m6A-driven SF3B1 translation control steers splicing to direct genome integrity and leukemogenesis.

SF3B1 is the most mutated splicing factor (SF) in myelodysplastic syndromes (MDSs), which are clonal hematopoietic disorders with variable risk of leukemic transformation. Although tumorigenic SF3B1 mutations have been extensively characterized, the role of "non-mutated" wild-type SF3B1 in cancer remains largely unresolved. Here, we identify a conserved epitranscriptomic program that steers SF3B1 levels to counteract leukemogenesis. Our analysis of human and murine pre-leukemic MDS cells reveals dynamic regulation of SF3B1 protein abundance, which affects MDS-to-leukemia progression in vivo. Mechanistically, ALKBH5-driven 5' UTR m6A demethylation fine-tunes SF3B1 translation directing splicing of central DNA repair and epigenetic regulators during transformation. This impacts genome stability and leukemia progression in vivo, supporting an integrative analysis in humans that SF3B1 molecular signatures may predict mutational variability and poor prognosis. These findings highlight a post-transcriptional gene expression nexus that unveils unanticipated SF3B1-dependent cancer vulnerabilities.

NRF2 Regulates Viability, Proliferation, Resistance to Oxidative Stress, and Differentiation of Murine Myoblasts and Muscle Satellite Cells.

Increased oxidative stress can slow down the regeneration of skeletal muscle and affect the activity of muscle satellite cells (mSCs). Therefore, we evaluated the role of the NRF2 transcription factor (encoded by the Nfe2l2 gene), the main regulator of the antioxidant response, in muscle cell biology. We used (i) an immortalized murine myoblast cell line (C2C12) with stable overexpression of NRF2 and (ii) primary mSCs isolated from wild-type and Nfe2l2 (transcriptionally)-deficient mice (Nfe2l2tKO). NRF2 promoted myoblast proliferation and viability under oxidative stress conditions and decreased the production of reactive oxygen species. Furthermore, NRF2 overexpression inhibited C2C12 cell differentiation by down-regulating the expression of myogenic regulatory factors (MRFs) and muscle-specific microRNAs. We also showed that NRF2 is indispensable for the viability of mSCs since the lack of its transcriptional activity caused high mortality of cells cultured in vitro under normoxic conditions. Concomitantly, Nfe2l2tKO mSCs grown and differentiated under hypoxic conditions were viable and much more differentiated compared to cells isolated from wild-type mice. Taken together, NRF2 significantly influences the properties of myoblasts and muscle satellite cells. This effect might be modulated by the muscle microenvironment.

Pseudouridine-modified tRNA fragments repress aberrant protein synthesis and predict leukaemic progression in myelodysplastic syndrome.

Transfer RNA-derived fragments (tRFs) are emerging small noncoding RNAs that, although commonly altered in cancer, have poorly defined roles in tumorigenesis1. Here we show that pseudouridylation (Ψ) of a stem cell-enriched tRF subtype2, mini tRFs containing a 5' terminal oligoguanine (mTOG), selectively inhibits aberrant protein synthesis programmes, thereby promoting engraftment and differentiation of haematopoietic stem and progenitor cells (HSPCs) in patients with myelodysplastic syndrome (MDS). Building on evidence that mTOG-Ψ targets polyadenylate-binding protein cytoplasmic 1 (PABPC1), we employed isotope exchange proteomics to reveal critical interactions between mTOG and functional RNA-recognition motif (RRM) domains of PABPC1. Mechanistically, this hinders the recruitment of translational co-activator PABPC1-interacting protein 1 (PAIP1)3 and strongly represses the translation of transcripts sharing pyrimidine-enriched sequences (PES) at the 5' untranslated region (UTR), including 5' terminal oligopyrimidine tracts (TOP) that encode protein machinery components and are frequently altered in cancer4. Significantly, mTOG dysregulation leads to aberrantly increased translation of 5' PES messenger RNA (mRNA) in malignant MDS-HSPCs and is clinically associated with leukaemic transformation and reduced patient survival. These findings define a critical role for tRFs and Ψ in difficult-to-treat subsets of MDS characterized by high risk of progression to acute myeloid leukaemia (AML).

