Spliceosomal disruption of the non-canonical BAF complex in cancer.

SF3B1 is the most commonly mutated RNA splicing factor in cancer1-4, but the mechanisms by which SF3B1 mutations promote malignancy are poorly understood. Here we integrated pan-cancer splicing analyses with a positive-enrichment CRISPR screen to prioritize splicing alterations that promote tumorigenesis. We report that diverse SF3B1 mutations converge on repression of BRD9, which is a core component of the recently described non-canonical BAF chromatin-remodelling complex that also contains GLTSCR1 and GLTSCR1L5-7. Mutant SF3B1 recognizes an aberrant, deep intronic branchpoint within BRD9 and thereby induces the inclusion of a poison exon that is derived from an endogenous retroviral element and subsequent degradation of BRD9 mRNA. Depletion of BRD9 causes the loss of non-canonical BAF at CTCF-associated loci and promotes melanomagenesis. BRD9 is a potent tumour suppressor in uveal melanoma, such that correcting mis-splicing of BRD9 in SF3B1-mutant cells using antisense oligonucleotides or CRISPR-directed mutagenesis suppresses tumour growth. Our results implicate the disruption of non-canonical BAF in the diverse cancer types that carry SF3B1 mutations and suggest a mechanism-based therapeutic approach for treating these malignancies.

A Massively Parallel Reporter Assay of 3' UTR Sequences Identifies In Vivo Rules for mRNA Degradation.

The stability of mRNAs is regulated by signals within their sequences, but a systematic and predictive understanding of the underlying sequence rules remains elusive. Here we introduce UTR-seq, a combination of massively parallel reporter assays and regression models, to survey the dynamics of tens of thousands of 3' UTR sequences during early zebrafish embryogenesis. UTR-seq revealed two temporal degradation programs: a maternally encoded early-onset program and a late-onset program that accelerated degradation after zygotic genome activation. Three signals regulated early-onset rates: stabilizing poly-U and UUAG sequences and destabilizing GC-rich signals. Three signals explained late-onset degradation: miR-430 seeds, AU-rich sequences, and Pumilio recognition sites. Sequence-based regression models translated 3' UTRs into their unique decay patterns and predicted the in vivo effect of sequence signals on mRNA stability. Their application led to the successful design of artificial 3' UTRs that conferred specific mRNA dynamics. UTR-seq provides a general strategy to uncover the rules of RNA cis regulation.

Quantitating translational control: mRNA abundance-dependent and independent contributions and the mRNA sequences that specify them.

Translation rate per mRNA molecule correlates positively with mRNA abundance. As a result, protein levels do not scale linearly with mRNA levels, but instead scale with the abundance of mRNA raised to the power of an 'amplification exponent'. Here we show that to quantitate translational control, the translation rate must be decomposed into two components. One, TRmD, depends on the mRNA level and defines the amplification exponent. The other, TRmIND, is independent of mRNA amount and impacts the correlation coefficient between protein and mRNA levels. We show that in Saccharomyces cerevisiae TRmD represents ∼20% of the variance in translation and directs an amplification exponent of 1.20 with a 95% confidence interval [1.14, 1.26]. TRmIND constitutes the remaining ∼80% of the variance in translation and explains ∼5% of the variance in protein expression. We also find that TRmD and TRmIND are preferentially determined by different mRNA sequence features: TRmIND by the length of the open reading frame and TRmD both by a ∼60 nucleotide element that spans the initiating AUG and by codon and amino acid frequency. Our work provides more appropriate estimates of translational control and implies that TRmIND is under different evolutionary selective pressures than TRmD.

Ribosome profiling reveals resemblance between long non-coding RNAs and 5' leaders of coding RNAs.

Large-scale genomics and computational approaches have identified thousands of putative long non-coding RNAs (lncRNAs). It has been controversial, however, as to what fraction of these RNAs is truly non-coding. Here, we combine ribosome profiling with a machine-learning approach to validate lncRNAs during zebrafish development in a high throughput manner. We find that dozens of proposed lncRNAs are protein-coding contaminants and that many lncRNAs have ribosome profiles that resemble the 5' leaders of coding RNAs. Analysis of ribosome profiling data from embryonic stem cells reveals similar properties for mammalian lncRNAs. These results clarify the annotation of developmental lncRNAs and suggest a potential role for translation in lncRNA regulation. In addition, our computational pipeline and ribosome profiling data provide a powerful resource for the identification of translated open reading frames during zebrafish development.

Short H2A histone variants are expressed in cancer.

