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1.
Science ; 286(5440): 768-71, 1999 Oct 22.
Article in English | MEDLINE | ID: mdl-10531061

ABSTRACT

Cryptochrome (CRY), a photoreceptor for the circadian clock in Drosophila, binds to the clock component TIM in a light-dependent fashion and blocks its function. In mammals, genetic evidence suggests a role for CRYs within the clock, distinct from hypothetical photoreceptor functions. Mammalian CRY1 and CRY2 are here shown to act as light-independent inhibitors of CLOCK-BMAL1, the activator driving Per1 transcription. CRY1 or CRY2 (or both) showed light-independent interactions with CLOCK and BMAL1, as well as with PER1, PER2, and TIM. Thus, mammalian CRYs act as light-independent components of the circadian clock and probably regulate Per1 transcriptional cycling by contacting both the activator and its feedback inhibitors.


Subject(s)
Biological Clocks , Circadian Rhythm , Drosophila Proteins , Eye Proteins , Flavoproteins/physiology , Gene Expression Regulation , Light , Nuclear Proteins/genetics , Photoreceptor Cells, Invertebrate , 3T3 Cells , ARNTL Transcription Factors , Animals , Basic Helix-Loop-Helix Transcription Factors , CLOCK Proteins , Cell Cycle Proteins , Cells, Cultured , Cryptochromes , Dimerization , Flavoproteins/metabolism , Genes, Reporter , Helix-Loop-Helix Motifs , Humans , Intracellular Signaling Peptides and Proteins , Mice , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism , Period Circadian Proteins , Receptors, G-Protein-Coupled , Trans-Activators/antagonists & inhibitors , Trans-Activators/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Transcriptional Activation , Transfection
2.
Science ; 285(5427): 553-6, 1999 Jul 23.
Article in English | MEDLINE | ID: mdl-10417378

ABSTRACT

Most organisms have circadian clocks consisting of negative feedback loops of gene regulation that facilitate adaptation to cycles of light and darkness. In this study, CRYPTOCHROME (CRY), a protein involved in circadian photoperception in Drosophila, is shown to block the function of PERIOD/TIMELESS (PER/TIM) heterodimeric complexes in a light-dependent fashion. TIM degradation does not occur under these conditions; thus, TIM degradation is uncoupled from abrogation of its function by light. CRY and TIM are part of the same complex and directly interact in yeast in a light-dependent fashion. PER/TIM and CRY influence the subcellular distribution of these protein complexes, which reside primarily in the nucleus after the perception of a light signal. Thus, CRY acts as a circadian photoreceptor by directly interacting with core components of the circadian clock.


Subject(s)
Biological Clocks , Circadian Rhythm , Drosophila Proteins , Eye Proteins , Flavoproteins/metabolism , Insect Proteins/metabolism , Light , Photoreceptor Cells, Invertebrate , Animals , Cell Line , Cell Nucleus/metabolism , Cryptochromes , Cytoplasm/metabolism , Darkness , Dimerization , Drosophila , Flavoproteins/genetics , Green Fluorescent Proteins , Insect Proteins/genetics , Luminescent Proteins , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Period Circadian Proteins , Receptors, G-Protein-Coupled , Recombinant Fusion Proteins/metabolism , Transfection , Yeasts/genetics , Yeasts/metabolism
3.
Neuron ; 21(5): 1101-13, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9856465

ABSTRACT

We report the cloning and mapping of mouse (mTim) and human (hTIM) orthologs of the Drosophila timeless (dtim) gene. The mammalian Tim genes are widely expressed in a variety of tissues; however, unlike Drosophila, mTim mRNA levels do not oscillate in the suprachiasmatic nucleus (SCN) or retina. Importantly, hTIM interacts with the Drosophila PERIOD (dPER) protein as well as the mouse PER1 and PER2 proteins in vitro. In Drosophila (S2) cells, hTIM and dPER interact and translocate into the nucleus. Finally, hTIM and mPER1 specifically inhibit CLOCK-BMAL1-induced transactivation of the mPer1 promoter. Taken together, these results demonstrate that mTim and hTIM are mammalian orthologs of timeless and provide a framework for a basic circadian autoregulatory loop in mammals.


