Glioblastoma (GBM) is a deadly brain tumor with a very poor prognosis. Development of new therapeutics is hindered by the lack of appropriate preclinical models that reflect the complexity of the tumor microenvironment, especially the crucial role of microglia. In this study, we investigated the impact of microglia on GBM models using humanized 3D spheroids. Homotypic and heterotypic spheroids were created out of a GBM-derived cell line (DKMG) or patient-derived glioma stem cells (GB22-13), along with a microglia cell line (HMC3). Heterotypic glioma-HMC3 spheroids exhibited increased proliferation and greater drug resistance to chemotherapy drug Temozolomide compared with homotypic spheroids. Heterotypic spheroids also grew larger, developed multinucleated structures within 7 days, and had a greater invasive potential. Additionally, a distinct core-shell structure emerged in the heterotypic spheroids, with glioma cells concentrated in the core and a surrounding layer of microglia forming a protective shell that appeared to hinder drug penetration to the tumor core. Further, heterotypic cells were able to induce migration and polarization of peripheral blood monocytes (THP-1) towards M2 phenotypes, increasing immune evasion. These findings highlight the critical role of microglia in GBM development and progression, demonstrating their contribution to both reduced drug diffusion and increased tumor growth.

Huntington disease (HD) is a progressive neurodegenerative disorder, caused by a CAG trinucleotide expansion in the first exon of the HTT gene. The full-penetrance threshold for HD is established at 40 CAG repeats, which are translated into a polyglutamine (polyQ) tract within the huntingtin (HTT) protein. Beyond this threshold, somatic repeat instability in the brain and CAG repeat purity can further modulate disease onset and severity. HTT can be processed into multiple N-terminal fragments. Among these, the exon 1 HTT fragment containing an expanded polyQ tract is considered the most pathogenic. While some studies propose this fragment is generated by HTT proteolytic cleavage, others indicate it is primarily generated by the choice of a premature, cryptic polyadenylation site within HTT intron 1. Here, we apply a targeted RNA sequencing approach to systematically characterize HTT cryptic polyadenylation in several HD mouse models, human cell lines, and HD postmortem brain tissue. This method, known as 3-end targeted RNA sequencing or 3TRS, enables an accurate, quantitative, and cost-effective measurement of alternative polyadenylation. We describe an experimental and computational protocol that can also be adapted to additional transcripts and disease models. We show that activation of HTT cryptic polyadenylation is highly selective for the distal polyadenylation site located 7.3kb downstream intron 1 and requires very long, uninterrupted CAG repeat expansions. In humanized YAC128 mice, cryptic HTT expression was detected across all inspected brain regions, whereas in knock-in mice it correlated strongly with tissue-specific somatic repeat instability, being most prominent in the striatum. In human cell lines, pure CAG tracts triggered cryptic polyadenylation, while CAA interruptions within the repeats completely suppressed HTT1a expression. In HD patient samples, cryptic HTT transcripts were detected only in a juvenile-onset fibroblast line carrying an ultralong CAG expansion and in brain regions exhibiting high somatic instability. Together, these data support a model in which long and unstable CAG repeat expansions drive HD pathogenesis by activating HTT cryptic polyadenylation. By enabling quantitative comparison of cryptic and canonical HTT isoforms, 3TRS provides a robust framework to investigate HTT1a biogenesis and to support the development and evaluation of HTT-lowering therapeutic strategies. ### Competing Interest Statement The authors have declared no competing interest. Instituto de Salud Carlos III, https://ror.org/00ca2c886, PI19/00468, PI22/00598 Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED) Spanish Ministry of Science and Innovation, CNS2023-144441, RYC2018-024397-I Education Department of the Basque Government, RF/2019/001, NEURODEGENPROT, PRE\_2021\_1_0171, PRE\_2023\_1_0126