Short H2A (sH2A) histone variants are primarily expressed in the testes of placental mammals. Their incorporation into chromatin is associated with nucleosome destabilization and modulation of alternate splicing. Here, we show that sH2As innately possess features similar to recurrent oncohistone mutations associated with nucleosome instability. Through analyses of existing cancer genomics datasets, we find aberrant sH2A upregulation in a broad array of cancers, which manifest splicing patterns consistent with global nucleosome destabilization. We posit that short H2As are a class of "ready-made" oncohistones, whose inappropriate expression contributes to chromatin dysfunction in cancer.

Minor intron retention drives clonal hematopoietic disorders and diverse cancer predisposition.

Most eukaryotes harbor two distinct pre-mRNA splicing machineries: the major spliceosome, which removes >99% of introns, and the minor spliceosome, which removes rare, evolutionarily conserved introns. Although hypothesized to serve important regulatory functions, physiologic roles of the minor spliceosome are not well understood. For example, the minor spliceosome component ZRSR2 is subject to recurrent, leukemia-associated mutations, yet functional connections among minor introns, hematopoiesis and cancers are unclear. Here, we identify that impaired minor intron excision via ZRSR2 loss enhances hematopoietic stem cell self-renewal. CRISPR screens mimicking nonsense-mediated decay of minor intron-containing mRNA species converged on LZTR1, a regulator of RAS-related GTPases. LZTR1 minor intron retention was also discovered in the RASopathy Noonan syndrome, due to intronic mutations disrupting splicing and diverse solid tumors. These data uncover minor intron recognition as a regulator of hematopoiesis, noncoding mutations within minor introns as potential cancer drivers and links among ZRSR2 mutations, LZTR1 regulation and leukemias.

Transgenic mice expressing tunable levels of DUX4 develop characteristic facioscapulohumeral muscular dystrophy-like pathophysiology ranging in severity.

BACKGROUND: All types of facioscapulohumeral muscular dystrophy (FSHD) are caused by the aberrant activation of the somatically silent DUX4 gene, the expression of which initiates a cascade of cellular events ultimately leading to FSHD pathophysiology. Typically, progressive skeletal muscle weakness becomes noticeable in the second or third decade of life, yet there are many individuals who are genetically FSHD but develop symptoms much later in life or remain relatively asymptomatic throughout their lives. Conversely, FSHD may clinically present prior to 5-10 years of age, ultimately manifesting as a severe early-onset form of the disease. These phenotypic differences are thought to be due to the timing and levels of DUX4 misexpression. METHODS: FSHD is a dominant gain-of-function disease that is amenable to modeling by DUX4 overexpression. We have recently created a line of conditional DUX4 transgenic mice, FLExDUX4, that develop a myopathy upon induction of human DUX4-fl expression in skeletal muscle. Here, we use the FLExDUX4 mouse crossed with the skeletal muscle-specific and tamoxifen-inducible line ACTA1-MerCreMer to generate a highly versatile bi-transgenic mouse model with chronic, low-level DUX4-fl expression and cumulative mild FSHD-like pathology that can be reproducibly induced to develop more severe pathology via tamoxifen induction of DUX4-fl in skeletal muscles. RESULTS: We identified conditions to generate FSHD-like models exhibiting reproducibly mild, moderate, or severe DUX4-dependent pathophysiology and characterized progression of pathology. We assayed DUX4-fl mRNA and protein levels, fitness, strength, global gene expression, and histopathology, all of which are consistent with an FSHD-like myopathic phenotype. Importantly, we identified sex-specific and muscle-specific differences that should be considered when using these models for preclinical studies. CONCLUSIONS: The ACTA1-MCM;FLExDUX4 bi-transgenic mouse model has mild FSHD-like pathology and detectable muscle weakness. The onset and progression of more severe DUX4-dependent pathologies can be controlled via tamoxifen injection to increase the levels of mosaic DUX4-fl expression, providing consistent and readily screenable phenotypes for assessing therapies targeting DUX4-fl mRNA and/or protein and are useful to investigate certain conserved downstream FSHD-like pathophysiology. Overall, this model supports that DUX4 expression levels in skeletal muscle directly correlate with FSHD-like pathology by numerous metrics.

Site-specific N- and C-terminal labeling of a single polypeptide using sortases of different specificity.

The unique reactivity of two sortase enzymes, SrtA(staph) from Staphylococcus aureus and SrtA(strep) from Streptococcus pyogenes, is exploited for site-specific labeling of a single polypeptide with different labels at its N and C termini. SrtA(strep) is used to label the protein's C terminus at an LPXTG site with a fluorescently labeled dialanine nucleophile. Selective N-terminal labeling of proteins containing N-terminal glycine residues is achieved using SrtA(staph) and LPXT derivatives. The generality of N-terminal labeling with SrtA(staph) is demonstrated by near-quantitative labeling of multiple protein substrates with excellent site specificity.

Quantitative principles of cis-translational control by general mRNA sequence features in eukaryotes.

