Advances in RNA therapeutics for modulation of 'undruggable' targets.

Over the past decades, drug discovery utilizing small pharmacological compounds, fragment-based therapeutics, and antibody therapy have significantly advanced treatment options for many human diseases. However, a major bottleneck has been that>70% of human proteins/genomic regions are 'undruggable' by the above-mentioned approaches. Many of these proteins constitute essential drug targets against complex multifactorial diseases like cancer, immunological disorders, and neurological diseases. Therefore, alternative approaches are required to target these proteins or genomic regions in human cells. RNA therapeutics is a promising approach for many of the traditionally 'undruggable' targets by utilizing methods such as antisense oligonucleotides, RNA interference, CRISPR/Cas-based genome editing, aptamers, and the development of mRNA therapeutics. In the following chapter, we will put emphasis on recent advancements utilizing these approaches against challenging drug targets, such as intranuclear proteins, intrinsically disordered proteins, untranslated genomic regions, and targets expressed in inaccessible tissues.

ShinyArchR.UiO: user-friendly,integrative and open-source tool for visualization of single-cell ATAC-seq data using ArchR.

MOTIVATION: Mapping of chromatin accessibility landscapes in single-cells and the integration with gene expression enables a better understanding of gene regulatory mechanisms defining cell identities and cell-fate determination in development and disease. Generally, raw data generated from single-cell Assay for Transposase-Accessible Chromatin sequencing (scATAC-seq) are deposited in repositories that are generally inaccessible due to lack of in-depth knowledge of computational programming. RESULTS: We have developed ShinyArchR.UiO, an R-based shiny app, that facilitates scATAC-seq data accessibility and visualization in a user-friendly, interactive and open-source web interface. ShinyArchR.UiO is an application that can streamline collaborative efforts for interpretation of massive chromatin accessibility datasets and allow for open access data sharing for wider audiences. AVAILABILITY AND IMPLEMENTATION: https://Github.com/EskelandLab/ShinyArchRUiO and a demo server with a hematopoietic tutorial dataset https://cancell.medisin.uio.no/ShinyArchR.UiO. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Optic Atrophy 1 Controls Human Neuronal Development by Preventing Aberrant Nuclear DNA Methylation.

Optic atrophy 1 (OPA1), a GTPase at the inner mitochondrial membrane involved in regulating mitochondrial fusion, stability, and energy output, is known to be crucial for neural development: Opa1 heterozygous mice show abnormal brain development, and inactivating mutations in OPA1 are linked to human neurological disorders. Here, we used genetically modified human embryonic and patient-derived induced pluripotent stem cells and reveal that OPA1 haploinsufficiency leads to aberrant nuclear DNA methylation and significantly alters the transcriptional circuitry in neural progenitor cells (NPCs). For instance, expression of the forkhead box G1 transcription factor, which is needed for GABAergic neuronal development, is repressed in OPA1+/- NPCs. Supporting this finding, OPA1+/- NPCs cannot give rise to GABAergic interneurons, whereas formation of glutamatergic neurons is not affected. Taken together, our data reveal that OPA1 controls nuclear DNA methylation and expression of key transcription factors needed for proper neural cell specification.

The pioneer factor activity of c-Myb involves recruitment of p300 and induction of histone acetylation followed by acetylation-induced chromatin dissociation.

BACKGROUND: The concept of pioneer transcription factors is emerging as an essential part of the epigenetic regulation, taking place during cell development and differentiation. However, the precise molecular mechanism underlying pioneer factor activity remains poorly understood. We recently reported that the transcription factor c-Myb acts as a pioneer factor in haematopoiesis, and a point mutation in its DNA binding domain, D152V, is able to abrogate this function. RESULTS: Here, we show that specific histone modifications, including H3K27ac, prevent binding of c-Myb to histone tails, representing a novel effect of histone modifications, namely restricting binding of a pioneer factor to chromatin. Furthermore, we have taken advantage of the pioneer-defect D152V mutant to investigate mechanisms of c-Myb's pioneer factor activity. We show that c-Myb-dependent transcriptional activation of a gene in inaccessible chromatin relies on c-Myb binding to histones, as well as on c-Myb interacting with the histone acetyltransferase p300. ChIP assays show that both wild type and the D152V mutant of c-Myb bind to a selected target gene at its promoter and enhancer, but only wild-type c-Myb causes opening and activation of the locus. Enhancement of histone acetylation restores activation of the same gene in the absence of c-Myb, suggesting that facilitating histone acetylation is a crucial part of the pioneer factor function of c-Myb. CONCLUSIONS: We suggest a pioneer factor model in which c-Myb binds to regions of closed chromatin and then recruits histone acetyltransferases. By binding to histones, c-Myb facilitates histone acetylation, acting as a cofactor for p300 at c-Myb bound sites. The resulting H3K27ac leads to chromatin opening and detachment of c-Myb from the acetylated chromatin. We propose that the latter phenomenon, acetylation-induced chromatin dissociation, represents a mechanism for controlling the dynamics of pioneer factor binding to chromatin.

