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1.
Nucleic Acids Res ; 49(16): 9424-9443, 2021 09 20.
Article in English | MEDLINE | ID: mdl-34365507

ABSTRACT

RNA provides the framework for the assembly of some of the most intricate macromolecular complexes within the cell, including the spliceosome and the mature ribosome. The assembly of these complexes relies on the coordinated association of RNA with hundreds of trans-acting protein factors. While some of these trans-acting factors are RNA-binding proteins (RBPs), others are adaptor proteins, and others still, function as both. Defects in the assembly of these complexes results in a number of human pathologies including neurodegeneration and cancer. Here, we demonstrate that Silencing Defective 2 (SDE2) is both an RNA binding protein and also a trans-acting adaptor protein that functions to regulate RNA splicing and ribosome biogenesis. SDE2 depletion leads to widespread changes in alternative splicing, defects in ribosome biogenesis and ultimately complete loss of cell viability. Our data highlight SDE2 as a previously uncharacterized essential gene required for the assembly and maturation of the complexes that carry out two of the most fundamental processes in mammalian cells.


Subject(s)
Alternative Splicing/genetics , DNA-Binding Proteins/genetics , RNA Splicing/genetics , Ribosomes/genetics , Genes, Essential/genetics , Humans , RNA-Binding Proteins/genetics , Spliceosomes/genetics
2.
Elife ; 102021 07 14.
Article in English | MEDLINE | ID: mdl-34259631

ABSTRACT

Tuberous sclerosis complex (TSC) is a genetic disorder that is associated with multiple neurological manifestations. Previously, we demonstrated that Tsc1 loss in cerebellar Purkinje cells (PCs) can cause altered social behavior in mice. Here, we performed detailed transcriptional and translational analyses of Tsc1-deficient PCs to understand the molecular alterations in these cells. We found that target transcripts of the Fragile X Mental Retardation Protein (FMRP) are reduced in mutant PCs with evidence of increased degradation. Surprisingly, we observed unchanged ribosomal binding for many of these genes using translating ribosome affinity purification. Finally, we found that multiple FMRP targets, including SHANK2, were reduced, suggesting that compensatory increases in ribosomal binding efficiency may be unable to overcome reduced transcript levels. These data further implicate dysfunction of FMRP and its targets in TSC and suggest that treatments aimed at restoring the function of these pathways may be beneficial.


Subject(s)
Fragile X Mental Retardation Protein/genetics , Fragile X Mental Retardation Protein/metabolism , Purkinje Cells/metabolism , Tuberous Sclerosis Complex 1 Protein/genetics , Tuberous Sclerosis Complex 1 Protein/metabolism , Animals , Disease Models, Animal , Gene Expression , Mice , Nerve Tissue Proteins/metabolism , Ribosomes/metabolism , Tuberous Sclerosis/genetics , Tuberous Sclerosis/metabolism
3.
Cell Rep ; 26(3): 594-607.e7, 2019 01 15.
Article in English | MEDLINE | ID: mdl-30650354

ABSTRACT

Alternative translation initiation and stop codon readthrough in a few well-studied cases have been shown to allow the same transcript to generate multiple protein variants. Because the brain shows a particularly abundant use of alternative splicing, we sought to study alternative translation in CNS cells. We show that alternative translation is widespread and regulated across brain transcripts. In neural cultures, we identify alternative initiation on hundreds of transcripts, confirm several N-terminal protein variants, and show the modulation of the phenomenon by KCl stimulation. We also detect readthrough in cultures and show differential levels of normal and readthrough versions of AQP4 in gliotic diseases. Finally, we couple translating ribosome affinity purification to ribosome footprinting (TRAP-RF) for cell-type-specific analysis of neuronal and astrocytic translational readthrough in the mouse brain. We demonstrate that this unappreciated mechanism generates numerous and diverse protein isoforms in a cell-type-specific manner in the brain.