Subject(s)
Circadian Rhythm/genetics , Drosophila Proteins , Insect Proteins/physiology , Nuclear Proteins/metabolism , Trans-Activators/genetics , Transcription Factors/genetics , Transcription Factors/physiology , ARNTL Transcription Factors , Alternative Splicing/genetics , Amino Acid Sequence , Animals , Basic Helix-Loop-Helix Transcription Factors , Biological Clocks/genetics , CLOCK Proteins , Cell Cycle Proteins , Cell Line , Chromosome Mapping , Chromosomes, Human, Pair 12/genetics , Cloning, Molecular , Drosophila , Female , Humans , Insect Proteins/genetics , Insect Proteins/metabolism , Intracellular Signaling Peptides and Proteins , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , Molecular Sequence Data , Nuclear Proteins/physiology , Period Circadian Proteins , Polymorphism, Genetic , RNA, Messenger/biosynthesis , Trans-Activators/antagonists & inhibitors , Transcription Factors/metabolism
4.
Science ; 280(5369): 1564-9, 1998 Jun 05.
Article in English | MEDLINE | ID: mdl-9616112

ABSTRACT

The mouse Clock gene encodes a bHLH-PAS protein that regulates circadian rhythms and is related to transcription factors that act as heterodimers. Potential partners of CLOCK were isolated in a two-hybrid screen, and one, BMAL1, was coexpressed with CLOCK and PER1 at known circadian clock sites in brain and retina. CLOCK-BMAL1 heterodimers activated transcription from E-box elements, a type of transcription factor-binding site, found adjacent to the mouse per1 gene and from an identical E-box known to be important for per gene expression in Drosophila. Mutant CLOCK from the dominant-negative Clock allele and BMAL1 formed heterodimers that bound DNA but failed to activate transcription. Thus, CLOCK-BMAL1 heterodimers appear to drive the positive component of per transcriptional oscillations, which are thought to underlie circadian rhythmicity.


Subject(s)
Circadian Rhythm/physiology , Nuclear Proteins/genetics , Trans-Activators/metabolism , Transcription Factors/metabolism , Transcriptional Activation , ARNTL Transcription Factors , Animals , Basic Helix-Loop-Helix Transcription Factors , Biological Clocks , CLOCK Proteins , Cell Cycle Proteins , Circadian Rhythm/genetics , Cloning, Molecular , Cricetinae , DNA/metabolism , Dimerization , Feedback , Gene Expression , Helix-Loop-Helix Motifs , Male , Mesocricetus , Mice , Mutation , Nuclear Proteins/metabolism , Period Circadian Proteins , Promoter Regions, Genetic , Retina/metabolism , Suprachiasmatic Nucleus/metabolism , Trans-Activators/genetics , Transcription Factors/genetics
5.
Science ; 280(5369): 1599-603, 1998 Jun 05.
Article in English | MEDLINE | ID: mdl-9616122

ABSTRACT

The circadian oscillator generates a rhythmic output with a period of about 24 hours. Despite extensive studies in several model systems, the biochemical mode of action has not yet been demonstrated for any of its components. Here, the Drosophila CLOCK protein was shown to induce transcription of the circadian rhythm genes period and timeless. dCLOCK functioned as a heterodimer with a Drosophila homolog of BMAL1. These proteins acted through an E-box sequence in the period promoter. The timeless promoter contains an 18-base pair element encompassing an E-box, which was sufficient to confer dCLOCK responsiveness to a reporter gene. PERIOD and TIMELESS proteins blocked dCLOCK's ability to transactivate their promoters via the E-box. Thus, dCLOCK drives expression of period and timeless, which in turn inhibit dCLOCK's activity and close the circadian loop.


Subject(s)
Circadian Rhythm/physiology , Drosophila Proteins , Insect Proteins/genetics , Nuclear Proteins/genetics , Trans-Activators/metabolism , Transcription Factors/metabolism , Transcriptional Activation , ARNTL Transcription Factors , Animals , Basic Helix-Loop-Helix Transcription Factors , Biological Clocks , CLOCK Proteins , Cell Line , Cell Nucleus/metabolism , Circadian Rhythm/genetics , Dimerization , Drosophila , Feedback , Gene Expression , Helix-Loop-Helix Motifs , Insect Proteins/metabolism , Nuclear Proteins/metabolism , Period Circadian Proteins , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , Trans-Activators/genetics , Transcription Factors/genetics , Transfection
6.
Nucleic Acids Res ; 23(20): 4081-6, 1995 Oct 25.
Article in English | MEDLINE | ID: mdl-7479068

ABSTRACT

We have characterized a monoclonal antibody (mAb) to the U1 snRNP component U1 70K. We find that this antibody recognizes several proteins, in addition to U1 70K, in purified spliceosomal complexes and in total HeLa cell nuclear extract preparations. The novel mAb U1 70K antigens can also be specifically immunoprecipitated by the antibody. Similarly to U1 70K, many of the mAb U1 70K antigens can be phosphorylated by a co-purifying kinase activity. The epitope recognized by mAb U1 70K was previously shown to be a repeating arginine/aspartate (RD) dipeptide. Thus we have designated the novel mAb U1 70K antigens the RD family. Comparison of mAb U1 70K with a recently characterized antibody, mAb 16H3, whose epitope is a repeating R/D or R/E motif, showed that a large subset of the antigens are common. In contrast, most of the mAb U1 70K antigens are distinct from the proteins detected by mAb 104, an antibody to the SR family of splicing factors.