OPA1-anchored PKA phosphorylates perilipin 1 on S522 and S497 in adipocytes differentiated from human adipose stem cells.

Optic atrophy 1 (OPA1) is the A-kinase anchoring protein targeting the pool of protein kinase A (PKA) responsible for perilipin 1 phosphorylation, a gatekeeper for lipolysis. However, the involvement of OPA1-bound PKA in the downstream regulation of lipolysis is unknown. Here we show up-regulation and relocation of OPA1 from mitochondria to lipid droplets during adipocytic differentiation of human adipose stem cells. We employed various biochemical and immunological approaches to demonstrate that OPA1-bound PKA phosphorylates perilipin 1 at S522 and S497 on lipolytic stimulation. We show that the first 30 amino acids of OPA1 are essential for its lipid droplet localization as is OMA1-dependent processing. Finally, our results indicate that presence of OPA1 is necessary for lipolytic phosphorylation of downstream targets. Our results show for the first time, to our knowledge, how OPA1 mediates adrenergic control of lipolysis in human adipocytes by regulating phosphorylation of perilipin 1.

Cytokinesis arrest and multiple centrosomes in B cell chronic lymphocytic leukaemia.

Cytokinesis failure leads to the emergence of tetraploid cells and multiple centrosomes. Chronic lymphocytic leukaemia (CLL) is the most common haematological malignancy in adults and is characterized by clonal B cell expansion. Here, we show that a significant number of peripheral blood CLL cells are arrested in cytokinesis and that this event occurred after nuclear envelope reformation and before cytoplasmic abscission. mRNA expression data showed that several genes known to be crucial for cell cycle regulation, checkpoint and centromere function, such as ING4, ING5, CDKN1A and CDK4, were significantly dysregulated in CLL samples. Our results demonstrate that CLL cells exhibit difficulties in completing mitosis, which is different from but may, at least in part, explain the previously reported accumulation of CLL cells in G0/1.

Cell signalling analyses in the functional genomics era.

The advancements in proteomics over the past decade have brought tremendous increases in sensitivity of mass spectrometry (MS) analyses and new technologies such as methods for quantitative MS and phosphoproteomics. The development of antibodies targeting a large fraction of the human proteome as well as specific antibodies that detect phosphorylations and other post-translational modifications now allows detection of a great variety of signalling marks. Combined with medium and high throughput methods for detecting many parallel signalling events such as phospho-flow cytometry analyses and MS-based analyses to identify signalling complexes, the available tools now allows analysis of whole signalling networks facilitating systems level understanding of cellular signalling. The even more recent advances in chemical biology and chemical proteomics are further enhancing the development in this area by providing a cache of small molecule compounds allowing perturbations of signal pathways further advancing our global understanding of the signal transduction dynamics at the single cell level as well as in cellular system, tissue and whole organs. This review highlights the recent advances of quantitative MS, phosphoflow cytometry and chemical biology with focus on the dynamic spatiotemporal phosphorylation events, and examples of their application.

G-patch domain and KOW motifs-containing protein, GPKOW; a nuclear RNA-binding protein regulated by protein kinase A.

BACKGROUND: Post-transcriptional processing of pre-mRNA takes place in several steps and requires involvement of a number of RNA-binding proteins. How pre-mRNA processing is regulated is in large enigmatic. The catalytic (C) subunit of protein kinase A (PKA) is a serine/threonine kinase, which regulates numerous cellular processes including pre-mRNA splicing. Despite that a significant fraction of the C subunit is found in splicing factor compartments in the nucleus, there are no indications of a direct interaction between RNA and PKA. Based on this we speculate if the specificity of the C subunit in regulating pre-mRNA splicing may be mediated indirectly through other nuclear proteins. RESULTS: Using yeast two-hybrid screening with the PKA C subunit Cbeta2 as bait, we identified the G-patch domain and KOW motifs-containing protein (GPKOW), also known as the T54 protein or MOS2 homolog, as an interaction partner for Cbeta2. We demonstrate that GPKOW, which contains one G-patch domain and two KOW motifs, is a nuclear RNA-binding protein conserved between species. GPKOW contains two sites that are phosphorylated by PKA in vitro. By RNA immunoprecipitation and site directed mutagenesis of the PKA phosphorylation sites we revealed that GPKOW binds RNA in vivo in a PKA sensitive fashion. CONCLUSION: GPKOW is a RNA-binding protein that binds RNA in a PKA regulated fashion. Together with our previous results demonstrating that PKA regulates pre-mRNA splicing, our results suggest that PKA phosphorylation is involved in regulating RNA processing at several steps.

