The mechanisms of gene expression regulation by miRNAs have been extensively studied. However, the regulation of miRNA function and decay has long remained enigmatic. Only recently, 3′ uridylation via LIN28A-TUT4/7 has been recognized as an essential component controlling the biogenesis of let-7 miRNAs in stem cells. Although uridylation has been generally implicated in miRNA degradation, the nuclease responsible has remained unknown. Here, we identify the Perlman syndrome-associated protein DIS3L2 as an oligo(U)-binding and processing exoribonuclease that specifically targets uridylated pre-let-7 in vivo. This study establishes DIS3L2 as the missing component of the LIN28-TUT4/7-DIS3L2 pathway required for the repression of let-7 in pluripotent cells.

MDM2 (HDM2) is a ubiquitin ligase that can target the p53 tumor suppressor protein for degradation. The RING domain is essential for the E3 activity of MDM2, and we show here that the extreme C‐terminal tail of MDM2 is also critical for efficient E3 activity. Loss of E3 function in MDM2 mutants deleted of the C‐terminal tail correlated with a failure of these mutants to oligomerize with MDM2, or with the related protein MDMX (HDMX). However, MDM2 containing point mutations within the C‐terminus that inactivated E3 function retained the ability to oligomerize with the wild‐type MDM2 RING domain and MDMX, and our results indicate that oligomers containing both wild‐type MDM2 and a C‐terminal mutant protein retain E3 function both in auto‐degradation and degradation of p53. Interestingly, the E3 activity of C‐terminal point mutants of MDM2 can also be supported by interaction with wild‐type MDMX, suggesting that MDMX can directly contribute to E3 function.

The plasma membrane calcium/calmodulin-dependent calcium ATPase (PMCA) (Shull, G.E., and J. Greeb. 1988. J. Biol. Chem. 263:8646–8657; Verma, A.K., A.G. Filoteo, D.R. Stanford, E.D. Wieben, J.T. Penniston, E.E. Strehler, R. Fischer, R. Heim, G. Vogel, S. Mathews, et al. 1988. J. Biol. Chem. 263:14152–14159; Carafoli, E. 1997. Basic Res. Cardiol. 92:59–61) has been proposed to be a regulator of calcium homeostasis and signal transduction networks of the cell. However, little is known about its precise mechanisms of action. Knock-out of (mainly neuronal) isoform 2 of the enzyme resulted in hearing loss and balance deficits due to severe inner ear defects, affecting formation and maintenance of otoconia (Kozel, P.J., R.A. Friedman, L.C. Erway, E.N. Yamoah, L.H. Liu, T. Riddle, J.J. Duffy, T. Doetschman, M.L. Miller, E.L. Cardell, and G.E. Shull. 1998. J. Biol. Chem. 273:18693–18696). Here we demonstrate that PMCA 4b is a negative regulator of nitric oxide synthase I (NOS-I, nNOS) in HEK293 embryonic kidney and neuro-2a neuroblastoma cell models. Binding of PMCA 4b to NOS-I was mediated by interaction of the COOH-terminal amino acids of PMCA 4b and the PDZ domain of NOS-I (PDZ: PSD 95/Dlg/ZO-1 protein domain). Increasing expression of wild-type PMCA 4b (but not PMCA mutants unable to bind PDZ domains or devoid of Ca2+-transporting activity) dramatically downregulated NO synthesis from wild-type NOS-I. A NOS-I mutant lacking the PDZ domain was not regulated by PMCA, demonstrating the specific nature of the PMCA–NOS-I interaction. Elucidation of PMCA as an interaction partner and major regulator of NOS-I provides evidence for a new dimension of integration between calcium and NO signaling pathways.

