Ribosome assembly in eukaryotes.

The gamma-aminobutyric acid type A (GABAA) receptor represents an elementary switching mechanism integral to the functioning of the central nervous system and a locus for the action of many mood- and emotion-altering agents such as benzodiazepines, barbiturates, steroids, and alcohol. Anxiety, sleep disorders, and convulsive disorders have been effectively treated with therapeutic agents that enhance the action of GABA at the GABAA receptor or increase the concentration of GABA in nervous tissue. The GABAA receptor is a multimeric membrane-spanning ligand-gated ion channel that admits chloride upon binding of the neurotransmitter GABA and is modulated by many endogenous and therapeutically important agents. Since GABA is the major inhibitory neurotransmitter in the CNS, modulation of its response has profound implications for brain functioning. The GABAA receptor is virtually the only site of action for the centrally acting benzodiazepines, the most widely prescribed of the anti-anxiety medications. Increasing evidence points to an important role for GABA in epilepsy and various neuropsychiatric disorders. Recent advances in molecular biology and complementary information derived from pharmacology, biochemistry, electrophysiology, anatomy and cell biology, and behavior have led to a phenomenal growth in our understanding of the structure, function, regulation, and evolution of the GABAA receptor. Benzodiazepines, barbiturates, steroids, polyvalent cations, and ethanol act as positive or negative modulators of receptor function. The description of a receptor gene superfamily comprising the subunits of the GABAA, nicotinic acetylcholine, and glycine receptors has led to a new way of thinking about gene expression and receptor assembly in the nervous system. Seventeen genetically distinct subunit subtypes (alpha 1-alpha 6, beta 1-beta 4, gamma 1-gamma 4, delta, p1-p2) and alternatively spliced variants contribute to the molecular architecture of the GABAA receptor. Mysteriously, certain preferred combinations of subunits, most notably the alpha 1 beta 2 gamma 2 arrangement, are widely codistributed, while the expression of other subunits, such as beta 1 or alpha 6, is severely restricted to specific neurons in the hippocampal formation or cerebellar cortex. Nervous tissue has the capacity to exert control over receptor number, allosteric uncoupling, subunit mRNA levels, and posttranslational modifications through cellular signal transduction mechanisms under active investigation. The genomic organization of the GABAA receptor genes suggests that the present abundance of subtypes arose during evolution through the duplication and translocations of a primordial alpha-beta-gamma gene cluster. This review describes these varied aspects of GABAA receptor research with special emphasis on contemporary cellular and molecular discoveries.

From ion currents to genomic analysis: Recent advances in GABAA receptor research

γ-Aminobutyric acidA (GABAA) receptors in the mammalian central nervous system (CNS) are members of a family of ligand-gated ion channels consisting of heterooligomeric glycoprotein complexes in synaptic and extrasynaptic membranes. Although molecular cloning studies have identified 5 subunits (with ∼40% amino acid homology) and isoforms thereof (∼70% homology), namely α1–6, β1–4, γ1–3, δ, and ρ, the subunit composition and stoichiometry of native receptors are not known.



The regional distribution and cellular expression of GABAA receptor messenger RNAs (mRNAs) in the rat CNS have now been investigated by in situ hybridization histochemistry with subunit-specific 35S-labelled oligonucleotide probes on adjacent cryostat sections. Whereas α1, β2, and γ2 transcripts were the most abundant and ubiquitous in the rat brain—correlating with the radioautographic distribution of GABAA receptors revealed by an ionophore ligand—others had a more restricted expression while often being abundant. For example, α2 transcripts were found only in the olfactory bulb, cerebral cortex, caudate putamen, hippocampal formation, and certain lower brain stem nuclei; α3 only in the olfactory bulb and cerebral cortex; α5 in the hippocampal formation; and α6 only in cerebellar granule cells. In addition, β1, β3, γ1, and δ mRNAs were also uniquely expressed in restricted brain regions. Moreover, in the spinal cord, α1–3, β2,3, and γ2 mRNAs were differently expressed in Rexed layers 2–9, with α2, β3, and γ2 transcripts most prominent in motoneurons of layer 9.



Although differential protein trafficking could lead to the incorporation of some subunits into somatic membranes and others into dendritic membranes, some tentative conclusions as to the probable composition of native proteins in various regions of the CNS may be drawn. For example, according to the strength of the hybridization signal the following subunits might be expected to play a prominent role in GABAergic neurotransmission in the corresponding regions: α1, β2 (1,3), γ2 in olfactory bulb mitral cells; α2, β3, γ2, δ in caudate putamen; α1,3 (2,5), β2,3, γ2 in cerebral cortex; α1, β2, γ1,2 in pallidum; α1,2,5, β1–3, γ2 in hippocampus; α1,5, β3, γ2, δ in dentate gyrus; α1, β2, γ1,2 in substantia nigra zona reticularis; α1, β2, γ2 in cerebellar Purkinje cells; α1,6, β2,3, γ2, δ in granule cells; α1,2, β2,3, γ2 in vestibular and facial nuclei; and finally α2, β3, γ2 in motoneurons of Rexed layer 9 in the spinal cord. © 1992 Wiley-Liss, Inc.