The small Cajal body-specific RNA 15 (SCARNA15) directs p53 and redox homeostasis via selective splicing in cancer cells.

Small Cajal body-specific RNAs (scaRNAs) guide post-transcriptional modification of spliceosomal RNA and, while commonly altered in cancer, have poorly defined roles in tumorigenesis. Here, we uncover that SCARNA15 directs alternative splicing (AS) and stress adaptation in cancer cells. Specifically, we find that SCARNA15 guides critical pseudouridylation (Ψ) of U2 spliceosomal RNA to fine-tune AS of distinct transcripts enriched for chromatin and transcriptional regulators in malignant cells. This critically impacts the expression and function of the key tumor suppressors ATRX and p53. Significantly, SCARNA15 loss impairs p53-mediated redox homeostasis and hampers cancer cell survival, motility and anchorage-independent growth. In sum, these findings highlight an unanticipated role for SCARNA15 and Ψ in directing cancer-associated splicing programs.

Oncogenic translation directs spliceosome dynamics revealing an integral role for SF3A3 in breast cancer.

Splicing is a central RNA-based process commonly altered in human cancers; however, how spliceosomal components are co-opted during tumorigenesis remains poorly defined. Here we unravel the core splice factor SF3A3 at the nexus of a translation-based program that rewires splicing during malignant transformation. Upon MYC hyperactivation, SF3A3 levels are modulated translationally through an RNA stem-loop in an eIF3D-dependent manner. This ensures accurate splicing of mRNAs enriched for mitochondrial regulators. Altered SF3A3 translation leads to metabolic reprogramming and stem-like properties that fuel MYC tumorigenic potential in vivo. Our analysis reveals that SF3A3 protein levels predict molecular and phenotypic features of aggressive human breast cancers. These findings unveil a post-transcriptional interplay between splicing and translation that governs critical facets of MYC-driven oncogenesis.

Heme Oxygenase-1 Influences Satellite Cells and Progression of Duchenne Muscular Dystrophy in Mice.

AIMS: Muscle damage in Duchenne muscular dystrophy (DMD) caused by the lack of dystrophin is strongly linked to inflammation. Heme oxygenase-1 (HO-1; Hmox1) is an anti-inflammatory and cytoprotective enzyme affecting myoblast differentiation by inhibiting myomiRs. The role of HO-1 has not been so far well addressed in DMD. RESULTS: In dystrophin-deficient mdx mice, expression of Hmox1 in limb skeletal muscles and diaphragm is higher than in wild-type animals, being consistently elevated from 8 up to 52 weeks, both in myofibers and inflammatory leukocytes. Accordingly, HO-1 expression is induced in muscles of DMD patients. Pharmacological inhibition of HO-1 activity or genetic ablation of Hmox1 aggravates muscle damage and inflammation in mdx mice. Double knockout animals (Hmox1-/-mdx) demonstrate impaired exercise capacity in comparison with mdx mice. Interestingly, in contrast to the effect observed in muscle fibers, in dystrophin-deficient muscle satellite cells (SCs) expression of Hmox1 is decreased, while MyoD, myogenin, and miR-206 are upregulated compared with wild-type counterparts. Mdx SCs demonstrate disturbed and enhanced differentiation, which is further intensified by Hmox1 deficiency. RNA sequencing revealed downregulation of Atf3, MafK, Foxo1, and Klf2 transcription factors, known to activate Hmox1 expression, as well as attenuation of nitric oxide-mediated cGMP-dependent signaling in mdx SCs. Accordingly, treatment with NO-donor induces Hmox1 expression and inhibits differentiation. Finally, differentiation of mdx SCs was normalized by CO, a product of HO-1 activity. Innovation and Conclusions: HO-1 is induced in DMD, and HO-1 inhibition aggravates DMD pathology. Therefore, HO-1 can be considered a therapeutic target to alleviate this disease. Antioxid. Redox Signal. 00, 000-000.