Subject(s)
Brain/metabolism , Protein Isoforms/metabolism , Proteomics/methods , Animals , Brain/pathology , Mice
4.
Endocrinology ; 158(11): 3954-3973, 2017 11 01.
Article in English | MEDLINE | ID: mdl-28973305

ABSTRACT

Defects in the biosynthesis of phospholipids and neutral lipids are associated with cell membrane dysfunction, disrupted energy metabolism, and diseases including lipodystrophy. In these pathways, the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) enzymes transfer a fatty acid to the sn-2 carbon of sn-1-acylglycerol-3-phosphate (lysophosphatidic acid) to form sn-1, 2-acylglycerol-3-phosphate [phosphatidic acid (PA)]. PA is a precursor for key phospholipids and diacylglycerol. AGPAT1 and AGPAT2 are highly homologous isoenzymes that are both expressed in adipocytes. Genetic defects in AGPAT2 cause congenital generalized lipodystrophy, indicating that AGPAT1 cannot compensate for loss of AGPAT2 in adipocytes. To further explore the physiology of AGPAT1, we characterized a loss-of-function mouse model (Agpat1-/-). The majority of Agpat1-/- mice died before weaning and had low body weight and low plasma glucose levels, independent of plasma insulin and glucagon levels, with reduced percentage of body fat but not generalized lipodystrophy. These mice also had decreased hepatic messenger RNA expression of Igf-1 and Foxo1, suggesting a decrease in gluconeogenesis. In male mice, sperm development was impaired, with a late meiotic arrest near the onset of round spermatid production, and gonadotropins were elevated. Female mice showed oligoanovulation yet retained responsiveness to gonadotropins. Agpat1-/- mice also demonstrated abnormal hippocampal neuron development and developed audiogenic seizures. In summary, Agpat1-/- mice developed widespread disturbances of metabolism, sperm development, and neurologic function resulting from disrupted phospholipid homeostasis. AGPAT1 appears to serve important functions in the physiology of multiple organ systems. The Agpat1-deficient mouse provides an important model in which to study the contribution of phospholipid and triacylglycerol synthesis to physiology and diseases.


Subject(s)
1-Acylglycerol-3-Phosphate O-Acyltransferase/genetics , Glycerol-3-Phosphate O-Acyltransferase/genetics , Infertility/genetics , Metabolic Diseases/genetics , Nervous System Diseases/genetics , Animals , Cells, Cultured , Female , Gluconeogenesis/genetics , Lipid Metabolism/genetics , Lipogenesis/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Reproduction/genetics
5.
Front Mol Neurosci ; 10: 9, 2017.
Article in English | MEDLINE | ID: mdl-28190998

ABSTRACT

Studies on regulation of gene expression have contributed substantially to understanding mechanisms for the long-term activity-dependent alterations in neural connectivity that are thought to mediate learning and memory. Most of these studies, however, have focused on the regulation of mRNA transcription. Here, we utilized high-throughput sequencing coupled with ribosome footprinting to globally characterize the regulation of translation in primary mixed neuronal-glial cultures in response to sustained depolarization. We identified substantial and complex regulation of translation, with many transcripts demonstrating changes in ribosomal occupancy independent of transcriptional changes. We also examined sequence-based mechanisms that might regulate changes in translation in response to depolarization. We found that these are partially mediated by features in the mRNA sequence-notably upstream open reading frames and secondary structure in the 5' untranslated region-both of which predict downregulation in response to depolarization. Translationally regulated transcripts are also more likely to be targets of FMRP and include genes implicated in autism in humans. Our findings support the idea that control of mRNA translation plays an important role in response to neural activity across the genome.

6.
Biol Psychiatry ; 81(3): 252-264, 2017 02 01.
Article in English | MEDLINE | ID: mdl-27113499

ABSTRACT

BACKGROUND: Studies in psychiatric genetics have identified >100 loci associated with disease risk, yet many of these loci are distant from protein coding genes. Recent characterization of the transcriptional landscape of cell lines and whole tissues has suggested widespread transcription in both coding and noncoding regions of the genome, including differential expression from loci that produce regulatory noncoding RNAs that function within the nucleus; however, the nuclear transcriptome of specific cell types in the brain has not been previously investigated. METHODS: We defined the nuclear transcriptional landscape of the three major cellular divisions of the nervous system using flow sorting of genetically labeled nuclei from bacTRAP mouse lines. Next, we characterized the unique expression of coding, noncoding, and intergenic RNAs in the mature mouse brain with RNA-Seq and validation with independent methods. RESULTS: We found diverse expression across the cell types of all classes of RNAs, including long noncoding RNAs, several of which were confirmed as highly enriched in the nuclei of specific cell types using anatomic methods. We also discovered several examples of cell type-specific expression of tandem gene fusions, and we report the first cell type-specific expression of circular RNAs-a neuron-specific and nuclear-enriched RNA arising from the gene Hnrnpu. CONCLUSIONS: These data provide an important resource for studies evaluating the function of various noncoding RNAs in the brain, including noncoding RNAs that may play a role in psychiatric disease.