TDP43 proteinopathy is a neuropathological hallmark of nearly all amyotrophic lateral sclerosis (ALS) and approximately half of frontotemporal dementia (FTD) cases. Nuclear loss of TDP43 leads to widespread RNA misprocessing, such as the inclusion of cryptic exons that are no longer repressed by TDP43. Notably, in-frame cryptic exons encode novel cryptic peptides that can be detected in biofluids, including that found in the HDGFL2 transcript. Here, we quantified HDGFL2 cryptic peptide and neurofilament light chain (NfL) in paired cerebrospinal fluid (CSF) and plasma samples from ALS and FTD patients. Cryptic HDGFL2 peptide was detected in the CSF of ALS patients, whereas no significant differences were observed between genetic and behavioral FTD subgroups. In contrast, NfL levels were elevated in both ALS and FTD, although this biomarker does not reflect TDP43 pathology. Notably, NfL:HDGFL2 cryptic peptide ratio outperformed either marker alone in discriminating ALS and FTD cases from controls, achieving high specificity. Moreover, this ratio correlated with disease progression in ALS, suggesting added prognostic value. Collectively, our findings support the NfL:HDGFL2 cryptic peptide ratio as a promising fluid biomarker that integrates neurodegeneration with TDP43 dysfunction, potentially improving diagnostic accuracy, disease stratification, and longitudinal monitoring in TDP43 associated neurodegenerative disorders. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study has been funded by Instituto de Salud Carlos III (ISCIII) through the project PI22/00598 and EITB Maratoia project BIO22/ALZ/005 awarded to L.B., and co-funded by the European Union) and Centro de Investigacion Biomedica en Red de Enfermedades Neurodegenerativas (CIBERNED). This research was also supported by the Spanish Ministry of Science and Innovation and the Education Department of the Basque Government, through Ramon y Cajal (RYC2018-024397-I) and IKERBASQUE (RF/2019/001) fellowships respectively, awarded to L.B. I.S.G is funded by ARISTOS Fellow Program (European Union Horizon Europe research and innovation program under the Marie Skłodowska-Curie grant agreement No. 101081334). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by the Euskadi Medicine Research Ethics Committee (CEIm-E) PI+CES-BIOEF 2023-11. All participants gave informed consent for sample collection and participation in the study. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript

OBJECTIVE:SNUPN-related muscular dystrophy or LGMDR29 is a new entity that covers from a congenital or childhood onset pure muscular dystrophy to more complex phenotypes combining neurodevelopmental features, cataracts, or spinocerebellar ataxia. So far, 12 different variants have been described. Here we report the first family with SNUPN-related muscular dystrophy presenting an adult-onset myopathy as well as novel ultrastructural findings. METHODS:Clinical evaluation, muscle and brain magnetic resonance imaging (MRI), and muscle histopathological and electron microscopy analysis were conducted. Functional studies including protein modelling and interaction, immunofluorescence and splicing analysis were also performed. RESULTS:Two siblings carrying two novel deleterious variants in the SNUPN gene (p.Arg27Cys and p.Cys174Tyr) showed adult-onset proximo-distal and axial muscle weakness with early respiratory involvement. One patient presented with asymptomatic cerebellar atrophy. Muscle MRI identified involvement in the paravertebral, triceps brachii, sartorius and gracilis muscles. The histopathology revealed dystrophic changes and an abnormal pattern of cytoskeletal and myofibrillar proteins, while electron microscopy disclosed the proliferation of granules and vesicles associated with features of nuclear envelope and sarcolemma remodelling. Functional studies showed that SNUPN variants impair snurportin-1 function through reduced binding affinity to importin-β and impaired folding, leading to disturbed nuclear import of small nuclear ribonucleoproteins and downstream splicing. INTERPRETATION:Our work expands the phenotype of SNUPN-related muscular dystrophy and provides more insights into their pathological profile. We advise SNUPN testing in patients with late-onset proximo-distal and axial weakness with early respiratory impairment and features reminding inclusion body myositis (IBM). Granular deposits suggestive of biomolecular condensates perturbed cell organelle traffic and membrane homeostasis, opening new avenues to understand the pathomechanisms involved in this novel disease.

