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The observation that BRCA1 - and BRCA2-deficient cells are sensitive to inhibitors of poly(ADP–ribose) polymerase (PARP) has spurred the development of cancer therapies that use these inhibitors to target deficiencies in homologous recombination 1 . The cytotoxicity of PARP inhibitors depends on PARP trapping, the formation of non-covalent protein–DNA adducts composed of inhibited PARP1 bound to DNA lesions of unclear origins 1 – 4 . To address the nature of such lesions and the cellular consequences of PARP trapping, we undertook three CRISPR (clustered regularly interspersed palindromic repeats) screens to identify genes and pathways that mediate cellular resistance to olaparib, a clinically approved PARP inhibitor 1 . Here we present a high-confidence set of 73 genes, which when mutated cause increased sensitivity to PARP inhibitors. In addition to an expected enrichment for genes related to homologous recombination, we discovered that mutations in all three genes encoding ribonuclease H2 sensitized cells to PARP inhibition. We establish that the underlying cause of the PARP-inhibitor hypersensitivity of cells deficient in ribonuclease H2 is impaired ribonucleotide excision repair 5 . Embedded ribonucleotides, which are abundant in the genome of cells deficient in ribonucleotide excision repair, are substrates for cleavage by topoisomerase 1, resulting in PARP-trapping lesions that impede DNA replication and endanger genome integrity. We conclude that genomic ribonucleotides are a hitherto unappreciated source of PARP-trapping DNA lesions, and that the frequent deletion of RNASEH2B in metastatic prostate cancer and chronic lymphocytic leukaemia could provide an opportunity to exploit these findings therapeutically. Mutations in all three genes encoding ribonuclease H2 sensitize cells to poly(ADP–ribose) polymerase inhibitors by compromising ribonucleotide excision repair.

IRE1, an ER-localized transmembrane protein, plays a central role in the unfolded protein response (UPR). IRE1 senses the accumulation of unfolded proteins in its luminal domain and transmits a signal to the cytosolic side through its kinase and RNase domains. Although the downstream pathways mediated by two mammalian IRE1s, IRE1[alpha] and IRE1[beta], are well documented, their luminal events have not been fully elucidated. In particular, there have been no reports on how IRE1[beta] senses the unfolded proteins. In this study, we performed a comparative analysis to clarify the luminal event mediated by the mammalian IRE1s. Confocal fluorescent microscopy using GFP-fused IRE1s revealed that IRE1[beta] clustered into discrete foci upon ER stress. Also, fluorescence correlation spectroscopy (FCS) analysis in living cells indicated that the size of the IRE1[beta] complex is robustly increased upon ER stress. Moreover, unlike IRE1[alpha], the luminal domain of IRE1[beta] showed anti-aggregation activity in vitro, and IRE1[beta] was coprecipitated with the model unfolded proteins in cells. Strikingly, association with BiP was drastically reduced in IRE1[beta], while IRE1[alpha] was associated with BiP and dissociated upon ER stress. This is the first report indicating that, differently from IRE1[alpha], the luminal event mediated by IRE1[beta] involves direct interaction with unfolded proteins rather than association/dissociation with BiP, implying an intrinsic diversity in the sensing mechanism of mammalian sensors.

Self-incompatibility (SI) is widespread in the angiosperms, but identifying the biochemical components of SI mechanisms has proven to be difficult in most lineages. Coffea (coffee; Rubiaceae) is a genus of old-world tropical understory trees in which the vast majority of diploid species utilize a mechanism of gametophytic self-incompatibility (GSI). The S-RNase GSI system was one of the first SI mechanisms to be biochemically characterized, and likely represents the ancestral Eudicot condition as evidenced by its functional characterization in both asterid (Solanaceae, Plantaginaceae) and rosid (Rosaceae) lineages. The S-RNase GSI mechanism employs the activity of class III RNase T2 proteins to terminate the growth of \"self\" pollen tubes. Here, we investigate the mechanism of Coffea GSI and specifically examine the potential for homology to S-RNase GSI by sequencing class III RNase T2 genes in populations of 14 African and Madagascan Coffea species and the closely related self-compatible species Psilanthus ebracteolatus. Phylogenetic analyses of these sequences aligned to a diverse sample of plant RNase T2 genes show that the Coffea genome contains at least three class III RNase T2 genes. Patterns of tissue-specific gene expression identify one of these RNase T2 genes as the putative Coffea S-RNase gene. We show that populations of SI Coffea are remarkably polymorphic for putative S-RNase alleles, and exhibit a persistent pattern of trans-specific polymorphism characteristic of all S-RNase genes previously isolated from GSI Eudicot lineages. We thus conclude that Coffea GSI is most likely homologous to the classic Eudicot S-RNase system, which was retained since the divergence of the Rubiaceae lineage from an ancient SI Eudicot ancestor, nearly 90 million years ago.