Mutually exclusive binding of PP1 and RNA to AKAP149 affects the mitochondrial network.

A-kinase-anchoring protein 149 (AKAP149) is a membrane protein of the mitochondrial and endoplasmic reticulum/nuclear envelope network. AKAP149 controls the subcellular localization and temporal order of protein phosphorylation by tethering protein kinases and phosphatases to these compartments. AKAP149 also includes an RNA-binding K homology (KH) domain, the loss of function of which has been associated in other proteins with neurodegenerative syndromes. We show here that protein phosphatase 1 (PP1) binding occurs through a conserved RVXF motif found in the KH domain of AKAP149 and that PP1 and RNA binding to this same site is mutually exclusive and controlled through a novel, phosphorylation-dependent mechanism. A collapse of the mitochondrial network is observed upon introduction of RNA-binding deficient mutants of AKAP149, pointing to the importance of RNA tethering to the mitochondrial membrane by AKAP149 for mitochondrial distribution.

The KH-Tudor domain of a-kinase anchoring protein 149 mediates RNA-dependent self-association.

A-Kinase anchoring proteins (AKAPs) control the subcellular localization and temporal specificity of protein phosphorylation mediated by cAMP-dependent protein kinase. AKAP149 (AKAP1) is found in mitochondria and in the endoplasmic reticulum-nuclear envelope network where it anchors protein kinases, phosphatases, and a phosphodiesterase. AKAP149 harbors in its COOH-terminal part one KH and one Tudor domain, both known to be involved in RNA binding. We investigated the properties of the COOH-terminal domain of AKAP149. We show here that AKAP149 is a self-associating protein with RNA binding features. The KH domain of AKAP149 is sufficient for self-association in a RNA-dependent manner. The Tudor domain is not necessary for self-association, but it is required together with the KH domain for targeting to well-defined nuclear foci. These foci are spatially closely related to nucleolar subcompartments. We also show that the KH-Tudor-containing domain of AKAP149 binds RNA in vitro and in RNA coprecipitation experiments. AKAP149 emerges as a scaffolding protein involved in the integration of intracellular signals and possibly in RNA metabolism.

Association of PP1 with its regulatory subunit AKAP149 is regulated by serine phosphorylation flanking the RVXF motif of AKAP149.

Reformation of the nuclear envelope at the end of mitosis involves the recruitment of the B-type lamin phosphatase PP1 to nuclear membranes by A-kinase anchoring protein AKAP149. PP1 remains associated to AKAP149 throughout G1 but dissociates from AKAP149 when AKAP149 is phosphorylated at the G1/S transition. We examine here the role of phosphorylation of serines flanking the RVXF PP1-binding motif of AKAP149, on PP1 anchoring. The use of AKAP149 peptides encompassing the RVXF motif and five flanking serines, either wild type (wt) or bearing S-->A or S-->D mutations, specifically shows that phosphorylation of S151 or S159 abolishes PP1 binding to immobilized AKAP149. Peptides with S151 or S159 as the only wt serine residue trigger dissociation of PP1 from immunoprecipitated AKAP149, whereas S151/159D mutants are ineffective. Furthermore, immunoprecipitated AKAP149 from purified G1-phase nuclear envelopes binds PKA and PKC in overlay assays. PKA binding to AKAP149 in vitro is unaffected by the presence of PKC or PP1, and similarly, PKC binding is independent of PKA or PP1. The immunoprecipitated AKAP149 complex contains PKA and PKC activities. Both AKAP149-associated PKA and PKC serine-phosphorylate immunoprecipitated AKAP149 in vitro; however, only PKC-mediated phosphorylation promotes dissociation of PP1 from the AKAP. The results suggest a putative temporally and spatially controlled mechanism promoting release of PP1 from AKAP149. AKAP149 emerges as a scaffolding protein for multiple protein kinases and phosphatases that may be involved in the integration of intracellular signals that converge at the nuclear envelope.