Alterations in RNA-splicing are a molecular hallmark of several neurological diseases, including muscular dystrophies, where mutations in genes involved in RNA metabolism or characterized by alterations in RNA splicing have been described. Here, we present five patients from two unrelated families with a limb-girdle muscular dystrophy (LGMD) phenotype carrying a biallelic variant in SNUPN gene. Snurportin-1, the protein encoded by SNUPN, plays an important role in the nuclear transport of small nuclear ribonucleoproteins (snRNPs), essential components of the spliceosome. We combine deep phenotyping, including clinical features, histopathology and muscle MRI, with functional studies in patient-derived cells and muscle biopsies to demonstrate that variants in SNUPN are the cause of a new type of LGMD according to current definition. Moreover, an in vivo model in Drosophila melanogaster further supports the relevance of Snurportin-1 in muscle. SNUPN patients show a similar phenotype characterized by proximal weakness starting in childhood, restrictive respiratory dysfunction and prominent contractures, although inter-individual variability in terms of severity even in individuals from the same family was found. Muscle biopsy showed myofibrillar-like features consisting of myotilin deposits and Z-disc disorganization. MRI showed predominant impairment of paravertebral, vasti, sartorius, gracilis, peroneal and medial gastrocnemius muscles. Conservation and structural analyses of Snurportin-1 p.Ile309Ser variant suggest an effect in nuclear-cytosol snRNP trafficking. In patient-derived fibroblasts and muscle, cytoplasmic accumulation of snRNP components is observed, while total expression of Snurportin-1 and snRNPs remains unchanged, which demonstrates a functional impact of SNUPN variant in snRNP metabolism. Furthermore, RNA-splicing analysis in patients' muscle showed widespread splicing deregulation, in particular in genes relevant for muscle development and splicing factors that participate in the early steps of spliceosome assembly. In conclusion, we report that SNUPN variants are a new cause of limb girdle muscular dystrophy with specific clinical, histopathological and imaging features, supporting SNUPN as a new gene to be included in genetic testing of myopathies. These results further support the relevance of splicing-related proteins in muscle disorders.

Alternative splicing allows multiple transcripts to be generated from the same gene to diversify the protein repertoire and gain new functions despite a limited coding genome. It can impact a wide spectrum of biological processes, including disease. However, its significance has long been underestimated due to limitations in dissecting the precise role of each splicing isoform in a physiological context. Furthermore, identifying key regulatory elements to correct deleterious splicing isoforms has proven equally challenging, increasing the difficulty of tackling the role of alternative splicing in cell biology. In this work, we take advantage of dCasRx, a catalytically inactive RNA targeting CRISPR-dCas13 ortholog, to efficiently switch alternative splicing patterns of endogenous transcripts without affecting overall gene expression levels cost-effectively. Additionally, we demonstrate a new application for the dCasRx splice-editing system to identify key regulatory RNA elements of specific splicing events. With this approach, we are expanding the RNA toolkit to better understand the regulatory mechanisms underlying alternative splicing and its physiological impact in various biological processes, including pathological conditions.

Liver kinase B1 (LKB1/STK11) is an important regulator of pancreatic β-cell identity and function. Elimination of Lkb1 from the β-cell results in improved glucose-stimulated insulin secretion and is accompanied by profound changes in gene expression, including the upregulation of several neuronal genes. The mechanisms through which LKB1 controls gene expression are, at present, poorly understood. Here, we explore the impact of β cell-selective deletion of Lkb1 on chromatin accessibility in mouse pancreatic islets. To characterize the role of LKB1 in the regulation of gene expression at the transcriptional level, we combine these data with a map of islet active transcription start sites and histone marks. We demonstrate that LKB1 elimination from β-cells results in widespread changes in chromatin accessibility, correlating with changes in transcript levels. Changes occurred in hundreds of promoter and enhancer regions, many of which were close to neuronal genes. We reveal that dysregulated enhancers are enriched in binding motifs for transcription factors (TFs) important for β-cell identity, such as FOXA, MAFA or RFX6, and we identify microRNAs (miRNAs) that are regulated by LKB1 at the transcriptional level. Overall, our study provides important new insights into the epigenetic mechanisms by which LKB1 regulates β-cell identity and function.