RNA conformational diversity has fundamental biological roles 1 , 2 , 3 , 4 – 5 , but direct visualization of its full conformational space in solution has not been possible using traditional biophysical techniques. Using solution atomic force microscopy, a deep neural network and statistical analyses, we show that the ribonuclease P (RNase P) RNA adopts heterogeneous conformations consisting of a conformationally invariant core and highly flexible peripheral structural elements that sample a broad conformational space, with amplitudes as large as 20–60 Å in a multitude of directions, with very low net energy cost. Increasing Mg 2+ drives compaction and enhances enzymatic activity, probably by narrowing the conformational space. Moreover, analyses of the correlations and anticorrelations between spatial flexibility and sequence conservation suggest that the functional roles of both the structure and dynamics of key regions are embedded in the primary sequence. These findings reveal the structure–dynamics basis for the embodiment of both enzymatic precision and substrate promiscuity in the RNA component of the RNase P. Mapping the conformational space of the RNase P RNA demonstrates a new general approach to studying RNA structure and dynamics. Using a deep neural network and statistical analyses of atomic force microscopy images of individual RNA molecules enables the mapping of RNA conformational space in solution.

The vault nanoparticle is a eukaryotic assembly consisting of 78 copies of the 99-kDa major vault protein. They generate two cup-shaped symmetrical halves, which in vivo enclose protein and RNA molecules. Overall, this assembly is mainly involved in pro-survival and cytoprotective functions. It also holds a remarkable biotechnological potential for drug/gene delivery, thanks to its huge internal cavity and the absence of toxicity/immunogenicity. The available purification protocols are complex, partly because they use higher eukaryotes as expression systems. Here, we report a simplified procedure that combines human vault expression in the yeast Komagataella phaffii, as described in a recent report, and a purification process we have developed. This consists of RNase pretreatment followed by size-exclusion chromatography, which is far simpler than any other reported to date. Protein identity and purity was confirmed by SDS-PAGE, Western blot and transmission electron microscopy. We also found that the protein displayed a significant propensity to aggregate. We thus investigated this phenomenon and the related structural changes by Fourier-transform spectroscopy and dynamic light scattering, which led us to determine the most suitable storage conditions. In particular, the addition of either trehalose or Tween-20 ensured the best preservation of the protein in native, soluble form.

Dicer has a key role in small RNA biogenesis, processing double-stranded RNAs (dsRNAs) 1 , 2 . Human DICER (hDICER, also known as DICER1) is specialized for cleaving small hairpin structures such as precursor microRNAs (pre-miRNAs) and has limited activity towards long dsRNAs—unlike its homologues in lower eukaryotes and plants, which cleave long dsRNAs. Although the mechanism by which long dsRNAs are cleaved has been well documented, our understanding of pre-miRNA processing is incomplete because structures of hDICER in a catalytic state are lacking. Here we report the cryo-electron microscopy structure of hDICER bound to pre-miRNA in a dicing state and uncover the structural basis of pre-miRNA processing. hDICER undergoes large conformational changes to attain the active state. The helicase domain becomes flexible, which allows the binding of pre-miRNA to the catalytic valley. The double-stranded RNA-binding domain relocates and anchors pre-miRNA in a specific position through both sequence-independent and sequence-specific recognition of the newly identified ‘GYM motif’ 3 . The DICER-specific PAZ helix is also reoriented to accommodate the RNA. Furthermore, our structure identifies a configuration of the 5′ end of pre-miRNA inserted into a basic pocket. In this pocket, a group of arginine residues recognize the 5′ terminal base (disfavouring guanine) and terminal monophosphate; this explains the specificity of hDICER and how it determines the cleavage site. We identify cancer-associated mutations in the 5′ pocket residues that impair miRNA biogenesis. Our study reveals how hDICER recognizes pre-miRNAs with stringent specificity and enables a mechanistic understanding of hDICER-related diseases. The active-state structure of human DICER bound to pre-miRNA reveals the structural basis for the specificity of DICER in how it selects substrates in a sequence dependent manner, and sheds light on DICER-related diseases.

