Transcription Factors, R-Loops and Deubiquitinating Enzymes: Emerging Targets in Myelodysplastic Syndromes and Acute Myeloid Leukemia.

Myeloid neoplasms encompass a very heterogeneous family of diseases characterized by the failure of the molecular mechanisms that ensure a balanced equilibrium between hematopoietic stem cells (HSCs) self-renewal and the proper production of differentiated cells. The origin of the driver mutations leading to preleukemia can be traced back to HSC/progenitor cells. Many properties typical to normal HSCs are exploited by leukemic stem cells (LSCs) to their advantage, leading to the emergence of a clonal population that can eventually progress to leukemia with variable latency and evolution. In fact, different subclones might in turn develop from the original malignant clone through accumulation of additional mutations, increasing their competitive fitness. This process ultimately leads to a complex cancer architecture where a mosaic of cellular clones-each carrying a unique set of mutations-coexists. The repertoire of genes whose mutations contribute to the progression toward leukemogenesis is broad. It encompasses genes involved in different cellular processes, including transcriptional regulation, epigenetics (DNA and histones modifications), DNA damage signaling and repair, chromosome segregation and replication (cohesin complex), RNA splicing, and signal transduction. Among these many players, transcription factors, RNA splicing proteins, and deubiquitinating enzymes are emerging as potential targets for therapeutic intervention.

The phase separation-dependent FUS interactome reveals nuclear and cytoplasmic function of liquid-liquid phase separation.

Liquid-liquid phase separation (LLPS) of proteins and RNAs has emerged as the driving force underlying the formation of membrane-less organelles. Such biomolecular condensates have various biological functions and have been linked to disease. The protein Fused in Sarcoma (FUS) undergoes LLPS and mutations in FUS have been causally linked to the motor neuron disease Amyotrophic Lateral Sclerosis (ALS-FUS). LLPS followed by aggregation of cytoplasmic FUS has been proposed to be a crucial disease mechanism. However, it is currently unclear how LLPS impacts the behaviour of FUS in cells, e.g. its interactome. Hence, we developed a method allowing for the purification of LLPS FUS-containing droplets from cell lysates. We observe substantial alterations in the interactome, depending on its biophysical state. While non-LLPS FUS interacts mainly with factors involved in pre-mRNA processing, LLPS FUS predominantly binds to proteins involved in chromatin remodelling and DNA damage repair. Interestingly, also mitochondrial factors are strongly enriched with LLPS FUS, providing a potential explanation for the observed changes in mitochondrial gene expression in mouse models of ALS-FUS. In summary, we present a methodology to investigate the interactomes of phase separating proteins and provide evidence that LLPS shapes the FUS interactome with implications for function and disease.

FUS-dependent liquid-liquid phase separation is important for DNA repair initiation.

RNA-binding proteins (RBPs) are emerging as important effectors of the cellular DNA damage response (DDR). The RBP FUS is implicated in RNA metabolism and DNA repair, and it undergoes reversible liquid-liquid phase separation (LLPS) in vitro. Here, we demonstrate that FUS-dependent LLPS is necessary for the initiation of the DDR. Using laser microirradiation in FUS-knockout cells, we show that FUS is required for the recruitment to DNA damage sites of the DDR factors KU80, NBS1, and 53BP1 and of SFPQ, another RBP implicated in the DDR. The relocation of KU80, NBS1, and SFPQ is similarly impaired by LLPS inhibitors, or LLPS-deficient FUS variants. We also show that LLPS is necessary for efficient γH2AX foci formation. Finally, using superresolution structured illumination microscopy, we demonstrate that the absence of FUS impairs the proper arrangement of γH2AX nanofoci into higher-order clusters. These findings demonstrate the early requirement for FUS-dependent LLPS in the activation of the DDR and the proper assembly of DSB repair complexes.