Subject(s)
Antibodies, Monoclonal , RNA-Binding Proteins/analysis , Ribonucleoprotein, U1 Small Nuclear/analysis , Spliceosomes/chemistry , Amino Acid Sequence , Antibody Specificity , Cell Extracts , Cell Nucleus/metabolism , Dipeptides , Epitopes/analysis , HeLa Cells , Humans , Molecular Sequence Data , RNA-Binding Proteins/chemistry
7.
Mol Cell Biol ; 14(11): 7670-82, 1994 Nov.
Article in English | MEDLINE | ID: mdl-7935481

ABSTRACT

We show that addition of SR proteins to in vitro splicing extracts results in a significant increase in assembly of the earliest prespliceosomal complex E and a corresponding decrease in assembly of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex H. In addition, SR proteins promote formation of the E5' and E3' complexes that assemble on RNAs containing only 5' and 3' splice sites, respectively. We conclude that SR proteins promote the earliest specific recognition of both the 5' and 3' splice sites and are limiting for this function in HeLa nuclear extracts. Using UV cross-linking, we demonstrate specific, splice site-dependent RNA-protein interactions of SR proteins in the E, E5', and E3' complexes. SR proteins do not UV cross-link in the H complex, and conversely, hnRNP cross-linking is largely excluded from the E-type complexes. We also show that a discrete complex resembling the E5' complex assembles on both purine-rich and non-purine-rich exonic splicing enhancers. This complex, which we have designated the Enhancer complex, contains U1 small nuclear RNP (snRNP) and is associated with different SR protein family members, depending on the sequence of the enhancer. We propose that both downstream 5' splice site enhancers and exonic enhancers function by establishing a network of pre-mRNA-protein and protein-protein interactions involving U1 snRNP, SR proteins, and U2AF that is similar to the interactions that bring the 5' and 3' splice sites together in the E complex.


Subject(s)
RNA Precursors/metabolism , RNA Splicing , RNA-Binding Proteins/metabolism , Ribonucleoprotein, U1 Small Nuclear/metabolism , Base Sequence , Binding Sites , Cross-Linking Reagents , DNA/genetics , Enhancer Elements, Genetic , HeLa Cells , Humans , Models, Biological , Molecular Sequence Data , RNA Precursors/genetics , RNA Precursors/radiation effects , RNA Splicing/genetics , RNA-Binding Proteins/radiation effects , Ribonucleoprotein, U1 Small Nuclear/genetics , Spliceosomes/metabolism , Ultraviolet Rays
8.
Mol Cell Biol ; 14(5): 2994-3005, 1994 May.
Article in English | MEDLINE | ID: mdl-8164655

ABSTRACT

Highly purified mammalian spliceosomal complex B contains more than 30 specific protein components. We have carried out UV cross-linking studies to determine which of these components directly contacts pre-mRNA in purified prespliceosomal and spliceosomal complexes. We show that heterogeneous nuclear ribonucleoproteins cross-link in the nonspecific complex H but not in the B complex. U2AF65, which binds to the 3' splice site, is the only splicing factor that cross-links in purified prespliceosomal complex E. U2AF65 and the U1 small nuclear ribonucleoprotein particle (snRNP) are subsequently destabilized, and a set of six spliceosome-associated proteins (SAPs) cross-links to the pre-mRNA in the prespliceosomal complex A. These proteins require the 3' splice site for binding and cross-link to an RNA containing only the branch site and 3' splice site. Significantly, all six of these SAPs are specifically associated with U2 snRNP. These proteins and a U5 snRNP component cross-link in the fully assembled B complex. Previous work detected an ATP-dependent, U2 snRNP-associated factor that protects a 30- to 40-nucleotide region surrounding the branchpoint sequence from RNase digestion. Our data indicate that the six U2 snRNP-associated SAPs correspond to this branchpoint protection factor. Four of the snRNP proteins that are in intimate contact with the pre-mRNA are conserved between Saccharomyces cerevisiae and humans, consistent with the possibility that these factors play key roles in mediating snRNA-pre-mRNA interactions during the splicing reaction.