Comparative molecular neuroanatomy of cloned GABAA receptor subunits in the rat CNS.

We have recently identified an RNA aptamer for the transcription factor NF-κB p50 homodimer [(p50)2], which exhibits little sequence resemblance to the consensus DNA target for (p50)2, but binds (p50)2 with an affinity similar to that of the optimal DNA target. We describe here the 2.45-A resolution x-ray crystal structure of the p50 RHR/RNA aptamer complex. The structure reveals that two RNA molecules bind independent of each other to the p50 N-terminal Ig-like domains. The RNA secondary structure is comprised of a stem and a stem–loop separated by an internal loop folded into a kinked helix because of the cross-strand stacking of three internal loop guanines. These guanines, placed at the edge of the 3′ helix, together with the major groove of the irregular 3′ helix, form the binding surface for p50. Each p50 monomer uses the same surface to recognize the distorted RNA major groove as observed in the κB DNA/p50 RHR complex, resulting in strikingly similar interfaces. The structure reveals how the aptamer specifically selects p50 and discriminates against p65. We also discuss the physiological implications of RNA binding by (p50)2.

https://www.pnas.org/content/pnas/100/16/9268.full.pdf

Crystal structure of NF-κB (p50)2 complexed to a high-affinity RNA aptamer

Multifunctional proteins challenge the conventional 'one protein-one function' paradigm. Here we note apparent multifunctional proteins with nucleic acid partners, tabulating eight examples. We then focus on eight additional cases of transcription factors that bind double-stranded DNA with sequence specificity, but that also appear to lead alternative lives as RNA-binding proteins. Exemplified by the prototypic Xenopus TFIIIA protein, and more recently by mammalian p53, this list of transcription factors includes WT-1, TRA-1, bicoid, the bacterial sigma(70) subunit, STAT1 and TLS/FUS. The existence of transcription factors that bind both DNA and RNA provides an interesting puzzle. Little is known concerning the biological roles of these alternative protein-nucleic acid interactions, and even less is known concerning the structural basis for dual nucleic acid specificity. We discuss how these natural examples have motivated us to identify artificial RNA sequences that competitively inhibit a DNA-binding transcription factor not known to have a natural RNA partner. The identification of such RNAs raises the possibility that RNA binding by DNA-binding proteins is more common than currently appreciated.

/pdf/having-it-both-ways-transcription-factors-that-bind-dna-and-54q44h1wmk.pdf

Having it both ways: transcription factors that bind DNA and RNA

Recent studies have suggested that the GABAA, receptor complex, the site of action of the inhibitory neurotransmitter gamma amino-butyric acid (GABAA) and the anxiolytic benzodiazepines, is heterogeneous. Moreover, its composition may change during development. To better understand the molecular basis of receptor heterogeneity, the levels and distribution of the mRNA encoding the alpha 1 receptor subunit were examined in the developing and adult rat brain with quantitative in situ hybridization histochemistry. Our studies demonstrate that alpha 1 subunit mRNA expression changes during ontogeny. At late embryonic stages and in the first postnatal week, low levels of the mRNA were detected in the cortex, inferior colliculus, and hippocampus. The mRNA levels in these regions increased during the second and third postnatal weeks. Furthermore, a dramatic change in the distribution of the alpha 1 subunit mRNA was seen in the second postnatal week when the message first became detectable in the cerebellar cortex. During subsequent development and in the mature brain, the alpha 1 subunit mRNA was most abundant in the cerebellum, olfactory bulb, and inferior colliculus, although the absolute levels of mRNA varied by as much as sixfold in selected brain regions. The mature distribution of alpha 1 subunit mRNA, along with its temporal appearance in the cerebellum, suggests that this subunit is a constituent of the Type 1 benzodiazepine site of the GABAA receptor complex. Furthermore, the onset of alpha 1 subunit mRNA expression in the cerebellar cortex coincides with a period of extensive synapse formation, raising the possibility that synaptic interactions modulate the appearance of this GABAA receptor subunit in the cerebellum.

Robin Haught Pittman

Papers

Developmental expression of the GABAA receptor α1 subunit mRNA in the rat brain

Functional analysis of the placenta-specific enhancer of the human glycoprotein hormone alpha subunit gene. Emergence of a new element.

A feedback loop coupling 5 S rRNA synthesis to accumulation of a ribosomal protein.