BACKGROUND: General translational cis-elements are present in the mRNAs of all genes and affect the recruitment, assembly, and progress of preinitiation complexes and the ribosome under many physiological states. These elements include mRNA folding, upstream open reading frames, specific nucleotides flanking the initiating AUG codon, protein coding sequence length, and codon usage. The quantitative contributions of these sequence features and how and why they coordinate to control translation rates are not well understood. RESULTS: Here, we show that these sequence features specify 42-81% of the variance in translation rates in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Arabidopsis thaliana, Mus musculus, and Homo sapiens. We establish that control by RNA secondary structure is chiefly mediated by highly folded 25-60 nucleotide segments within mRNA 5' regions, that changes in tri-nucleotide frequencies between highly and poorly translated 5' regions are correlated between all species, and that control by distinct biochemical processes is extensively correlated as is regulation by a single process acting in different parts of the same mRNA. CONCLUSIONS: Our work shows that general features control a much larger fraction of the variance in translation rates than previously realized. We provide a more detailed and accurate understanding of the aspects of RNA structure that directs translation in diverse eukaryotes. In addition, we note that the strongly correlated regulation between and within cis-control features will cause more even densities of translational complexes along each mRNA and therefore more efficient use of the translation machinery by the cell.

DUX4 Suppresses MHC Class I to Promote Cancer Immune Evasion and Resistance to Checkpoint Blockade.

Advances in cancer immunotherapies make it critical to identify genes that modulate antigen presentation and tumor-immune interactions. We report that DUX4, an early embryonic transcription factor that is normally silenced in somatic tissues, is re-expressed in diverse solid cancers. Both cis-acting inherited genetic variation and somatically acquired mutations in trans-acting repressors contribute to DUX4 re-expression in cancer. Although many DUX4 target genes encode self-antigens, DUX4-expressing cancers were paradoxically characterized by reduced markers of anti-tumor cytolytic activity and lower major histocompatibility complex (MHC) class I gene expression. We demonstrate that DUX4 expression blocks interferon-γ-mediated induction of MHC class I, implicating suppressed antigen presentation in DUX4-mediated immune evasion. Clinical data in metastatic melanoma confirmed that DUX4 expression was associated with significantly reduced progression-free and overall survival in response to anti-CTLA-4. Our results demonstrate that cancers can escape immune surveillance by reactivating a normal developmental pathway and identify a therapeutically relevant mechanism of cell-intrinsic immune evasion.

Conservation of uORF repressiveness and sequence features in mouse, human and zebrafish.

Upstream open reading frames (uORFs) are ubiquitous repressive genetic elements in vertebrate mRNAs. While much is known about the regulation of individual genes by their uORFs, the range of uORF-mediated translational repression in vertebrate genomes is largely unexplored. Moreover, it is unclear whether the repressive effects of uORFs are conserved across species. To address these questions, we analyse transcript sequences and ribosome profiling data from human, mouse and zebrafish. We find that uORFs are depleted near coding sequences (CDSes) and have initiation contexts that diminish their translation. Linear modelling reveals that sequence features at both uORFs and CDSes modulate the translation of CDSes. Moreover, the ratio of translation over 5' leaders and CDSes is conserved between human and mouse, and correlates with the number of uORFs. These observations suggest that the prevalence of vertebrate uORFs may be explained by their conserved role in repressing CDS translation.

Toddler: an embryonic signal that promotes cell movement via Apelin receptors.

It has been assumed that most, if not all, signals regulating early development have been identified. Contrary to this expectation, we identified 28 candidate signaling proteins expressed during zebrafish embryogenesis, including Toddler, a short, conserved, and secreted peptide. Both absence and overproduction of Toddler reduce the movement of mesendodermal cells during zebrafish gastrulation. Local and ubiquitous production of Toddler promote cell movement, suggesting that Toddler is neither an attractant nor a repellent but acts globally as a motogen. Toddler drives internalization of G protein-coupled APJ/Apelin receptors, and activation of APJ/Apelin signaling rescues toddler mutants. These results indicate that Toddler is an activator of APJ/Apelin receptor signaling, promotes gastrulation movements, and might be the first in a series of uncharacterized developmental signals.

A straight path to circular proteins.

Folding and stability are parameters that control protein behavior. The possibility of conferring additional stability on proteins has implications for their use in vivo and for their structural analysis in the laboratory. Cyclic polypeptides ranging in size from 14 to 78 amino acids occur naturally and often show enhanced resistance toward denaturation and proteolysis when compared with their linear counterparts. Native chemical ligation and intein-based methods allow production of circular derivatives of larger proteins, resulting in improved stability and refolding properties. Here we show that circular proteins can be made reversibly with excellent efficiency by means of a sortase-catalyzed cyclization reaction, requiring only minimal modification of the protein to be circularized.