RNA–protein interactions are crucial for most biological processes in all organisms. However, it appears that the complexity of RNA-based regulation increases with the complexity of the organism, creating additional regulatory circuits, the scope of which is only now being revealed. It is becoming apparent that previously unappreciated features, such as disordered structural regions in proteins or non-coding regions in DNA leading to higher plasticity and pliability in RNA–protein complexes, are in fact essential for complex, precise and fine-tuned regulation. This review addresses the issue of the role of RNA–protein interactions in generating eukaryotic complexity, focusing on the newly characterized disordered RNA-binding motifs, moonlighting of metabolic enzymes, RNA-binding proteins interactions with different RNA species and their participation in regulatory networks of higher order.

Aberrant expression of DNA polymerase β, a key enzyme involved in base excision repair, leads to genetic instability and carcinogenesis. Pol β expression has been previously shown to be regulated at the level of transcription, but there is also evidence of post-transcriptional regulation, since rat transcripts undergo alternative polyadenylation, and the resulting 3′UTR contain at least one regulatory element. Data presented here indicate that RNA of the short 3′UTR folds to form a strong secondary structure (hairpin). Its regulatory role was established utilizing a luciferase-based reporter system. Further studies led to the identification of a protein factor, which binds to this element—the anti-apoptotic, cytoskeleton-related protein Hax-1. The results of in vitro binding analysis indicate that the formation of the RNA–protein complex is significantly impaired by disruption of the hairpin motif. We demonstrate that Hax-1 binds to Pol β mRNA exclusively in the form of a dimer. Biochemical analysis revealed the presence of Hax-1 in mitochondria, but also in the nuclear matrix, which, along with its transcript-binding properties, suggests that Hax-1 plays a role in post-transcriptional regulation of expression of Pol β.

HAX-1 is a multi-functional protein that is involved in the regulation of apoptosis, cell motility and calcium homeostasis. It is also reported to bind RNA: it associates with structural motifs present in the 3′ untranslated regions of at least two transcripts, but the functional significance of this binding remains unknown. Although HAX-1 has been detected in various cellular compartments, it is predominantly cytoplasmic. Our detailed localization studies of HAX-1 isoforms revealed partial nuclear localization, the extent of which depends on the protein isoform. Further studies demonstrated that HAX-1 is in fact a nucleocytoplasmic shuttling protein, dependent on the exportin 1 nuclear export receptor. Systematic mutagenesis allowed identification of the two nuclear export signals in the HAX-1 sequence. HAX-1 nuclear accumulation was observed after inhibition of nuclear export by leptomycin B, but also after specific cellular stress. The biological role of HAX-1 nuclear localization and shuttling remains to be established, but the HAX-1 transcript-binding properties suggest that it may be connected to mRNA processing and surveillance. In this study, HAX-1 status was shown to influence mRNA levels of DNA polymerase b, one of the HAX-1 mRNA targets, although this effect becomes pronounced only after specific stress is applied. Moreover, HAX-1 tethering to the reporter transcript caused a significant decrease in its expression. Additionally, the HAX-1 co-localization with P-body markers, reported here, implies a role in mRNA processing. These results suggest that HAX-1 may be involved in the regulation of expression of bound transcripts, possibly as part of the stress response. Structured digital abstract• HAX1 and DCP1A, colocalize by fluorescence microscopy (View Interaction: 1, 2) • HAX1 physically interacts with XPO1 by anti tagcoimmunoprecipitation (View interaction) Abbreviations CPEB, cytoplasmic polyadenylation element-binding protein; Crm1, chromosome region maintenance 1; Dcp1a, mRNA-decapping enzyme 1A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HuR, human antigen R, ELAV-like protein 1; LMB, leptomycin B; NES, nuclear export signal; POLB, DNA polymerase b; Pat1b, protein PAT1 homolog 1; rck/p54, ATP dependent RNA helicase DDX6; TG, thapsigargin; XPO1, exportin1.

diabetes; endothelial nitric oxide synthase ACCELERATED ATHEROSCLEROSIS, one of the major vascular complications of diabetes, is attributed to hyperglycemia mediatedendothelial dysfunction (6). Endothelial dysfunction is characterized by the loss of nitric oxide (NO) bioactivity in the vessel wall with concomitant increase in superoxide release, which impairs vasodilatation, inhibits protection against leukocyteendothelial interactions, platelet aggregation, and adhesion and smooth muscle cell proliferation (3,17,31). The optimal generation of NO from endothelial nitric oxide synthase (eNOS) activity is dependent on several factors, including availability of the substrate L-arginine (9) and the cofactor tetrahydrobiopterin (BH4) (1). The activation of eNOS is a complex process and may be regulated by 1) both transcriptional and posttranslational mechanisms, 2) site-specific phosphorylation of eNOS, 3) protein-protein interactions, 4) prosthetic groups, and 5) calcium and calmodulin (8).eNOS has been shown to interact with several regulatory proteins such as heat shock protein-90 (Hsp-90), caveolin-1, G protein-coupled receptors, NO-interacting protein (NOSIP), Dynamin-2, and Porin (8). In endothelial cells, both BH 4 and Hsp-90 have been shown to be important effectors in regulating eNOS activity. Hsp-90 is known to regulate calciumdependent dissociation of eNOS from caveolin-1, enzyme activation, maturation, and trafficking, followed by the Aktdependent activation and phosphorylation of serine 1177 (human eNOS) or serine 1179 (bovine eNOS) (8). Phosphorylation of eNOS is a key posttranslational modification that was believed to be a key determinant of eNOS activity associated with NO generation. The binding of Hsp-90 to eNOS ensures the transition from the early Ca 2ϩ -dependent to the late phosphorylation-dependent activation of eNOS (18). Failure of this binding has been demonstrated to cause eNOS uncoupling and increased eNOS-dependent superoxide anion production (20,28,30).A recent study in the diabetes literature has demonstrated that chronic exposure of endothelial cells to high glucose (HG) conditions downregulate both protein interaction between eNOS and Hsp-90 and the recruitment of activated Akt. The end result is the deactivation of eNOS and imbalance in NO versus reactive oxygen species (ROS) levels (18). Evidence of metformin-stimulated NOS in vivo (5) by promoting the association of Hsp-90 further supports the results of an earlier study (18) and confirms the concept that elevated glucose downregulates the interaction of eNOS and Hsp-90.Parallel studies investigating NO production in endothelial cells under conditions of HG suggested that HG-mediated endothelial dysfunction involves activation of IKK␤, which inhibits insulin receptor substrate (IRS-1)/phosphatidylinositol 3-kinase (PI3 kinase) signaling and therefore attenuates NO production (14). This has been implicated in insulin resistance, a common complication of diabetes.Requirement of Hsp-90 for the activation of IKK complex consisting of ...

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