miR-129-5p: A key factor and therapeutic target in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a relentless and fatal neurological disease characterized by the selective degeneration of motor neurons. No effective therapy is available for this disease. Several lines of evidence indicate that alteration of RNA metabolism, including microRNA (miRNA) processing, is a relevant pathogenetic factor and a possible therapeutic target for ALS. Here, we showed that the abundance of components in the miRNA processing machinery is altered in a SOD1-linked cellular model, suggesting consequent dysregulation of miRNA biogenesis. Indeed, high-throughput sequencing of the small RNA fraction showed that among the altered miRNAs, miR-129-5p was increased in different models of SOD1-linked ALS and in peripheral blood cells of sporadic ALS patients. We demonstrated that miR-129-5p upregulation causes the downregulation of one of its targets: the RNA-binding protein ELAVL4/HuD. ELAVL4/HuD is predominantly expressed in neurons, where it controls several key neuronal mRNAs. Overexpression of pre-miR-129-1 inhibited neurite outgrowth and differentiation via HuD silencing in vitro, while its inhibition with an antagomir rescued the phenotype. Remarkably, we showed that administration of an antisense oligonucleotide (ASO) inhibitor of miR-129-5p to an ALS animal model, SOD1 (G93A) mice, result in a significant increase in survival and improved the neuromuscular phenotype in treated mice. These results identify miR-129-5p as a therapeutic target that is amenable to ASO modulation for the treatment of ALS patients.

SOX6 blocks the proliferation of BCR-ABL1+ and JAK2V617F+ leukemic cells.

SOX6 is a HMG-box transcription factor expressed in a wide range of tissues. Recent data show that SOX6 expression is altered in different cancers, in the majority of cases being downregulated. To date, no data are available about SOX6 role in hematological malignancies. Here we demonstrate that SOX6 overexpressing BCR-ABL1+ B-ALL cells are unable to promote leukemia in a mouse model. Starting from this observation, we extended our study to a panel of human leukemic cells carrying genetic lesions distinctive of different types of leukemias and myeloproliferative disorders (the BCR-ABL1 translocation and the JAK2V617F amino acid substitution) to dissect the cellular events induced by SOX6. The inhibition of proliferation is the invariant outcome of SOX6 overexpression but it is achieved via two different cellular responses: terminal differentiation in erythroid-biased cells, irrespectively of their mutation, and apoptosis in megakaryocytic-primed and lymphoid cells. Within this context, cells carrying the highest copy number of the JAK2V617F allele better counteract the SOX6-imposed growth arrest. The interrogation of the GEPIA (Gene Expression Profiling Interactive Analysis) human dataset reveals that SOX6 is downregulated in a cohort of AML patients, uncovering a wide anti-proliferative role of SOX6 in a variety of mutant backgrounds.

MicroRNA expression analysis identifies a subset of downregulated miRNAs in ALS motor neuron progenitors.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder that is characterized by a progressive degeneration of motor neurons (MNs). The pathomechanism underlying the disease is largely unknown, even though increasing evidence suggests that RNA metabolism, including microRNAs (miRNAs) may play an important role. In this study, human ALS induced pluripotent stem cells were differentiated into MN progenitors and their miRNA expression profiles were compared to those of healthy control cells. We identified 15 downregulated miRNAs in patients' cells. Gene ontology and molecular pathway enrichment analysis indicated that the predicted target genes of the differentially expressed miRNAs were involved in neurodegeneration-related pathways. Among the 15 examined miRNAs, miR-34a and miR504 appeared particularly relevant due to their involvement in the p53 pathway, synaptic vesicle regulation and general involvement in neurodegenerative diseases. Taken together our results demonstrate that the neurodegenerative phenotype in ALS can be associated with a dysregulation of miRNAs involved in the control of disease-relevant genetic pathways, suggesting that targeting entire gene networks can be a potential strategy to treat complex diseases such as ALS.

Oxidative stress controls the choice of alternative last exons via a Brahma-BRCA1-CstF pathway.