Amyotrophic Lateral Sclerosis (ALS) is a multisystemic neurodegenerative disorder, with accumulating evidence indicating metabolic disruptions in the skeletal muscle preceding disease symptoms, rather than them manifesting as a secondary consequence of motor neuron (MN) degeneration. Hence, energy homeostasis is deeply implicated in the complex physiopathology of ALS and skeletal muscle has emerged as a key therapeutic target. Here, we describe intrinsic abnormalities in ALS skeletal muscle, both in patient-derived muscle cells and in muscle cell lines with genetic knockdown of genes related to familial ALS, such as TARDBP (TDP-43) and FUS. We found a functional impairment of myogenesis that parallels defects of glucose oxidation in ALS muscle cells. We identified FOXO1 transcription factor as a key mediator of these metabolic and functional features in ALS muscle, via gene expression profiling and biochemical surveys in TDP-43 and FUS-silenced muscle progenitors. Strikingly, inhibition of FOXO1 mitigated the impaired myogenesis in both the genetically modified and the primary ALS myoblasts. In addition, specific in vivo conditional knockdown of TDP-43 or FUS orthologs (TBPH or caz) in Drosophila muscle precursor cells resulted in decreased innervation and profound dysfunction of motor nerve terminals and neuromuscular synapses, accompanied by motor abnormalities and reduced lifespan. Remarkably, these phenotypes were partially corrected by foxo inhibition, bolstering the potential pharmacological management of muscle intrinsic abnormalities associated with ALS. The findings demonstrate an intrinsic muscle dysfunction in ALS, which can be modulated by targeting FOXO factors, paving the way for novel therapeutic approaches that focus on the skeletal muscle as complementary target tissue.

Synthetic riboswitches are promising regulatory devices due to their small size, lack of immunogenicity, and ability to fine-tune gene expression in the absence of exogenous trans-acting factors. Based on a gene inhibitory system developed at our lab, termed U1snRNP interference (U1i), we developed tetracycline (TC)-inducible riboswitches that modulate mRNA polyadenylation through selective U1 snRNP recruitment. First, we engineered different TC-U1i riboswitches, which repress gene expression unless TC is added, leading to inductions of gene expression of 3-to-4-fold. Second, we developed a technique called Systematic Evolution of Riboswitches by Exponential Enrichment (SEREX), to isolate riboswitches with enhanced U1 snRNP binding capacity and activity, achieving inducibilities of up to 8-fold. Interestingly, by multiplexing riboswitches we increased inductions up to 37-fold. Finally, we demonstrated that U1i-based riboswitches are dose-dependent and reversible and can regulate the expression of reporter and endogenous genes in culture cells and mouse models, resulting in attractive systems for gene therapy applications. Our work probes SEREX as a much-needed technology for the in vitro identification of riboswitches capable of regulating gene expression in vivo.

The exon junction complex (EJC) plays key roles throughout the lifespan of RNA and is particularly relevant in the nervous system. We investigated the roles of two EJC members, the paralogs MAGOH and MAGOHB, with respect to brain tumour development. High MAGOH/MAGOHB expression was observed in 14 tumour types; glioblastoma (GBM) showed the greatest difference compared to normal tissue. Increased MAGOH/MAGOHB expression was associated with poor prognosis in glioma patients, while knockdown of MAGOH/MAGOHB affected different cancer phenotypes. Reduced MAGOH/MAGOHB expression in GBM cells caused alterations in the splicing profile, including re-splicing and skipping of multiple exons. The binding profiles of EJC proteins indicated that exons affected by MAGOH/MAGOHB knockdown accumulated fewer complexes on average, providing a possible explanation for their sensitivity to MAGOH/MAGOHB knockdown. Transcripts (genes) showing alterations in the splicing profile are mainly implicated in cell division, cell cycle, splicing, and translation. We propose that high MAGOH/MAGOHB levels are required to safeguard the splicing of genes in high demand in scenarios requiring increased cell proliferation (brain development and GBM growth), ensuring efficient cell division, cell cycle regulation, and gene expression (splicing and translation). Since differentiated neuronal cells do not require increased MAGOH/MAGOHB expression, targeting these paralogs is a potential option for treating GBM.

Cells must coordinate the activation of thousands of replication origins dispersed throughout their genome. Active transcription is known to favor the formation of mammalian origins, although the role that RNA plays in this process remains unclear. We show that the ORC1 subunit of the human Origin Recognition Complex interacts with RNAs transcribed from genes with origins in their transcription start sites (TSSs), displaying a positive correlation between RNA binding and origin activity. RNA depletion, or the use of ORC1 RNA-binding mutant, result in inefficient activation of proximal origins, linked to impaired ORC1 chromatin release. ORC1 RNA binding activity resides in its intrinsically disordered region, involved in intra- and inter-molecular interactions, regulation by phosphorylation, and phase-separation. We show that RNA binding favors ORC1 chromatin release, by regulating its phosphorylation and subsequent degradation. Our results unveil a non-coding function of RNA as a dynamic component of the chromatin, orchestrating the activation of replication origins.