R/S Ratio Variability in Lead V1 Observed between Selected Four 1-Minute ECG Fragments of 24-Hour ECG as a Predictor of Incomplete Resynchronization during Full 24-Hour ECG: Pilot Study.

BACKGROUND: About 30% of patients do not have an effective cardiac resynchronization therapy (CRT). Routine assessment of CRT by devices interrogation (DI) is not entirely reliable. Additional information provide detailed QRS analysis in 24-hour ECG, however it is time-consuming. The aim of the study was the assessment of R/S ratio variability in lead V1 between selected fragments of 24-hour ECG as a predictor of incomplete biventricular pacing (BIVP) during full 24-hour ECG. METHODS: The 12-lead 24-hour ECGs of 43 patients with sinus rhythm were studied. During 24-hour ECG the 6-minute walk test (6MWT) was performed. The CRT was assessed by analyzing DI and full 24-hour ECG and four 1-minute fragments of these ECG recordings: during the minimum and the maximum heart rate and at the 1st and last minute of 6MWT. RESULTS: During DI the effective (>95%) BIVP was present in 36 patients (83.7%). Analysis of full 24-hour ECG confirmed appropriate BIVP in 31 patients (72%) and suspected incomplete BIVP (≤95%) in 12 patients (28%). In 9/12 patients the R/S ratio variability in lead V1 was visible between selected ECG fragments of 24-hour ECG. These results were not associated with the results of DI but were significantly associated with full 24-hour analysis of QRS. CONCLUSIONS: R/S variability in lead V1 between selected fragments of 24-hour ECG can be considered a predictor of potentially incomplete BIVP confirmed by further complete 24-hour ECG analysis in patients with appropriate pacing reported during DI.

Analysis of the QRS morphology in lead V1 during 24-hour Holter electrocardiogram monitoring to evaluate function of a cardiac resynchronisation therapy device in patients with sinus rhythm: a pilot study.

BACKGROUND: Cardiac resynchronisation therapy (CRT) is an important advance in the treatment of chronic heart failure. The aim of CRT is biventricular capture in all beats. However, inadequate delivery of biventricular pacing is still seen in about 30% of patients with an implanted CRT device. Device interrogation is a routine approach to assess CRT delivery. However, some reports indicate that analysis of 24-h electrocardiogram (ECG) may provide additional and important information regarding CRT function. AIM: Assessment of the adequacy of CRT delivery based on device interrogation and analysis of QRS morphology during 24-h ECG recording in patients with preserved sinus rhythm (SR). METHODS: We analysed 24-h Holter ECG recordings and data from device interrogation devices in 43 patients with preserved SR (age 56 ± 23 years, 9 women and 34 men). The obtained results were compared in an independent manner. Assessment of adequacy of CRT delivery by 24-h ECG was based on the occurrence of QRS variability, defined as a change in R wave amplitude in lead V1 by > 3 mm and/or change in QRS duration by > 40 ms and/or change in the R/S ratio. Adequate CRT delivery, i.e. complete resynchronisation, was defined as more than 95% of pacing without the defined QRS variability. RESULTS: Both methods allowed independent assessment of CRT delivery (p < 0.05 by the Fisher's exact test). In multivariate analysis, factors that were independently associated with incomplete resynchronisation included ventricular arrhythmias (each 100 ventricular beats per day increased the risk of incomplete resynchronisation 1.14-fold; confidence interval [CI] 1.036-1.25, p = 0.007), maximum heart rate (HR) (each increase by 10 bpm increased the risk 3.3-fold; CI 1.36-7.9, p = 0.008), QRS duration at the minimum HR (each increase by 10 ms increased the risk 1.74-fold; CI 1.075-2.8, p = 0.024), and the programmed atrioventricular delay (each increase by 10 ms increased the risk 2.15-fold, CI 1.18-3.9, p = 0.013). CONCLUSIONS: In patients with preserved SR, device interrogation and evaluation of 24-h ECG are complementary methods to evaluate adequate CRT delivery. Therefore, both methods should be taken into account when assessing CRT function.