Risk alleles of genes with monoallelic expression are enriched in gain-of-function variants and depleted in loss-of-function variants for neurodevelopmental disorders.

Over 3000 human genes can be expressed from a single allele in one cell, and from the other allele-or both-in neighboring cells. Little is known about the consequences of this epigenetic phenomenon, monoallelic expression (MAE). We hypothesized that MAE increases expression variability, with a potential impact on human disease. Here, we use a chromatin signature to infer MAE for genes in lymphoblastoid cell lines and human fetal brain tissue. We confirm that across clones MAE status correlates with expression level, and that in human tissue data sets, MAE genes show increased expression variability. We then compare mono- and biallelic genes at three distinct scales. In the human population, we observe that genes with polymorphisms influencing expression variance are more likely to be MAE (P<1.1 × 10-6). At the trans-species level, we find gene expression differences and directional selection between humans and chimpanzees more common among MAE genes (P<0.05). Extending to human disease, we show that MAE genes are under-represented in neurodevelopmental copy number variants (CNVs) (P<2.2 × 10-10), suggesting that pathogenic variants acting via expression level are less likely to involve MAE genes. Using neuropsychiatric single-nucleotide polymorphism (SNP) and single-nucleotide variant (SNV) data, we see that genes with pathogenic expression-altering or loss-of-function variants are less likely MAE (P<7.5 × 10-11) and genes with only missense or gain-of-function variants are more likely MAE (P<1.4 × 10-6). Together, our results suggest that MAE genes tolerate a greater range of expression level than biallelic expression (BAE) genes, and this information may be useful in prediction of pathogenicity.

Olfactory receptor trafficking involves conserved regulatory steps.

Olfactory receptors are difficult to functionally express in heterologous cells. They are typically retained in the endoplasmic reticulum of cells commonly used for functional expression studies and are only released to the plasma membrane in mature cells of the olfactory receptor neuron lineage. A recently developed olfactory cell line, odora, traffics olfactory receptors to the plasma membrane when differentiated. We found that undifferentiated odora cells do not traffic olfactory receptors to their surface, even though they release the receptors to the Golgi apparatus and endosomes. This behavior differs from other cell lines tested thus far. Differentiated odora cells also properly traffic vomeronasal receptors of the VN1 type, which lack sequence similarity to olfactory receptors. ODR-4, a protein that is necessary for plasma membrane trafficking of a chemosensory receptor in nematodes, facilitates trafficking of rat olfactory receptor U131 in odora and Chinese hamster ovary cells. Olfactory receptor trafficking from the endoplasmic reticulum to the plasma membrane involves at least two steps whose regulation depends on the maturation state of cells in the olfactory receptor neuron lineage. These results also indicate that some components of the regulatory mechanism are conserved.

Truncation releases olfactory receptors from the endoplasmic reticulum of heterologous cells.

Olfactory receptors are difficult to express functionally in heterologous cells. We found that olfactory receptors traffic poorly to the plasma membrane even in cells with neuronal phenotypes, including cell lines derived from the olfactory epithelium. Other than mature olfactory receptor neurons, few cells appear able to traffic olfactory receptors to the plasma membrane. In human embryonic kidney 293 cells and Xenopus fibroblasts, olfactory receptor immunoreactivity overlapped with a marker for the endoplasmic reticulum (ER) but not with markers for the Golgi apparatus or endosomes. Except for the ER, olfactory receptors were therefore absent from organelles normally involved in the plasma membrane trafficking of receptors. Olfactory receptors truncated prior to transmembrane domain VI were expressed in the plasma membrane, however. Co-expression of the missing C-terminal fragment with these truncated receptors prevented their expression in the plasma membrane. Intramolecular interactions between N- and C-terminal domains joined by the third cytoplasmic loop appear to be responsible for retention of olfactory receptors in the ER of heterologous cells. Our results are consistent with misfolding of the receptors but could also be explained by altered trafficking of the receptors.

Consensus translational initiation sites of marine invertebrate phyla.

The efficiency of translational initiation depends upon the sequence context surrounding the AUG codon (1, 2, 3). A purine at position -3 contributes critically to context, but other neighboring nucleotides are also important. Nucleotide frequencies at these neighboring positions vary among distant taxa (4, 5). We have analyzed the translational initiation sites of cnidarian, echinoderm, molluscan, annelid, and crustacean sequences in nucleotide sequence databases. These taxa conform to the pattern of a strong preference for a purine at -3, but the frequencies of nucleotides at neighboring positions are characteristic for each taxon. The consensus translational initiation sequences of the marine invertebrate taxa are also different from those of vertebrates and single-celled eukaryotes. These consensus sequences are useful guides for predicting translational initiation sites in cDNA clones.

Functional expression of olfactory-adrenergic receptor chimeras and intracellular retention of heterologously expressed olfactory receptors.

Replacing the G-protein-coupling domains of the beta 2-adrenergic receptor with homologous domains of putative olfactory receptors produced chimeric receptors which were able to stimulate pigment dispersion in Xenopus melanophores, a G-protein-mediated pathway. A multiple replacement chimera containing the second, third and C-terminal cytoplasmic domains of receptor OR5 elevated cyclic adenosine 3':5'-monophosphate (cAMP) and suppressed production of inositol phosphates. Co-expression of G alpha olf did not alter the strength of response of this chimera. A novel rat olfactory receptor cDNA (U131) was isolated and sequenced. Expression of U131 and OR5 constructs containing an N-terminal epitope-tag or C-terminal fusion to green fluorescent protein occurred in an intracellular network but not in the plasma membrane of heterologous cells. Similarly treated beta 2-adrenergic receptors were functional and were observed in the plasma membrane and the intracellular network. These results demonstrate that the putative cytoplasmic domains of olfactory receptors are capable of functional interaction with heterologous G-proteins of the G alpha s subtype. Instead, the absence of these receptors from the plasma membrane of heterologous cells appears to explain our inability to determine if odorants can activate the olfactory receptor clones. We hypothesize that the olfactory receptors have requirements for maturation and targeting to the plasma membrane that are different from most other G-protein-coupled receptors.

Recoverin mediates the calcium effect upon rhodopsin phosphorylation and cGMP hydrolysis in bovine retina rod cells.

Rhodopsin phosphorylation and in consequence cGMP hydrolysis in bovine rod outer segments are Ca2+ dependent in the presence of ATP. The level of rhodopsin phosphorylation decreases and the lifetime of active phosphodiesterase increases when the free [Ca2+] is raised from < 1 nM to about 1 microM; in both cases the half-maximal effect was observed at 140-170 nM of free Ca2+. Antibodies to recoverin reverse both effects at high [Ca2+] but have no influence at low [Ca2+]. We conclude that the Ca2+ effects observed are mediated by recoverin which inhibits rhodopsin kinase at a high Ca2+ level.