Here, we show that direct recruitment of U1A to target transcripts can increase gene expression. This is a new regulatory role, in addition to previous knowledge showing that U1A decreases the levels of U1A mRNA and other specific targets. In fact, genome-wide, U1A more often increases rather than represses gene expression and many U1A-upregulated transcripts are directly bound by U1A according to individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) studies. Interestingly, U1A-mediated positive regulation can be transferred to a heterologous system for biotechnological purposes. Finally, U1A-bound genes are enriched for those involved in cell cycle and adhesion. In agreement with this, higher U1A mRNA expression associates with lower disease-free survival and overall survival in many cancer types, and U1A mRNA levels positively correlate with those of some oncogenes involved in cell proliferation. Accordingly, U1A depletion leads to decreased expression of these genes and the migration-related gene CCN2/CTGF, which shows the strongest regulation by U1A. A decrease in U1A causes a strong drop in CCN2 expression and CTGF secretion and defects in the expression of CTGF EMT targets, cell migration, and proliferation. These results support U1A as a putative therapeutic target for cancer treatment. In addition, U1A-binding sequences should be considered in biotechnological applications.

During autophagy, the coordinated actions of autophagosomes and lysosomes result in the controlled removal of damaged intracellular organelles and superfluous substrates. The evolutionary conservation of this process and its requirement for maintaining cellular homeostasis emphasizes the need to better dissect the pathways governing its molecular regulation. In our previously performed high-content screen, we assessed the effect of 1530 RNA-binding proteins on autophagy. Among the top regulators, we identified the eukaryotic translation initiation factor 4A-3 (eIF4A3). Here we show that depletion of eIF4A3 leads to a potent increase in autophagosome and lysosome biogenesis and an enhanced autophagic flux. This is mediated by the key autophagy transcription factor, TFEB, which becomes dephosphorylated and translocates from the cytoplasm to the nucleus where it elicits an integrated transcriptional response. We further identified an exon-skipping event in the transcript encoding for the direct TFEB kinase, GSK3B, which leads to a reduction in GSK3B expression and activity. Through analysis of TCGA data, we found a significant upregulation of eIF4A3 expression across several cancer types and confirmed the potential relevance of this newly identified signaling axis in human tumors. Hence, our data suggest a previously unrecognized role for eIF4A3 as a gatekeeper of autophagy through the control of TFEB activation, revealing a new mechanism for autophagy regulation.

Abstract Epiphrenic diverticula are acquired mucosal out-pouchings of the distal third of the oesophagus; vastly undiagnosed due to their usual asymptomatic status, with limited number in all published series, and a still ongoing debate both in their surgical indication and technique. This video shows all steps we think that are necessary when operating an epiphrenic diverticulum, in order to illustrate them all and to show the feasibility of performing such operation with minimally invasive procedures. Methods A 52-year-old male was diagnosed of a symptomatic giant epiphrenic diverticulum after previous complaints of dysphagia and regurgitation of undigested food. Preoperative barium-swallow and upper endoscopy confirmed the diagnosis, excluding any other secondary aetiology, and conventional oesophageal manometry did not show any relevant findings. Results Surgery was indicated for symptomatic disease, and a laparoscopic procedure was performed, with pneumoperitoneum created through a Veress needle and trocars placed similar to those used for a hiatal hernia repair. After opening the hiatus, access was granted to the mediastinum and the diverticulum was identified and dissected, and when its neck was completely exposed, diverticulectomy was performed using endostapler loads reinforced with Peri-strips Dry®. Aferwards, both an oesphageal myotomy (from the oesophago-gastric junction to the distal point of the diveticulum’s neck) and a partial Dor fundoplication (despite the absence of relevant findings in the conventional manometry) were performed. Conclusion Surgical management of epiphrenic diverticula should entail both resection of the pouch (diverticulectomy, to avoid postoperative persistent symptoms) and treatment of the underlying motility disorder that has been demonstrated in other studies in nearly 100% of the patients using high-resolution manometry (myotomy and fundoplication, both to avoid recurrences and leakages of the staple line). The laparoscopic approach is feasible without video-assisted thoracoscopy in diverticula with their neck up to 10 cm from the oesophagogastric junction. Video https://www.dropbox.com/s/hugbemgi6wl55kp/EpiphrenicDiverticulumx2.mp4?dl=0