Subject(s)
RNA Precursors/metabolism , RNA Splicing , Ribonucleoprotein, U2 Small Nuclear/metabolism , Spliceosomes/metabolism , Animals , Cell Fractionation , Chromatography, Affinity , Chromatography, Gel , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Macromolecular Substances , Mammals , Models, Biological , RNA Precursors/isolation & purification , Ribonucleoprotein, U2 Small Nuclear/isolation & purification , Spliceosomes/radiation effects , Spliceosomes/ultrastructure , Transcription, Genetic , Ultraviolet Rays
9.
J Virol ; 67(9): 5419-25, 1993 Sep.
Article in English | MEDLINE | ID: mdl-8394456

ABSTRACT

During herpes simplex virus infection, expression of the viral DNA polymerase (pol) gene is regulated temporally as an early (beta) gene and is additionally down-regulated at late times at the level of translation (D. R. Yager, A. I. Marcy, and D. M. Coen, J. Virol. 64:2217-2225, 1990). To examine the role of viral DNA synthesis in pol regulation, we studied pol expression during infections in which viral DNA synthesis was blocked, either by using drugs that inhibit Pol or ribonucleotide reductase or by using viral mutants with lesions in either the pol or a primase-helicase subunit gene. Under any of these conditions, the level of cytoplasmic pol mRNA was reduced. This reduction was first seen at approximately the time DNA synthesis begins and, when normalized to levels of other early mRNAs, became as great as 20-fold late in infection. The reduction was also observed in the absence of the adjacent origin of replication, oriL. Thus, although pol mRNA accumulated as expected for an early gene in terms of temporal regulation, it behaved more like that of a late (gamma) gene in its response to DNA synthesis inhibition. Surprisingly, despite the marked decrease in pol mRNA in the absence of DNA synthesis, the accumulation of Pol polypeptide was unaffected. This was accompanied by loss of the normal down-regulation of translation of pol mRNA at late times. We suggest a model to explain these findings.


Subject(s)
DNA-Directed DNA Polymerase/genetics , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Viral , Genes, pol , Simplexvirus/enzymology , Simplexvirus/genetics , Animals , Aphidicolin/pharmacology , DNA Replication/drug effects , DNA-Directed DNA Polymerase/biosynthesis , Ganciclovir/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Viral/drug effects , Hydroxyurea/pharmacology , Kinetics , Phosphonoacetic Acid/pharmacology , Protein Biosynthesis/drug effects , RNA, Messenger/biosynthesis , RNA, Messenger/metabolism , RNA, Viral/antagonists & inhibitors , RNA, Viral/biosynthesis , Simplexvirus/drug effects , Thymidine Kinase/biosynthesis , Transcription, Genetic/drug effects , Vero Cells
10.
Mol Cell Biol ; 12(7): 3165-75, 1992 Jul.
Article in English | MEDLINE | ID: mdl-1620124

ABSTRACT

We have investigated the composition of the earliest detectable complex (H) assembled on pre-mRNA during the in vitro splicing reaction. We show that most of the proteins in this complex correspond to heterogeneous nuclear ribonucleoproteins (hnRNP), a set of abundant RNA-binding proteins that bind nascent RNA polymerase II transcripts in vivo. Thus, these studies establish a direct parallel between the initial events of RNA processing in vitro and in vivo. In contrast to previous studies, in which total hnRNP particles were isolated from mammalian nuclei, we determined the hnRNP composition of complexes assembled on individual RNAs of defined sequence. We found that a unique combination of hnRNP proteins is associated with each RNA. Thus, our data provide direct evidence for transcript-dependent assembly of pre-mRNA in hnRNP complexes. The observation that pre-mRNA is differentially bound by hnRNP proteins prior to spliceosome assembly suggests the possibility that RNA packaging could play a central role in the mechanism of splice site selection, as well as other posttranscriptional events.


Subject(s)
RNA Precursors/metabolism , RNA Splicing , RNA, Messenger/metabolism , Ribonucleoproteins/metabolism , Adenosine Triphosphate/pharmacology , Animals , Cell Nucleus/chemistry , Cell Nucleus/metabolism , Globins/genetics , Heterogeneous-Nuclear Ribonucleoproteins , Macromolecular Substances , RNA Splicing/drug effects , RNA, Heterogeneous Nuclear/metabolism , Ribonucleoproteins/chemistry , Tropomyosin/genetics
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