Recent studies in invertebrates indicate that a rapid genomic response to neuronal stimulation has a critical role in long-term changes in synaptic efficacy. Because several of the genes (immediately early genes; IEGs) that respond rapidly to growth factor stimulation of vertebrate cells in vitro are also activated by neuronal stimulation in vivo, attention has focused on the possibility that they play a part in synaptic plasticity in vertebrate nervous systems. Four IEGs thought to encode transcription factors, zif/268 (also termed Egr-1, NGFI-A, Krox 24), c-fos, c-jun, and jun-B are rapidly induced in the brain by seizure activity, and we have now studied the induction of these genes in a well-characterized model of synaptic plasticity in the vertebrate brain--long-term potentiation (LTP) of the perforant pathgranule cell (pp-gc) synapse in vivo. We found that high-frequency (but not low-frequency) stimulation of the pp-gc synapse markedly increases zif/268 messenger RNA (mRNA) levels in the ipsilateral granule cell neurons; mRNA of c-fos, c-jun and jun-B is less consistently increased. The stimulus frequency and intensity required to increase zif/268 mRNA levels are similar to those required to induce LTP, which is also seen only ipsilaterally, and both responses are blocked by NMDA-receptor antagonists as well as by convergent synaptic inhibitory inputs already known to block LTP. Accordingly, zif/268 mRNA levels and LTP seem to be regulated by similar synaptic mechanisms.

Administration of the convulsants pentylenetetrazole (Metrazole) or picrotoxin to rats caused a dramatic increase in mRNAs of four putative transcription factor genes,

Recent studies have demonstrated that several transcription factor genes are rapidly activated by neuronal stimulation. For example, we have found that prolonged and repeated seizure activity produced by administration of chemical convulsants induces a rapid and transient increase in mRNA levels of four immediate early genes in rat brain. These genes, zif/268, c-fos, c-jun, and jun-B, encode sequence specific DNA binding proteins thought to act as transcription regulatory factors. To ascertain whether a brief electrically induced seizure discharge of the type utilized in clinical electroconvulsive treatment is sufficient to induce a similar genomic response, we have examined the response of these mRNAs in rat brain following single and repeated electroshock-induced seizures. After electroshock, mRNA levels of each of these genes increase within 15 min, and all except c-jun return to near baseline levels within 4 h. Although this response is most prominent in granule cell neurons of the hippocampus, increases are also apparent in neocortex and pyriform cortex. The rapid mRNA response persists in animals receiving a chronic electroshock protocol similar to that used in clinical electroconvulsive therapy. Intrahippocampal infusion of the sodium channel antagonist tetrodotoxin blocks hippocampal mRNA responses without blocking seizures, indicating a role for electrical excitation in the electroshock-induced mRNA response. By contrast, pretreatment with anticonvulsants or selective NMDA antagonists, which reduce seizure intensity and block hindlimb extension, fails to alter mRNA responses, suggesting that seizure induction, rather than spread, is linked to these mRNA responses. Because electroshock induces robust, highly reproducible mRNA responses, it may be useful to study the neuronal genomic response to stimulation.

Tryptophan hydroxylase isoform 2 (TPH2) is expressed in serotonergic neurons in the raphe nuclei, where it catalyzes the rate-limiting step in the synthesis of the neurotransmitter serotonin. In search for functional polymorphisms within the TPH2 gene locus, we measured allele-specific expression of TPH2 mRNA in sections of human pons containing the dorsal and median raphe nuclei. Differences in allelic mRNA expression -referred to as allelic expression imbalance (AEI) -are a measure of cis-acting regulation of gene expression and mRNA processing. Two marker SNPs, located in exons 7 and 9 of TPH2 (rs7305115 and rs4290270, respectively), served for quantitative allelic mRNA measurements in pons RNA samples from 27 individuals heterozygous for one or both SNPs. Significant AEI (ranging from 1.2-to 2.5-fold) was detected in 19 out of the 27 samples, implying the presence of cis-acting polymorphisms that differentially affect TPH2 mRNA levels in pons. For individuals heterozygous for both marker SNPs, the results correlated well (r = 0.93), validating the AEI analysis. AEI is tightly associated with the exon 7 marker SNP, in 17 of 18 subjects. Remarkably, expression from the minor allele exceeded that of the major allele in each case, possibly representing a gain-of-function. Genotyping of 20 additional TPH2 SNPs identified a haplotype block of five tightly linked SNPs for which heterozygosity is highly correlated with AEI and overall expression of TPH2 mRNA. These results reveal the presence of a functional cis-acting polymorphism, with high frequency in normal human subjects, resulting in increased TPH2 expression levels. The SNPs that correlate with AEI are closely linked to TPH2 SNPs previously shown to associate with major depression and suicide.

