(G)Patching up mis-splicing in cancer.

Benbarche, Pineda, Galvis, et al. delineate an essential role for the G-patch motif-containing protein GPATCH8 in mis-splicing associated with cancer-driving mutations of the splicing factor SF3B1. GPATCH8 cooperates with SF3B1 mutants, affecting the splicing machinery. Targeting GPATCH8 reveals therapeutic opportunities for SF3B1 mutant cancers and other splicing-related diseases.

Roles and regulation of tRNA-derived small RNAs in animals.

A growing class of small RNAs, known as tRNA-derived RNAs (tdRs), tRNA-derived small RNAs or tRNA-derived fragments, have long been considered mere intermediates of tRNA degradation. These small RNAs have recently been implicated in an evolutionarily conserved repertoire of biological processes. In this Review, we discuss the biogenesis and molecular functions of tdRs in mammals, including tdR-mediated gene regulation in cell metabolism, immune responses, transgenerational inheritance, development and cancer. We also discuss the accumulation of tRNA-derived stress-induced RNAs as a distinct adaptive cellular response to pathophysiological conditions. Furthermore, we highlight new conceptual advances linking RNA modifications with tdR activities and discuss challenges in studying tdR biology in health and disease.

Alternative promoters and splicing create multiple functionally distinct isoforms of oestrogen receptor alpha in breast cancer and healthy tissues.

BACKGROUND: Oestrogen receptor alpha (ER) is involved in cell growth and proliferation and functions as a transcription factor, a transcriptional coregulator, and in cytoplasmic signalling. It affects, for example, bone, endometrium, ovaries and mammary epithelium. It is a key biomarker in clinical management of breast cancer, where it is used as a prognostic and treatment-predictive factor, and a therapeutical target. Several ER isoforms have been described, but transcript annotation in public databases is incomplete and inconsistent, and functional differences are not well understood. METHODS: We have analysed short- and long-read RNA sequencing data from breast tumours, breast cancer cell lines, and normal tissues to create a comprehensive annotation of ER transcripts and combined it with experimental studies of full-length protein and six alternative isoforms. RESULTS: The isoforms have varying transcription factor activity, subcellular localisation, and response to the ER-targeting drugs tamoxifen and fulvestrant. Antibodies differ in ability to detect alternative isoforms, which raises concerns for the interpretation of ER-status in routine pathology. CONCLUSIONS: Future work should investigate the effects of alternative isoforms on patient survival and therapy response. An accurate annotation of ER isoforms will aid in interpretation of clinical data and inform functional studies to improve our understanding of the ER in health and disease.

Enhanced protein synthesis is a defining requirement for neonatal B cell development.

The LIN28B RNA binding protein exhibits an ontogenically restricted expression pattern and is a key molecular regulator of fetal and neonatal B lymphopoiesis. It enhances the positive selection of CD5+ immature B cells early in life through amplifying the CD19/PI3K/c-MYC pathway and is sufficient to reinitiate self-reactive B-1a cell output when ectopically expressed in the adult. In this study, interactome analysis in primary B cell precursors showed direct binding by LIN28B to numerous ribosomal protein transcripts, consistent with a regulatory role in cellular protein synthesis. Induction of LIN28B expression in the adult setting is sufficient to promote enhanced protein synthesis during the small Pre-B and immature B cell stages, but not during the Pro-B cell stage. This stage dependent effect was dictated by IL-7 mediated signaling, which masked the impact of LIN28B through an overpowering stimulation on the c-MYC/protein synthesis axis in Pro-B cells. Importantly, elevated protein synthesis was a distinguishing feature between neonatal and adult B cell development that was critically supported by endogenous Lin28b expression early in life. Finally, we used a ribosomal hypomorphic mouse model to demonstrate that subdued protein synthesis is specifically detrimental for neonatal B lymphopoiesis and the output of B-1a cells, without affecting B cell development in the adult. Taken together, we identify elevated protein synthesis as a defining requirement for early-life B cell development that critically depends on Lin28b. Our findings offer new mechanistic insights into the layered formation of the complex adult B cell repertoire.

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.

A fetal tumor suppressor axis abrogates MLL-fusion-driven acute myeloid leukemia.

MLL-rearrangements (MLL-r) are recurrent genetic events in acute myeloid leukemia (AML) and frequently associate with poor prognosis. In infants, MLL-r can be sufficient to drive transformation. However, despite the prenatal origin of MLL-r in these patients, congenital leukemia is very rare with transformation usually occurring postnatally. The influence of prenatal signals on leukemogenesis, such as those mediated by the fetal-specific protein LIN28B, remains controversial. Here, using a dual-transgenic mouse model that co-expresses MLL-ENL and LIN28B, we investigate the impact of LIN28B on AML. LIN28B impedes the progression of MLL-r AML through compromised leukemia-initiating cell activity and suppression of MYB signaling. Mechanistically, LIN28B directly binds to MYBBP1A mRNA, resulting in elevated protein levels of this MYB co-repressor. Functionally, overexpression of MYBBP1A phenocopies the tumor-suppressor effects of LIN28B, while its perturbation omits it. Thereby, we propose that developmentally restricted expression of LIN28B provides a layer of protection against MYB-dependent AML.

