Magnesium (Mg) is an essential enzyme cofactor and a key structural component of biological molecules, but relatively little is known about the molecular components required for Mg homeostasis in eukaryotic cells. The yeast genome encodes four characterized members of the CorA Mg transporter superfamily located in the plasma membrane (Alr1 and Alr2) or the mitochondrial inner membrane (Mrs2 and Lpe10). We describe a fifth yeast CorA homolog (Mnr2) required for Mg homeostasis. MNR2 gene inactivation was associated with an increase in both the Mg requirement and the Mg content of yeast cells. In Mg-replete conditions, wild-type cells accumulated an intracellular store of Mg that supported growth under deficient conditions. An mnr2 mutant was unable to access this store, suggesting that Mg was trapped in an intracellular compartment. Mnr2 was localized to the vacuole membrane, implicating this organelle in Mg storage. The mnr2 mutant growth and Mg-content phenotypes were dependent on vacuolar proton-ATPase activity, but were unaffected by the loss of mitochondrial Mg uptake, indicating a specific dependence on vacuole function. Overexpression of Mnr2 suppressed the growth defect of an alr1 alr2 mutant, indicating that Mnr2 could function independently of the ALR genes. Together, our results implicate a novel eukaryotic CorA homolog in the regulation of intracellular Mg storage.

Background Studies in psychiatric genetics have identified over 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 non-coding regions of the genome, including differential expression from loci that produce regulatory non-coding RNAs which function within the nucleus; however, the nuclear transcriptome of specific cell types in the brain has not been previously investigated. Methods Here we have 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. This was followed by characterization of the unique expression of coding, non-coding and intergenic RNAs in the mature mouse brain with RNAseq and validation with independent methods. Results Our findings reveal diverse expression across the cell-types of all classes of RNAs, including long non-coding RNAs – several of which were confirmed as highly enriched in the nuclei of specific cell-types using anatomical methods. Finally, we also discovered several examples of cell-type specific expression of tandem gene fusions, and report the first cell-type specific expression of circular RNAs, notably a neuron-specific and nuclear-enriched RNA arising from the gene Hnrnpu. Conclusion These data will provide an important resource for studies evaluating the function of a variety of ncRNAs in the brain, including those that may play a role in psychiatric disease.

Summary The regulation of transcription and translation by specific cell types is essential to generate the cellular diversity that typifies complex multicellular organisms. Tagging and purification of ribosomal proteins has been shown to be an innovative and effective means of characterizing the ribosome bound transcriptome of highly specific cell populations in vivo. To test the feasibility of using translating ribosome affinity purification (TRAP) in zebrafish, we have generated both a ubiquitous TRAP line and a melanocyte-specific TRAP line using the native zebrafish rpl10a ribosomal protein. We have demonstrated the capacity to capture mRNA transcripts bound to ribosomes, and confirmed the expected enrichment of melanocyte specific genes and depletion of non-melanocyte genes when expressing the TRAP construct with a cell specific promoter. We have also generated a generic EGFP-rpl10a Tol2 plasmid construct (Tol2-zTRAP) that can be readily modified to target any additional cell populations with characterized promoters in zebrafish.

Mg homeostasis is critical to eukaryotic cells, but the contribution of Mg transporter activity to homeostasis is not fully understood. In yeast, Mg uptake is primarily mediated by the Alr1 transporter, which also allows low affinity uptake of other divalent cations such as Ni2+, Mn2+, Zn2+ and Co2+. Using Ni2+ uptake to assay Alr1 activity, we observed approximately nine-fold more activity under Mg-deficient conditions. The mnr2 mutation, which is thought to block release of vacuolar Mg stores, was associated with increased Alr1 activity, suggesting Alr1 was regulated by intracellular Mg supply. Consistent with a previous report of the regulation of Alr1 expression by Mg supply, Mg deficiency and the mnr2 mutation both increased the accumulation of a carboxy-terminal epitope-tagged version of the Alr1 protein (Alr1-HA). However, Mg supply had little effect on ALR1 promoter activity or mRNA levels. In addition, while Mg deficiency caused a seven-fold increase in Alr1-HA accumulation, the N-terminally tagged and untagged Alr1 proteins increased less than two-fold. These observations argue that the Mg-dependent accumulation of the C-terminal epitope-tagged protein was primarily an artifact of its modification. Plasma membrane localization of YFP-tagged Alr1 was also unaffected by Mg supply, indicating that a change in Alr1 location did not explain the increased activity we observed. We conclude that variation in Alr1 protein accumulation or location does not make a substantial contribution to its regulation by Mg supply, suggesting Alr1 activity is directly regulated via as yet unknown mechanisms.