In this study, we report the molecular cloning of cDNAs encoding three distinct isoforms of rat (r) TRP6 Ca 2؉ channels. The longest isoform, rTRP6A, contains 930 amino acid residues; rTRP6B lacks 54 amino acids (3-56) at the N terminus, and rTRP6C is missing an additional 68 amino acids near the C terminus. influx. Glycosylation analysis indicated that rTRP6A and rTRP6B are glycosylated in COS cells, but that rTRP6C is mostly not glycosylated. Together these results suggest that the N terminus (3-56 amino acids) is crucial for the activation of rTRP6A by diacylglycerol and that the 735-802 amino acid segment located just downstream from the 6th transmembrane segment may be required for processing of the rTRP6 protein.

RBM10 is an RNA splicing regulator that is frequently mutated in lung adenocarcinoma (LUAD) and has recently been proposed to be a cancer gene. How RBM10 mutations observed in LUAD affect its normal functions, however, remains largely unknown. Here integrative analysis of RBM10 mutation and RNA expression data revealed that LUAD-associated RBM10 mutations exhibit a mutational spectrum similar to that of tumor suppressor genes. In addition, this analysis showed that RBM10 mutations identified in LUAD patients lacking canonical oncogenes are associated with significantly reduced RBM10 expression. To systematically investigate RBM10 mutations, we developed an experimental pipeline for elucidating their functional effects. Among six representative LUAD-associated RBM10 mutations, one nonsense and one frameshift mutation caused loss-of-function as expected, whereas four missense mutations differentially affected RBM10-mediated splicing. Importantly, changes in proliferation rates of LUAD-derived cells caused by these RBM10 missense mutants correlated with alterations in RNA splicing of RBM10 target genes. Together, our data implies that RBM10 mutations contribute to LUAD pathogenesis, at least in large part, by deregulating splicing. The methods described in this study should be useful for analyzing mutations in additional cancer-associated RNA splicing regulators.

Genetic variation in mRNA expression plays a critical role in human phenotypic diversity, but it has proven difficult to detect regulatory polymorphisms -mostly single nucleotide polymorphisms (rSNPs). Additionally, variants in the transcribed region, termed here 'structural RNA SNPs' (srSNPs), can affect mRNA processing and turnover. Both rSNPs and srSNPs cause allelic mRNA expression imbalance (AEI) in heterozygous individuals. We have applied a rapid and accurate AEI methodology for testing 42 genes implicated in human diseases and drug response, specifically cardiovascular and CNS diseases, and affecting drug metabolism and transport. Each gene was analyzed in physiologically relevant human autopsy tissues, including brain, heart, liver, intestines, and lymphocytes. Substantial AEI was observed in ∼55% of the surveyed genes. Focusing on cardiovascular candidate genes in human hearts, AEI analysis revealed frequent cis-acting regulatory factors in SOD2 and ACE mRNA expression, having potential clinical significance. SNP scanning to locate regulatory polymorphisms in a number of genes failed to support several previously proposed promoter SNPs discovered with use of reporter gene assays in heterologous tissues, while srSNPs appear more frequent than expected. Computational analysis of mRNA folding indicates that ∼90% of srSNPs affects mRNA folding, and hence potentially function. Our results indicate that both rSNPs and srSNPs represent a still largely untapped reservoir of variants that contribute to human phenotypic diversity.

An insertion/deletion polymorphism in the SERT linked promoter region (SERTLPR), previously reported to regulate mRNA expression in vitro, has been associated with mental disorders and response to psychotropic drugs. Contradictory evidence, however, has raised questions about the role of SERTLPR in regulating mRNA expression in vivo. We have used analysis of allelic expression imbalance (AEI) of SERT mRNA to assess quantitatively the contribution of SERTLPR to mRNA expression in human post-mortem pons tissue sections containing serotonergic neurons of the dorsal and median raphe nuclei. Any difference in the expression of one allele over the other indicates the presence of cis-acting elements that differentially affect transcription and/or mRNA processing and turnover. Using a marker SNP in the 3 0 untranslated region of SERT mRNA, statistically significant differences in allelic mRNA levels were detected in nine out of 29 samples heterozygous for the marker SNP. While the allelic expression differences were relatively small (15-25%), they could nevertheless be physiologically relevant. Although previous results had suggested that the long form of SERTLPR yields higher mRNA levels than the short form, we did not observe a correlation between SERTLPR and allelic expression ratios. Also in contrast to previous results, we found no correlation between SERTLPR and allelic expression ratios or SERT mRNA levels in Blymphocytes. This study demonstrates that regulation of SERT mRNA is independent of SERTLPR, but could be associated with polymorphisms in partial linkage disequilibrium with SERTLPR.