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).

Ribosomal biogenesis regulator DIMT1 controls ß-cell protein synthesis, mitochondrial function, and insulin secretion.

We previously reported that loss of mitochondrial transcription factor B1 (TFB1M) leads to mitochondrial dysfunction and is involved in the pathogenesis of type 2 diabetes (T2D). Whether defects in ribosomal processing impact mitochondrial function and could play a pathogenetic role in ß-cells and T2D is not known. To this end, we explored expression and the functional role of dimethyladenosine transferase 1 homolog (DIMT1), a homolog of TFB1M and a ribosomal RNA (rRNA) methyltransferase implicated in the control of rRNA. Expression of DIMT1 was increased in human islets from T2D donors and correlated positively with expression of insulin mRNA, but negatively with insulin secretion. We show that silencing of DIMT1 in insulin-secreting cells impacted mitochondrial function, leading to lower expression of mitochondrial OXPHOS proteins, reduced oxygen consumption rate, dissipated mitochondrial membrane potential, and a slower rate of ATP production. In addition, the rate of protein synthesis was retarded upon DIMT1 deficiency. Consequently, we found that DIMT1 deficiency led to perturbed insulin secretion in rodent cell lines and islets, as well as in a human ß-cell line. We observed defects in rRNA processing and reduced interactions between NIN1 (RPN12) binding protein 1 homolog (NOB-1) and pescadillo ribosomal biogenesis factor 1 (PES-1), critical ribosomal subunit RNA proteins, the dysfunction of which may play a part in disturbing protein synthesis in ß-cells. In conclusion, DIMT1 deficiency perturbs protein synthesis, resulting in mitochondrial dysfunction and disrupted insulin secretion, both potential pathogenetic processes in T2D.

Aberrant chromatin landscape following loss of the H3.3 chaperone Daxx in haematopoietic precursors leads to Pu.1-mediated neutrophilia and inflammation.

Defective silencing of retrotransposable elements has been linked to inflammageing, cancer and autoimmune diseases. However, the underlying mechanisms are only partially understood. Here we implicate the histone H3.3 chaperone Daxx, a retrotransposable element repressor inactivated in myeloid leukaemia and other neoplasms, in protection from inflammatory disease. Loss of Daxx alters the chromatin landscape, H3.3 distribution and histone marks of haematopoietic progenitors, leading to engagement of a Pu.1-dependent transcriptional programme for myelopoiesis at the expense of B-cell differentiation. This causes neutrophilia and inflammation, predisposing mice to develop an autoinflammatory skin disease. While these molecular and phenotypic perturbations are in part reverted in animals lacking both Pu.1 and Daxx, haematopoietic progenitors in these mice show unique chromatin and transcriptome alterations, suggesting an interaction between these two pathways. Overall, our findings implicate retrotransposable element silencing in haematopoiesis and suggest a cross-talk between the H3.3 loading machinery and the pioneer transcription factor Pu.1.

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.

Stressin' and slicin': stress-induced tRNA fragmentation codon-adapts translation to repress cell growth.

Transfer RNAs (tRNAs) are central adaptors that decode genetic information during translation and have been long considered static cellular components. However, whether dynamic changes in tRNAs and tRNA-derived fragments actively contribute to gene regulation remains debated. In this issue, Huh et al (2020) highlight tyrosine tRNAGUA fragmentation at the nexus of an evolutionarily conserved adaptive codon-based stress response that fine-tunes translation to restrain growth in human cells.

Novel insights into the emerging roles of tRNA-derived fragments in mammalian development.

ABTRACT tRNA-derived fragments or tRFs were long considered merely degradation intermediates of full-length tRNAs; however, emerging research is highlighting unanticipated new and highly distinct functions in epigenetic control, metabolism, immune activity and stem cell fate commitment. Importantly, recent studies suggest that RNA epitranscriptomic modifications may provide an additional regulatory layer that dynamically directs tRF activity in stem and cancer cells. In this review, we explore current work illustrating unanticipated roles of tRFs in mammalian stem cells with a focus on the impact of post-transcriptional RNA modifications for the biogenesis and function of this growing class of small noncoding RNAs.

Human sperm displays rapid responses to diet.

The global rise in obesity and steady decline in sperm quality are two alarming trends that have emerged during recent decades. In parallel, evidence from model organisms shows that paternal diet can affect offspring metabolic health in a process involving sperm tRNA-derived small RNA (tsRNA). Here, we report that human sperm are acutely sensitive to nutrient flux, both in terms of sperm motility and changes in sperm tsRNA. Over the course of a 2-week diet intervention, in which we first introduced a healthy diet followed by a diet rich in sugar, sperm motility increased and stabilized at high levels. Small RNA-seq on repeatedly sampled sperm from the same individuals revealed that tsRNAs were up-regulated by eating a high-sugar diet for just 1 week. Unsupervised clustering identified two independent pathways for the biogenesis of these tsRNAs: one involving a novel class of fragments with specific cleavage in the T-loop of mature nuclear tRNAs and the other exclusively involving mitochondrial tsRNAs. Mitochondrial involvement was further supported by a similar up-regulation of mitochondrial rRNA-derived small RNA (rsRNA). Notably, the changes in sugar-sensitive tsRNA were positively associated with simultaneous changes in sperm motility and negatively associated with obesity in an independent clinical cohort. This rapid response to a dietary intervention on tsRNA in human sperm is attuned with the paternal intergenerational metabolic responses found in model organisms. More importantly, our findings suggest shared diet-sensitive mechanisms between sperm motility and the biogenesis of tsRNA, which provide novel insights about the interplay between nutrition and male reproductive health.

