly of determined cerebral cortex regions, involved not only in attentional, perceptual, cognitive and psychomotor functioning, but also in information processing (memory recording mechanism). Hence, the negative influence of prolonged temporal lobe or meso-diencephalic epileptic discharges on these structures may explain the almost complete amnesia that patients generally show at the termination of this kind of twilight state.