Subject(s)
Brain/metabolism , Neuroglia/metabolism , Neurons/metabolism , RNA, Nuclear/metabolism , Transcriptome , Animals , Female , Heterogeneous-Nuclear Ribonucleoprotein U/metabolism , Male , Mice , Oligodendroglia/metabolism , RNA, Long Noncoding/metabolism , Sequence Analysis, RNA
7.
J Lipid Res ; 57(4): 616-30, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26880786

ABSTRACT

Reducing triacylglycerol (TAG) in the liver continues to pose a challenge in states of nonalcoholic hepatic steatosis. MonoacylglycerolO-acyltransferase (MOGAT) enzymes convert monoacylglycerol (MAG) to diacylglycerol, a precursor for TAG synthesis, and are involved in a major pathway of TAG synthesis in selected tissues, such as small intestine. MOGAT1 possesses MGAT activity in in vitro assays, but its physiological function in TAG metabolism is unknown. Recent studies suggest a role for MOGAT1 in hepatic steatosis in lipodystrophic [1-acylglycerol-3-phosphateO-acyltransferase (Agpat)2(-/-)] and obese (ob/ob) mice. To test this, we deletedMogat1in theAgpat2(-/-)andob/obgenetic background to generateMogat1(-/-);Agpat2(-/-)andMogat1(-/-);ob/obdouble knockout (DKO) mice. Here we report that, despite the absence ofMogat1in either DKO mouse model, we did not find any decrease in liver TAG by 16 weeks of age. Additionally, there were no measureable changes in plasma glucose (diabetes) and insulin resistance. Our data indicate a minimal role, if any, of MOGAT1 in liver TAG synthesis, and that TAG synthesis in steatosis associated with lipodystrophy and obesity is independent of MOGAT1. Our findings suggest that MOGAT1 likely has an alternative function in vivo.


Subject(s)
Acyltransferases/deficiency , Acyltransferases/genetics , Gene Deletion , Lipodystrophy/genetics , Non-alcoholic Fatty Liver Disease/enzymology , Non-alcoholic Fatty Liver Disease/genetics , Animals , Female , Insulin/blood , Insulin Resistance , Lipodystrophy/complications , Lipodystrophy/metabolism , Liver/metabolism , Male , Mice , Mice, Obese , Non-alcoholic Fatty Liver Disease/blood , Non-alcoholic Fatty Liver Disease/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism
8.
Genes Dev ; 27(5): 565-78, 2013 Mar 01.
Article in English | MEDLINE | ID: mdl-23431030

ABSTRACT

Hypocretin (orexin; Hcrt)-containing neurons of the hypothalamus are essential for the normal regulation of sleep and wake behaviors and have been implicated in feeding, anxiety, depression, and reward. The absence of these neurons causes narcolepsy in humans and model organisms. However, little is known about the molecular phenotype of these cells; previous attempts at comprehensive profiling had only limited sensitivity or were inaccurate. We generated a Hcrt translating ribosome affinity purification (bacTRAP) line for comprehensive translational profiling of all ribosome-bound transcripts in these neurons in vivo. From this profile, we identified >6000 transcripts detectably expressed above background and 188 transcripts that are highly enriched in these neurons, including all known markers of the cells. Blinded analysis of in situ hybridization databases suggests that ~60% of these are expressed in a Hcrt marker-like pattern. Fifteen of these were confirmed with double labeling and microscopy, including the transcription factor Lhx9. Ablation of this gene results in a >30% loss specifically of Hcrt neurons, without a general disruption of hypothalamic development. Polysomnography and activity monitoring revealed a profound hypersomnolence in these mice. These data provide an in-depth and accurate profile of Hcrt neuron gene expression and suggest that Lhx9 may be important for specification or survival of a subset of these cells.