Lin28b controls a neonatal to adult switch in B cell positive selection.

The ability of B-1 cells to become positively selected into the mature B cell pool, despite being weakly self-reactive, has puzzled the field since its initial discovery. Here, we explore changes in B cell positive selection as a function of developmental time by exploiting a link between CD5 surface levels and the natural occurrence of self-reactive B cell receptors (BCRs) in BCR wild-type mice. We show that the heterochronic RNA binding protein Lin28b potentiates a neonatal mode of B cell selection characterized by enhanced overall positive selection in general and the developmental progression of CD5+ immature B cells in particular. Lin28b achieves this by amplifying the CD19/PI3K/c-Myc positive feedback loop, and ectopic Lin28b expression restores both positive selection and mature B cell numbers in CD19-/- adult mice. Thus, the temporally restricted expression of Lin28b relaxes the rules for B cell selection during ontogeny by modulating tonic signaling. We propose that this neonatal mode of B cell selection represents a cell-intrinsic cue to accelerate the de novo establishment of the adaptive immune system and incorporate a layer of natural antibody-mediated immunity throughout life.

The X-Linked DDX3X RNA Helicase Dictates Translation Reprogramming and Metastasis in Melanoma.

The X-linked DDX3X gene encodes an ATP-dependent DEAD-box RNA helicase frequently altered in various human cancers, including melanomas. Despite its important roles in translation and splicing, how DDX3X dysfunction specifically rewires gene expression in melanoma remains completely unknown. Here, we uncover a DDX3X-driven post-transcriptional program that dictates melanoma phenotype and poor disease prognosis. Through an unbiased analysis of translating ribosomes, we identified the microphthalmia-associated transcription factor, MITF, as a key DDX3X translational target that directs a proliferative-to-metastatic phenotypic switch in melanoma cells. Mechanistically, DDX3X controls MITF mRNA translation via an internal ribosome entry site (IRES) embedded within the 5' UTR. Through this exquisite translation-based regulatory mechanism, DDX3X steers MITF protein levels dictating melanoma metastatic potential in vivo and response to targeted therapy. Together, these findings unravel a post-transcriptional layer of gene regulation that may provide a unique therapeutic vulnerability in aggressive male melanomas.

Pseudouridylation of tRNA-Derived Fragments Steers Translational Control in Stem Cells.

Pseudouridylation (Ψ) is the most abundant and widespread type of RNA epigenetic modification in living organisms; however, the biological role of Ψ remains poorly understood. Here, we show that a Ψ-driven posttranscriptional program steers translation control to impact stem cell commitment during early embryogenesis. Mechanistically, the Ψ "writer" PUS7 modifies and activates a novel network of tRNA-derived small fragments (tRFs) targeting the translation initiation complex. PUS7 inactivation in embryonic stem cells impairs tRF-mediated translation regulation, leading to increased protein biosynthesis and defective germ layer specification. Remarkably, dysregulation of this posttranscriptional regulatory circuitry impairs hematopoietic stem cell commitment and is common to aggressive subtypes of human myelodysplastic syndromes. Our findings unveil a critical function of Ψ in directing translation control in stem cells with important implications for development and disease.

H/ACA small RNA dysfunctions in disease reveal key roles for noncoding RNA modifications in hematopoietic stem cell differentiation.

Noncoding RNAs control critical cellular processes, although their contribution to disease remains largely unexplored. Dyskerin associates with hundreds of H/ACA small RNAs to generate a multitude of functionally distinct ribonucleoproteins (RNPs). The DKC1 gene, encoding dyskerin, is mutated in the multisystem disorder X-linked dyskeratosis congenita (X-DC). A central question is whether DKC1 mutations affect the stability of H/ACA RNPs, including those modifying ribosomal RNA (rRNA). We carried out comprehensive profiling of dyskerin-associated H/ACA RNPs, revealing remarkable heterogeneity in the expression and function of subsets of H/ACA small RNAs in X-DC patient cells. Using a mass spectrometry approach, we uncovered single-nucleotide perturbations in dyskerin-guided rRNA modifications, providing functional readouts of small RNA dysfunction in X-DC. In addition, we identified that, strikingly, the catalytic activity of dyskerin is required for accurate hematopoietic stem cell differentiation. Altogether, these findings reveal that small noncoding RNA dysfunctions may contribute to the pleiotropic manifestation of human disease.