Poor therapeutic outcomes and serious side effects, together with acquired resistance to multiple drugs, are common problems of current cancer therapies. Therefore, there is an urgent need for new cancer-targeted drugs, which has led (inter alia) to the development of molecules that can specifically inhibit cyclin-dependent kinases (CDKs). In addition to their cell cycle regulatory functions, CDKs, especially CDK7 and CDK9, play important roles in the regulation of RNA polymerase II-mediated transcription. Here, we report on progress in the preclinical development of CDK inhibitors and their anticancer activities. Special attention is paid to the action mechanisms of the pan-specific CDK inhibitors flavopiridol and roscovitine, which have already entered phase II clinical trials as treatments for various tumours. The links between their ability to inhibit transcription and sensitisation of some types of cancer to apoptosis, mechanisms leading to p53 activation, and their synergistic cooperation with common DNA damaging drugs are also discussed. It has been demonstrated that drug-resistant cancer cells can arise during therapeutic application of small molecule protein kinase inhibitors. Clinical resistance to CDK inhibitors has not yet been described, but by comparing CDKs to other kinases, and CDK inhibitors to other clinically used protein kinase inhibitors, we also discuss possible mechanisms that could lead to resistance to CDK inhibitors.

Cardiovascular diseases remain the leading cause of death worldwide; hence there is an increasing focus on developing physiologically relevant in vitro cardiovascular tissue models suitable for studying personalized medicine and pre-clinical tests. Despite recent advances, models that reproduce both tissue complexity and maturation are still limited. We have established a scaffold-free protocol to generate multicellular, beating human cardiac microtissues in vitro from hiPSCs—namely human organotypic cardiac microtissues (hOCMTs)—that show some degree of self-organization and can be cultured for long term. This is achieved by the differentiation of hiPSC in 2D monolayer culture towards cardiovascular lineage, followed by further aggregation on low-attachment culture dishes in 3D. The generated hOCMTs contain multiple cell types that physiologically compose the heart and beat without external stimuli for more than 100 days. We have shown that 3D hOCMTs display improved cardiac specification, survival and metabolic maturation as compared to standard monolayer cardiac differentiation. We also confirmed the functionality of hOCMTs by their response to cardioactive drugs in long-term culture. Furthermore, we demonstrated that they could be used to study chemotherapy-induced cardiotoxicity. Due to showing a tendency for self-organization, cellular heterogeneity, and functionality in our 3D microtissues over extended culture time, we could also confirm these constructs as human cardiac organoids (hCOs). This study could help to develop more physiologically-relevant cardiac tissue models, and represent a powerful platform for future translational research in cardiovascular biology.

9-Substituted adenine derivatives with protected phosphoryl groups were synthesized and tested as inhibitors of adenylyl cyclase in isolated enzyme and intact cell systems. Protected 3′-phosphoryl derivatives of 2′,5′-dideoxyadenosine (2′,5′-dd-Ado) and β-l-2′,5′-dd-Ado, protected 5′-phosphoryl derivatives of β-l-2′,3′-dd-Ado, and protected phosphoryl derivatives of two 9-(2-phosphonomethoxy-acyl)-adenines were synthesized. Protection was afforded by two cyclosaligenyl- or three S-acyl-2-thioethyl-substituents. These pro-nucleotides were tested for their capacity to block forskolin-induced increases in [³H]cAMP in OB1771 and F442A preadipocytes and human macrophages prelabeled with [³H]adenine. A striking selectivity for 2′,5′-dd-Ado-3′-phosphoryl derivatives was observed. Cyclosaligenyl-derivatives (IC₅₀ ∼2 μm) were much less potent than S-acyl-2-thioethyl-derivatives. Best studied of these was 2′,5′-dd-Ado-3′-O-bis(S-pivaloyl-2-thioethyl)-phosphate, which blocked [³H]cAMP formation in preadipocytes (IC₅₀ ∼30 nm) and suppressed opening of cAMP-dependent Cl^– channels in cardiac myocytes (IC₅₀ ∼800 nm). None of the pro-nucleotides inhibited adenylyl cyclase per se, whether isolated from rat brain or OB1771 cells. These compounds exhibit the hallmarks of prodrugs. Data suggest they are taken up, are deprotected, and are converted to a potent inhibitory form to inhibit adenylyl cyclase, but only by intact cells. The availability and characteristics of these prodrugs should make them useful for blocking cAMP-mediated pathways in intact cell systems, in biochemical, pharmacological, and potentially therapeutic contexts.