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.

Growing tumors avoid recognition and destruction by the immune system. During continuous stimulation of tumor-infiltrating lymphocytes (TILs) by tumors, TILs become functionally exhausted; thus, they become unable to kill tumor cells and to produce certain cytokines and lose their ability to proliferate. This collectively results in the immune escape of cancer cells. Here, we show that breast cancer cells expressing PD-L1 can accelerate exhaustion of persistently activated human effector CD4⁺ T cells, manifesting in high PD-1 and PD-L1 expression level son T cell surfaces, decreased glucose metabolism genes, strong downregulation of SWI/SNF chromatin remodeling complex subunits, and p21 cell cycle inhibitor upregulation. This results in inhibition of T cell proliferation and reduction of T cell numbers. The RNAseq analysis on exhausted CD4⁺ T cells indicated strong overexpression of IDO1 and genes encoding pro-inflammatory cytokines and chemokines. Some interleukins were also detected in media from CD4⁺ T cells co-cultured with cancer cells. The PD-L1 overexpression was also observed in CD4⁺ T cells after co-cultivation with other cell lines overexpressing PD-L1, which suggested the existence of a general mechanism of CD4⁺ T cell exhaustion induced by cancer cells. The ChIP analysis on the PD-L1 promoter region indicated that the BRM recruitment in control CD4⁺ T cells was replaced by BRG1 and EZH2 in CD4⁺ T cells strongly exhausted by cancer cells. These findings suggest that epi-drugs such as EZH2 inhibitors may be used as immunomodulators in cancer treatment.

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 β, 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 HAX1andDCP1A,colocalizebyfluorescence microscopy(View Interaction:1,2) HAX1 physically interacts with XPO1 by anti tagcoimmunoprecipitation (View interaction)

A decline in the bioavailability of nitric oxide (NO) that causes endothelial dysfunction is a hallmark of diabetes. The availability of NO to the vasculature is regulated by endothelial nitric oxide synthase (eNOS) activity and the involvement of heat shock protein-90 (Hsp-90) in the regulation of eNOS activity has been demonstrated. Hsp-90 has been shown to interact with upstream kinases [inhibitor κB kinases (IKK)α, β, and γ] in nonvascular cells. In this study, we have investigated the interaction of Hsp-90-IKKβ in endothelial cells under conditions of high glucose (HG) as a possible mechanism that diminishes Hsp-90-eNOS interaction, which could contribute to reduced bioavailability of NO. We report for the first time that IKKβ interacts with Hsp-90, and this interaction is augmented by HG in vascular endothelial cells. HG also augments transcriptional (3.5 ± 0.65-fold) and translational (1.97 ± 0.17-fold) expression as well as the catalytic activity of IKKβ (2.45 ± 0.4-fold). Both IKKβ and eNOS could be coimmunoprecipitated with Hsp-90. Inhibition of Hsp-90 with geldanamycin (2 μM) or Radicicol (20 μM) mitigated (0.45 ± 0.04-fold and 0.93 ± 0.16-fold, respectively) HG induced-IKKβ activity (2.5 ± 0.42-fold). Blocking of IKKβ expression by IKK inhibitor II (15 μM wedelolactone) or small interferring RNA (siRNA) improved Hsp-90-eNOS interaction and NO production under conditions of HG. These results illuminate a possible mechanism for the declining eNOS activity reported under conditions of HG.