Subject(s)
Gene Expression Profiling , Gene Expression Regulation , Intracellular Signaling Peptides and Proteins , Neurons/metabolism , Neuropeptides , Sleep/physiology , Animals , Female , Hypothalamus/cytology , Hypothalamus/metabolism , LIM-Homeodomain Proteins/genetics , LIM-Homeodomain Proteins/metabolism , Male , Mice , Mice, Inbred C57BL , Orexins , Promoter Regions, Genetic/genetics , Protein Array Analysis , Sleep/genetics , Transcription Factors/genetics , Transcription Factors/metabolism
9.
Mol Cell Neurosci ; 50(2): 201-10, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22579729

ABSTRACT

Interaction of the cell adhesion molecule L1 with the cytoskeletal adaptor ankyrin is essential for topographic mapping of retinal ganglion cell (RGC) axons to synaptic targets in the superior colliculus (SC). Mice mutated in the L1 ankyrin-binding motif (FIGQY(1229)H) display abnormal mapping of RGC axons along the mediolateral axis of the SC, resembling mouse mutant phenotypes in EphB receptor tyrosine kinases. To investigate whether L1 functionally interacts with EphBs, we investigated the role of EphB kinases in phosphorylating L1 using a phospho-specific antibody to the tyrosine phosphorylated FIGQY(1229) motif. EphB2, but not an EphB2 kinase dead mutant, induced tyrosine phosphorylation of L1 at FIGQY(1229) and perturbed ankyrin recruitment to the membrane in L1-transfected HEK293 cells. Src family kinases mediated L1 phosphorylation at FIGQY(1229) by EphB2. Other EphB receptors that regulate medial-lateral retinocollicular mapping, EphB1 and EphB3, also mediated phosphorylation of L1 at FIGQY(1229). Tyrosine(1176) in the cytoplasmic domain of L1, which regulates AP2/clathrin-mediated endocytosis and axonal trafficking, was not phosphorylated by EphB2. Accordingly mutation of Tyr(1176) to Ala in L1-Y(1176)A knock-in mice resulted in normal retinocollicular mapping of ventral RGC axons. Immunostaining of the mouse SC during retinotopic mapping showed that L1 colocalized with phospho-FIGQY in RGC axons in retinorecipient layers. Immunoblotting of SC lysates confirmed that L1 was phosphorylated at FIGQY(1229) in wild type but not L1-FIGQY(1229)H (L1Y(1229)H) mutant SC, and that L1 phosphorylation was decreased in the EphB2/B3 mutant SC. Inhibition of ankyrin binding in L1Y(1229)H mutant RGCs resulted in increased neurite outgrowth compared to WT RGCs in retinal explant cultures, suggesting that L1-ankyrin binding serves to constrain RGC axon growth. These findings are consistent with a model in which EphB kinases phosphorylate L1 at FIGQY(1229) in retinal axons to modulate L1-ankyrin binding important for mediolateral retinocollicular topography.


Subject(s)
Brain Mapping , Neural Cell Adhesion Molecule L1/metabolism , Receptor, EphB2/metabolism , Retinal Ganglion Cells/physiology , Superior Colliculi/physiology , Animals , Ankyrins/metabolism , Cells, Cultured , HEK293 Cells , Humans , Mice , Mice, Mutant Strains , Mutation , Neural Cell Adhesion Molecule L1/chemistry , Neural Cell Adhesion Molecule L1/genetics , Phosphorylation , Protein Interaction Domains and Motifs , Receptor, EphB2/genetics , Receptors, Eph Family/metabolism , Retinal Ganglion Cells/metabolism , Tyrosine/genetics
10.
J Neurochem ; 119(4): 772-84, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21895655

ABSTRACT

As class III unconventional myosins are motor proteins with an N-terminal kinase domain, it seems likely they play a role in both signaling and actin based transport. A growing body of evidence indicates that the motor functions of human class IIIA myosin, which has been implicated in progressive hearing loss, are modulated by intermolecular autophosphorylation. However, the phosphorylation sites have not been identified. We studied the kinase activity and phosphorylation sites of mouse class III myosins, mMyo3A and 3B, which are highly similar to their human orthologs. We demonstrate that the kinase domains of mMyo3A and 3B are active kinases, and that they have similar, if not identical, substrate specificities. We show that the kinase domains of these proteins autophosphorylate, and that they can phosphorylate sites within their myosin and tail domains. Using liquid chromatography-mass spectrometry, we identified phosphorylated sites in the kinase, myosin motor and tail domains of both mMyo3A and 3B. Most of the phosphorylated sites we identified and their consensus phosphorylation motifs are highly conserved among vertebrate class III myosins, including human class III myosins. Our findings are a major step toward understanding how the functions of class III myosins are regulated by phosphorylation.


