Altered Gene Expression Associated with microRNA Binding Site Polymorphisms

Allele-specific gene expression associated with genetic variation in regulatory regions can play an important role in the development of complex traits. We hypothesized that polymorphisms in microRNA (miRNA) response elements (MRE-SNPs) that either disrupt a miRNA binding site or create a new miRNA binding site can affect the allele-specific expression of target genes. By integrating public expression quantitative trait locus (eQTL) data, miRNA binding site predictions, small RNA sequencing, and Argonaute crosslinking immunoprecipitation (AGO-CLIP) datasets, we identified genetic variants that can affect gene expression by modulating miRNA binding efficiency. We also identified MRE-SNPs located in regions associated with complex traits, indicating possible causative mechanisms associated with these loci. The results of this study expand the current understanding of gene expression regulation and help to interpret the mechanisms underlying eQTL effects.     

IgG from patients with liver diseases inhibit mitochondrial respiration in permeabilized oxidative muscle cells: Impaired function of intracellular energetic units?

The effect of IgG purified from the sera of healthy persons and patients with primary biliary cirrhosis (PBC) and chronic hepatitis (CH) on ADP dependent respiration (oxidative phosphorylation) in skinned muscle fibers from rat oxidative muscles (heart and M. soleus) and glycolytic skeletal muscle (M. gastrocnemius) was studied. The results show that IgG from three different sources inhibited the rate of respiration by 13, 44 and 42%, respectively, these effects being equally expressed in both types of oxidative muscles, whereas no inhibition was observed in glycolytic muscle. The following washout of unbound IgG did not abolish the inhibition of respiration suggesting that the specific interaction of IgG with antigens had taken place. Laser confocal analysis revealed binding of IgG predominantly to the sarcomeric structures such as Z-disk and M-lines in the cardiomyocytes. The staining of IgG within Z-disks and intermitochondrial space coincided throughout the muscle cells so that transversally serial spaces, each containing mitochondria and adjacent sarcomere, became clearly visible. When the IgG from a CH patient was incubated with the skinned myocardial fibers of the desmin knockout mice, its binding to Z-disks and the sarcomeric area was found to be similar to that in normal cardiac muscle. However, the transversal staining pattern was disintegrated, because of the slippage of the myofibrils in relation to each other and accumulation of mitochondria between them. These observations support the recent hypothesis that in oxidative muscles the mitochondria and adjacent sarcomeres form complexes, termed as the intracellular energetic units, ICEUs. Moreover, they indicate that human autoantibodies can be useful tools for localizing the proteins responsible for formation of ICEUs and modulation of their function. Thus, it appears that the proteins associated with the Z-disks and M-lines may participate in formation of ICEUs and that binding of IgG to these proteins decreases the access of exogenous adenine nucleotides to mitochondria, which manifests as decreased rate of ADP-dependent respiration.     

Macrocompartmentation of total creatine in cardiomyocytes revisited

Distribution of total creatine (free creatine + phosphocreatine) between two subcellular macrocompartments – mitochondrial matrix space and cytoplasm – in heart and skeletal muscle cells was reinvestigated by using a permeabilized cell technique. Isolated cardiomyocytes were treated with saponin (50 g/ml for 30 min or 600 g/ml for 1 min) to open the outer cellular membrane and release the metabolites from cytoplasm (cytoplasmic fraction, CF). All mitochondrial population in permeabilized cells remained intact: the outer membrane was impermeable for exogenous cytochrome c, the acceptor control index of respiration exceeded 10, the mitochondrial creatine kinase reaction was fully coupled to the adenine nucleotide translocator. Metabolites were released from mitochondrial fraction (MF) by 2–5% Triton X100. Total cellular pool of free creatine + phosphocreatine (69.6 ± 2.1 nmoles per mg of protein) was found exclusively in CF and was practically absent in MF. When fibers were prepared from perfused rat hearts, cellular distribution of creatine was not dependent on functional state of the heart and only slightly modified by ischemia. It is concluded that there is no stable pool of creatine or phosphocreatine in the mitochondrial matrix in the intact muscle cells, and the total creatine pool is localized in only one macrocompartment – cytoplasm.     

Developmental changes in regulation of mitochondrial respiration by ADP and creatine in rat heart in vivo

In saponin-skinned muscle fibers from adult rat heart and m. soleus the apparent affinity of the mitochondrial oxidative phosphorylation system for ADP (K_m = 200-400 M) is much lower than in isolated mitochondria (K_m = 10-20 M). This suggests a limited permeability of the outer mitochondrial membrane (OMM) to adenine nucleotides in slow-twitch muscle cells. We have studied the postnatal changes in the affinity of mitochondrial respiration for ADP, in relation to morphological alterations and expression of mitochondrial creatine kinase (mi-CK) in rat heart in vivo. Analysis of respiration of skinned fibers revealed a gradual decrease in the apparent affinity of mitochondria to ADP throughout 6 weeks post partum that indicates the development of mechanism which increasingly limits the access of ADP to mitochondria. The expression of mi-CK started between the 1st and 2nd weeks and reached the adult levels after 6 weeks. This process was associated with increases in creatine-activated respiration and affinity of oxidative phosphorylation to ADP thus reflecting the progressive coupling of mi-CK to adenine nucleotide translocase. Laser confocal microscopy revealed significant changes in rearrangement of mitochondria in cardiac cells: while the mitochondria of variable shape and size appeared to be random-clustered in the cardiomyocytes of 1 day old rat, they formed a fine network between the myofibrils by the age of 3 weeks. These results allow to conclude that in early period of development, i.e. within 2-3 weeks, the diffusion of ADP to mitochondria becomes progressively restricted, that appears to be related to significant structural rearrangements such as formation of the mitochondrial network. Later (after 3 weeks) the control shifts to mi-CK, which by coupling to adenine nucleotide translocase, allows to maximally activate the processes of oxidative phosphorylation despite limited access of ADP through the OMM.     

Human Disease-Associated Genetic Variation Impacts Large Intergenic Non-Coding RNA Expression

Recently it has become clear that only a small percentage (7%) of disease-associated single nucleotide polymorphisms (SNPs) are located in protein-coding regions, while the remaining 93% are located in gene regulatory regions or in intergenic regions. Thus, the understanding of how genetic variations control the expression of non-coding RNAs (in a tissue-dependent manner) has far-reaching implications. We tested the association of SNPs with expression levels (eQTLs) of large intergenic non-coding RNAs (lincRNAs), using genome-wide gene expression and genotype data from five different tissues. We identified 112 cis-regulated lincRNAs, of which 45% could be replicated in an independent dataset. We observed that 75% of the SNPs affecting lincRNA expression (lincRNA cis-eQTLs) were specific to lincRNA alone and did not affect the expression of neighboring protein-coding genes. We show that this specific genotype-lincRNA expression correlation is tissue-dependent and that many of these lincRNA cis-eQTL SNPs are also associated with complex traits and diseases.