Background: The evaluation of minimal residual disease (MRD) using flow cytometry (MFC) is an essential tool in decision making in acute myeloblastic leukemia (AML). Here we present a reproducible analysis strategy based on automatic separation of cell clusters analyzed by fully standardized methods (EuroFlow standards). Aims: 1) To develop a reproducible strategy to identify and quantify leukemic clusters using standardized procedures throughout the follow-up 2) To study its reproducibility among different investigators 3) To validate the strategy analyzing the correlation between MFC-MRD and PCR-MRD as well as between MFC-MRD and disease free survival (DFS). Methods: Patients and samples: 101 AML patients (May 2014 to December 2018) in first complete remission. Normal databases: Bone marrow normal samples of 25 patients. The reference images were created in CD117+ and CD117- compartments according to different routes of the early/intermediate myeloid maturation. Diagnostic phase: Leukemic clusters were first identified and then compared with the normal maturational counterpart (APS Infinicyt). The leukemia-associated immunophenotype (LAIP) specificity was successively calculated by quantifying the percentage of normal events that were indistinguishable from leukemic cells using the planned monitoring strategy. The most informative combinations were chosen for monitoring. Evaluation of the MFC-MRD: A mean of 4.1 million cells per tube were analyzed (IC90% 2.9–5.3). The MFC-MRD was determined on day +30 by three independent investigators considered positive when a suspect cluster was clearly differentiable from the normal counterpart but not necessarily identical to the original leukemic population. Statistical methodology: Kendall rank correlation coefficient (KCC) was used to evaluate concordance and survival curves were plotted using the Kaplan-Meier method and the log-rank test (SPSS Version 15.0 software). Results: 1) The strategy was applicable in 92% of the patients whereas in only 8% of patients none of the standardized combinations were clearly informative of LAIPs. 2) An average of 3.7 clusters (CI 90% 1.7–5.8) were identified in each patient. The specificity of LAIPs ranged between 7.8x10–2 and 1.0x10–5. The highest sensitivity reached was 10–5 (50 events over 5 million cells analyzed CV 15%) (Fig. 1 A). 3) During the follow-up the most frequently used combinations (41%) were tubes 1 + 4 (granulocytic lineage + TdT, CD7, CD56, CD19) although combinations of up to 5 tubes were sometimes necessary. 4) The results showed a high reproducibility within the investigators (KCC = 0.91 & Dev < 0.1). 5) Molecular follow up was evaluable in 46 PCR+ patients (7 RUNX1-RUNX1T1, 5 CBF-MYH11, 7 KMT2A MLL-T3, 23 NPM1, 4 CEBPA). We found a positive/negative concordance between MFC-MRD and PCR of 89%. 6) Patients in the MFC-MRD<0.01% group had a significantly longer DFS than the remaining patients after a follow-up of 30 months (79.3% DFS vrs 32.4% Fig. 1 B).Summary/Conclusion: 1) The reproduction of experiments performed at diagnosis and the automatic characterization of each cluster using specific databases is a promising way to achieve consensus in AML-MRD. 2) The sensitivity threshold of MFC/MDR is individual and depends on the number and specific characteristics of the leukemic clusters detected in each particular case. 3) The threshold of 0.1% is clearly insufficient and should be reconsidered in order to establish new risk stratifications in the disease.

Productive splicing of human precursor messenger RNAs (pre-mRNAs) requires the correct selection of authentic splice sites (SS) from the large pool of potential SS. Although SS consensus sequence and splicing regulatory proteins are known to influence SS usage, the mechanisms ensuring the effective suppression of cryptic SS are insufficiently explored. Here, we find that many aberrant exonic SS are efficiently silenced by the exon junction complex (EJC), a multi-protein complex that is deposited on spliced mRNA near the exon-exon junction. Upon depletion of EJC proteins, cryptic SS are de-repressed, leading to the mis-splicing of a broad set of mRNAs. Mechanistically, the EJC-mediated recruitment of the splicing regulator RNPS1 inhibits cryptic 5'SS usage, while the deposition of the EJC core directly masks reconstituted 3'SS, thereby precluding transcript disintegration. Thus, the EJC protects the transcriptome of mammalian cells from inadvertent loss of exonic sequences and safeguards the expression of intact, full-length mRNAs.