Subject(s)
Myosin Type III/chemistry , Myosin Type III/metabolism , Protein Kinases/metabolism , Amino Acid Sequence , Amino Acids , Animals , Humans , Mass Spectrometry , Mice , Myosin Type III/classification , Myosin Type III/genetics , Peptides/metabolism , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Analysis, Protein , Substrate Specificity
11.
J Neurosci ; 31(4): 1545-58, 2011 Jan 26.
Article in English | MEDLINE | ID: mdl-21273439

ABSTRACT

NrCAM is a neural cell adhesion molecule of the L1 family that has been linked to autism spectrum disorders, a disease spectrum in which abnormal thalamocortical connectivity may contribute to visual processing defects. Here we show that NrCAM interaction with neuropilin-2 (Npn-2) is critical for semaphorin 3F (Sema3F)-induced guidance of thalamocortical axon subpopulations at the ventral telencephalon (VTe), an intermediate target for thalamic axon sorting. Genetic deletion of NrCAM or Npn-2 caused contingents of embryonic thalamic axons to misproject caudally in the VTe. The resultant thalamocortical map of NrCAM-null mutants showed striking mistargeting of motor and somatosensory thalamic axon contingents to the primary visual cortex, but retinogeniculate targeting and segregation were normal. NrCAM formed a molecular complex with Npn-2 in brain and neural cells, and was required for Sema3F-induced growth cone collapse in thalamic neuron cultures, consistent with a vital function for NrCAM in Sema3F-induced axon repulsion. NrCAM-null mice displayed reduced responses to visual evoked potentials recorded from layer IV in the binocular zone of primary visual cortex (V1), particularly when evoked from the ipsilateral eye, indicating abnormal visual acuity and ocularity. These results demonstrate that NrCAM is required for normal maturation of cortical visual acuity, and suggest that the aberrant projection of thalamic motor and somatosensory axons to the visual cortex in NrCAM-null mutant mice impairs cortical functions.


Subject(s)
Axons/physiology , Cell Adhesion Molecules/physiology , Motor Cortex/ultrastructure , Somatosensory Cortex/ultrastructure , Thalamus/ultrastructure , Visual Acuity , Visual Cortex/ultrastructure , Animals , Cell Adhesion Molecules/genetics , Evoked Potentials, Visual , Female , Growth Cones/physiology , Male , Membrane Proteins/physiology , Mice , Mice, Knockout , Motor Cortex/embryology , Motor Cortex/growth & development , Nerve Tissue Proteins/physiology , Neuropilin-2/genetics , Neuropilin-2/physiology , Somatosensory Cortex/embryology , Somatosensory Cortex/growth & development , Thalamus/embryology , Thalamus/growth & development , Visual Cortex/embryology , Visual Cortex/growth & development
12.
J Neurosci ; 29(50): 15630-41, 2009 Dec 16.
Article in English | MEDLINE | ID: mdl-20016077

ABSTRACT

ALCAM [activated leukocyte cell adhesion molecule (BEN/SC-1/DM-GRASP)] is a transmembrane recognition molecule of the Ig superfamily (IgSF) containing five Ig domains (two V-type, three C2-type). Although broadly expressed in the nervous and immune systems, few of its developmental functions have been elucidated. Because ALCAM has been suggested to interact with the IgSF adhesion molecule L1, a determinant of retinocollicular mapping, we hypothesized that ALCAM might direct topographic targeting to the superior colliculus (SC) by serving as a substrate within the SC for L1 on incoming retinal ganglion cell (RGC) axons. ALCAM was expressed in the SC during RGC axon targeting and on RGC axons as they formed the optic nerve; however, it was downregulated distally on RGC axons as they entered the SC. Axon tracing with DiI revealed pronounced mistargeting of RGC axons from the temporal retina half of ALCAM null mice to abnormally lateral sites in the contralateral SC, in which these axons formed multiple ectopic termination zones. ALCAM null mutant axons were specifically compromised in medial orientation of interstitial branches, which is known to require the ankyrin binding function of L1. As a substrate, ALCAM-Fc protein promoted L1-dependent attachment of acutely dissociated retinal cells and an L1-expressing, ALCAM-negative cell line, consistent with an ALCAM-L1 heterophilic molecular interaction. Together, these results suggest a model in which ALCAM in the SC interacts with L1 on RGC axons to promote medial extension of RGC axon branches important for mediolateral axon targeting in the formation of retinocollicular maps.