Alternative splicing of terminal exons increases transcript and protein diversity. How physiological and pathological stimuli regulate the choice between alternative terminal exons is, however, largely unknown. Here, we show that Brahma (BRM), the ATPase subunit of the hSWI/SNF chromatin-remodeling complex interacts with BRCA1/BARD1, which ubiquitinates the 50 kDa subunit of the 3' end processing factor CstF. This results in the inhibition of transcript cleavage at the proximal poly(A) site and a shift towards inclusion of the distal terminal exon. Upon oxidative stress, BRM is depleted, cleavage inhibition is released, and inclusion of the proximal last exon is favoored. Our findings elucidate a novel regulatory mechanism, distinct from the modulation of transcription elongation by BRM that controls alternative splicing of internal exons.

Minor intron splicing is regulated by FUS and affected by ALS-associated FUS mutants.

Fused in sarcoma (FUS) is a ubiquitously expressed RNA-binding protein proposed to function in various RNA metabolic pathways, including transcription regulation, pre-mRNA splicing, RNA transport and microRNA processing. Mutations in the FUS gene were identified in patients with amyotrophic lateral sclerosis (ALS), but the pathomechanisms by which these mutations cause ALS are not known. Here, we show that FUS interacts with the minor spliceosome constituent U11 snRNP, binds preferentially to minor introns and directly regulates their removal. Furthermore, a FUS knockout in neuroblastoma cells strongly disturbs the splicing of minor intron-containing mRNAs, among them mRNAs required for action potential transmission and for functional spinal motor units. Moreover, an ALS-associated FUS mutant that forms cytoplasmic aggregates inhibits splicing of minor introns by trapping U11 and U12 snRNAs in these aggregates. Collectively, our findings suggest a possible pathomechanism for ALS in which mutated FUS inhibits correct splicing of minor introns in mRNAs encoding proteins required for motor neuron survival.

The intriguing case of motor neuron disease: ALS and SMA come closer.

MNDs (motor neuron diseases) form a heterogeneous group of pathologies characterized by the progressive degeneration of motor neurons. More and more genetic factors associated with MND encode proteins that have a function in RNA metabolism, suggesting that disturbed RNA metabolism could be a common underlying problem in several, perhaps all, forms of MND. In the present paper we review recent developments showing a functional link between SMN (survival of motor neuron), the causative factor of SMA (spinal muscular atrophy), and FUS (fused in sarcoma), a genetic factor in ALS (amyotrophic lateral sclerosis). SMN is long known to have a crucial role in the biogenesis and localization of the spliceosomal snRNPs (small nuclear ribonucleoproteins), which are essential assembly modules of the splicing machinery. Now we know that FUS interacts with SMN and pathogenic FUS mutations have a significant effect on snRNP localization. Together with other recently published evidence, this finding potentially links ALS pathogenesis to disturbances in the splicing machinery, and implies that pre-mRNA splicing may be the common weak point in MND, although other steps in mRNA metabolism could also play a role. Certainly, further comparison of the RNA metabolism in different MND will greatly help our understanding of the molecular causes of these devastating diseases.

RNA splicing: a new player in the DNA damage response.

It is widely accepted that tumorigenesis is a multistep process characterized by the sequential accumulation of genetic alterations. However, the molecular basis of genomic instability in cancer is still partially understood. The observation that hereditary cancers are often characterized by mutations in DNA repair and checkpoint genes suggests that accumulation of DNA damage is a major contributor to the oncogenic transformation. It is therefore of great interest to identify all the cellular pathways that contribute to the response to DNA damage. Recently, RNA processing has emerged as a novel pathway that may contribute to the maintenance of genome stability. In this review, we illustrate several different mechanisms through which pre-mRNA splicing and genomic stability can influence each other. We specifically focus on the role of splicing factors in the DNA damage response and describe how, in turn, activation of the DDR can influence the activity of splicing factors.

Proteomics strategies to identify SUMO targets and acceptor sites: a survey of RNA-binding proteins SUMOylation.

SUMOylation is a protein posttranslational modification that participates in the regulation of numerous biological processes within the cells. Small ubiquitin-like modifier (SUMO) proteins are members of the ubiquitin-like protein family and, similarly to ubiquitin, are covalently linked to a lysine residue on a target protein via a multi-enzymatic cascade. To assess the specific mechanism triggered by SUMOylation, the identification of SUMO protein substrates and of the precise acceptor site to which SUMO is bound is of critical relevance. Despite hundreds of mammalian proteins have been described as targets of SUMOylation, the identification of the precise acceptor sites still represents an important analytical challenge because of the relatively low stoichiometry in vivo and the highly dynamic nature of this modification. Moreover, mass spectrometry-based identification of SUMOylated sites is hampered by the large peptide remnant of SUMO proteins that are left on the modified lysine residue upon tryptic digestion. The present review provides a survey of the strategies that have been exploited in order to enrich, purify and identify SUMOylation substrates and acceptor sites in human cells on a large-scale format. The success of the presented strategies helped to unravel the numerous activities of this modification, as it was shown by the exemplary case of the RNA-binding protein family, whose SUMOylation is here reviewed.

Mutant SOD1 and mitochondrial damage alter expression and splicing of genes controlling neuritogenesis in models of neurodegeneration.

Mitochondrial dysfunction has been implicated in the pathogenesis of a number of neurodegenerative disorders including Parkinson, Alzheimer, and Amyotrophic Lateral Sclerosis (ALS). In addition, aberrant mRNA splicing has been documented in neurodegeneration. To characterize the cellular response to mitochondrial perturbations at the level of gene expression and alternative pre-mRNA splicing we used splicing-sensitive microarrays to profile human neuroblastoma SH-SY5Y cells treated with paraquat, a neurotoxic herbicide that induces the formation of reactive oxygen species and causes mitochondrial damage in animal models, and SH-SY5Y cells stably expressing the mutant G93A-SOD1 protein, one of the genetic causes of ALS. In both models we identified a common set of genes whose expression and alternative splicing are deregulated. Pathway analysis of the deregulated genes revealed enrichment in genes involved in neuritogenesis, axon growth and guidance, and synaptogenesis. Alterations in transcription and pre-mRNA splicing of candidate genes were confirmed experimentally in the cell line models as well as in brain and spinal cord of transgenic mice carrying the G93A-SOD1 mutation. Our findings expand the realm of the pathways implicated in neurodegeneration and suggest that alterations of axonal function may descend directly from mitochondrial damage.

A soluble form of the receptor for advanced glycation endproducts (RAGE) is produced by proteolytic cleavage of the membrane-bound form by the sheddase a disintegrin and metalloprotease 10 (ADAM10).

The receptor for advanced glycation endproducts (RAGE) mediates responses to cell danger and stress. When bound by its many ligands (which include advanced glycation endproducts, certain members of the S100/calgranulin family, extracellular high-mobility group box 1, the integrin Mac-1, amyloid beta-peptide and fibrils), RAGE activates programs responsible for acute and chronic inflammation. RAGE is therefore also involved in cancer progression, diabetes, atherosclerosis, and Alzheimer's disease. RAGE has several isoforms deriving from alternative splicing, including a soluble form called endogenous secretory RAGE (esRAGE). We show here that most soluble RAGE, either produced by cell lines or present in human blood, is not recognized by an anti-esRAGE antibody. Cells transfected with the cDNA for full-length RAGE, and thus not expressing esRAGE, produce a form of soluble RAGE, cleaved RAGE (cRAGE) that derives from proteolytic cleavage of the membrane-bound molecules and acts as a decoy receptor. By screening chemical inhibitors and genetically modified mouse embryonic fibroblasts (MEFs), we identify the sheddase ADAM10 as a membrane protease responsible for RAGE cleavage. Binding of its ligand HMGB1 promotes RAGE shedding. Our data do not disprove the interpretation that high levels of soluble forms of RAGE protect against chronic inflammation, but rather suggest that they correlate with high levels of ongoing inflammation.