Subject(s)
Activated-Leukocyte Cell Adhesion Molecule/physiology , Brain Mapping , Retina/physiology , Superior Colliculi/physiology , Activated-Leukocyte Cell Adhesion Molecule/genetics , Activated-Leukocyte Cell Adhesion Molecule/metabolism , Animals , Animals, Newborn , Brain Mapping/methods , Cell Line, Tumor , Functional Laterality/physiology , Humans , Mice , Mice, Knockout , Mice, Mutant Strains , Neural Cell Adhesion Molecule L1/metabolism , Neural Cell Adhesion Molecule L1/physiology , Retinal Ganglion Cells/physiology , Visual Pathways/physiology
13.
Exp Eye Res ; 89(2): 224-37, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19332056

ABSTRACT

Class III myosins are important for the function and survival of photoreceptors and ciliary hair cells. Although vertebrates possess two class III myosin genes, myo3A and myo3B, recent studies have focused on Myo3A because mutations in the human gene are implicated in progressive hearing loss. Myo3B may compensate for defects in Myo3A, yet little is known about its distribution and function. This study focuses on Myo3B expression in the mouse retina. We cloned two variants of myo3B from mouse retina and determined that they are expressed early in retinal development. In this study we show for the first time in a mammal that both Myo3B and Myo3A proteins are present in inner segments of all photoreceptors. Myo3B is also present in outer segments of S opsin-immunoreactive cones but not M opsin dominant cones. Myo3B is also detected in rare cells of the inner nuclear layer and some ganglion cells. Myo3B may have diverse roles in retinal neurons. In photoreceptor inner segments Myo3B is positioned appropriately to prevent photoreceptor loss of function caused by Myo3A defects.


Subject(s)
Eye Proteins/metabolism , Myosin Heavy Chains/metabolism , Myosin Type III/metabolism , Retina/metabolism , Amino Acid Sequence , Animals , Cloning, Molecular , Eye Proteins/genetics , Eye Proteins/immunology , Immune Sera , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Myosin Heavy Chains/genetics , Myosin Heavy Chains/immunology , Myosin Type III/genetics , Myosin Type III/immunology , Retina/growth & development , Retinal Cone Photoreceptor Cells/metabolism , Retinal Ganglion Cells/metabolism , Retinal Photoreceptor Cell Outer Segment/metabolism , Tissue Distribution
14.
Vis Neurosci ; 20(5): 523-34, 2003.
Article in English | MEDLINE | ID: mdl-14977331

ABSTRACT

Much has been learned from studies of Limulus photoreceptors about the role of the circadian clock and light in the removal of photosensitive membrane. However, little is known in this animal about mechanisms regulating photosensitive membrane renewal, including the synthesis of proteins in, and associated with, the photosensitive membrane. To begin to understand renewal, this study examines diurnal changes in the levels of mRNAs encoding opsin, the integral membrane protein component of visual pigment, and the relative roles of light and the circadian clock in producing these changes. We show that at least two distinct opsin genes encoding very similar proteins are expressed in both the lateral and ventral eyes, and that during the day and night in the lateral eye, the average level of mRNA encoding opsinl is consistently higher than that encoding opsin2. Northern blot assays showed further that total opsin mRNA in the lateral eyes of animals maintained under natural illumination increases during the afternoon (9 & 12 h after sunrise) in the light and falls at night in the dark. This diurnal change occurs whether or not the eyes receive input from the circadian clock, but it is eliminated in eyes maintained in the dark. Thus, it is regulated by light and darkness, not by the circadian clock, with light stimulating an increase in opsin mRNA levels. The rise in opsin mRNA levels observed under natural illumination was seasonal; it occurred during the summer but not the spring and fall. However, a significant increase in opsin mRNA levels could be achieved in the fall by exposing lateral eyes to 3 h of natural illumination followed by 9 h of artificial light. The diurnal regulation of opsin mRNA levels contrasts sharply with the circadian regulation of visual arrestin mRNA levels (Battelle et al., 2000). Thus, in Limulus, distinctly different mechanisms regulate the levels of mRNA encoding two proteins critical for the photoresponse.


Subject(s)
Circadian Rhythm/physiology , Gene Expression Regulation/physiology , Rod Opsins/metabolism , Animals , Autoradiography , Blotting, Northern/methods , Blotting, Southern , Denervation/methods , Eye/cytology , Eye/metabolism , Functional Laterality , Horseshoe Crabs , Molecular Sequence Data , Photoreceptor Cells, Invertebrate/metabolism , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Rod Opsins/genetics , Time Factors
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