Despite critical roles in diseases, human pathways acting on strictly nuclear non-coding RNAs have been refractory to forward genetics. To enable their forward genetic discovery, we developed a single-cell approach that “Mirrors” activities of nuclear pathways with cytoplasmic fluorescence. Application of Mirror to two nuclear pathways targeting MALAT1’s 3′ end, the pathway of its maturation and the other, the degradation pathway blocked by the triple-helical Element for Nuclear Expression (ENE), identified nearly all components of three complexes: Ribonuclease P and the RNA Exosome, including nuclear DIS3 , EXOSC10 , and C1D , as well as the Nuclear Exosome Targeting (NEXT) complex. Additionally, Mirror identified DEAD-box helicase DDX59 associated with the genetic disorder Oral-Facial-Digital syndrome (OFD), yet lacking known substrates or roles in nuclear RNA degradation. Knockout of DDX59 exhibits stabilization of the full-length MALAT1 with a stability-compromised ENE and increases levels of 3′-extended forms of small nuclear RNAs. It also exhibits extensive retention of minor introns, including in OFD-associated genes, suggesting a mechanism for DDX59 association with OFD. Mirror efficiently identifies pathways acting on strictly nuclear non-coding RNAs, including essential and indirectly-acting components, and as a result can uncover unexpected links to human disease. Human pathways acting on nuclear ncRNAs have been refractory to forward genetics. Here, the authors develop a forward genetic approach that identifies such pathways and show DDX59 is required for minor intron splicing, suggesting a mechanism for its association with Oral-Facial-Digital syndrome.

Key Points Recent progress in high-throughput sequencing has uncovered an astounding landscape of small RNAs in eukaryotic cells. Various small RNAs of distinctive characteristics have been found and can be classified into three classes based on their biogenesis mechanism and the type of Argonaute (Ago) protein that they are associated with. MicroRNAs (miRNAs) are generated from local hairpin structures by the action of two RNase III-type proteins, Drosha and Dicer. Mature ∼22-nucleotide (nt) miRNAs are then bound by Ago-subfamily proteins. miRNAs target mRNAs and thereby function as post-transcriptional regulators. Piwi-interacting RNAs (piRNAs), which are 24–31 nt in length, are associated with Piwi-subfamily proteins. The biogenesis of piRNAs does not depend on Dicer. At least some piRNAs are involved in transposon silencing through heterochromatin formation or RNA destabilization. Endogenous small interfering RNAs (endo-siRNAs), such as miRNAs, associate with Ago-subfamily proteins. However, endo-siRNAs differ from miRNAs in that they are derived from long double-stranded RNAs and are dependent only on Dicer but not on Drosha. They are also slightly shorter (∼21 nt) than miRNAs. At least some of the endo-siRNAs have been shown to function as post-transcriptional regulators that target RNAs. There are numerous other small RNAs that are generated through non-canonical pathways. Many of them are difficult to classify and their biogenesis pathways remain poorly understood, but they may have species-specific functions that are not yet fully appreciated. Recent progress in high-throughput sequencing has uncovered an astounding landscape of small RNAs in eukaryotic cells. Various small RNAs can be classified into three classes based on their biogenesis mechanism and the type of Argonaute protein that they are associated with. Small RNAs of 20–30 nucleotides can target both chromatin and transcripts, and thereby keep both the genome and the transcriptome under extensive surveillance. Recent progress in high-throughput sequencing has uncovered an astounding landscape of small RNAs in eukaryotic cells. Various small RNAs of distinctive characteristics have been found and can be classified into three classes based on their biogenesis mechanism and the type of Argonaute protein that they are associated with: microRNAs (miRNAs), endogenous small interfering RNAs (endo-siRNAs or esiRNAs) and Piwi-interacting RNAs (piRNAs). This Review summarizes our current knowledge of how these intriguing molecules are generated in animal cells.

RNA–protein interaction networks govern many biological processes but are difficult to examine comprehensively. We devised ribonucleoprotein networks analyzed by mutational profiling (RNP-MaP), a live-cell chemical probing strategy that maps cooperative interactions among multiple proteins bound to single RNA molecules at nucleotide resolution. RNP-MaP uses a hetero-bifunctional crosslinker to freeze interacting proteins in place on RNA and then maps multiple bound proteins on single RNA strands by read-through reverse transcription and DNA sequencing. RNP-MaP revealed that RNase P and RMRP, two sequence-divergent but structurally related non-coding RNAs, share RNP networks and that network hubs define functional sites in these RNAs. RNP-MaP also identified protein interaction networks conserved between mouse and human XIST long non-coding RNAs and defined protein communities whose binding sites colocalize and form networks in functional regions of XIST. RNP-MaP enables discovery and efficient validation of functional protein interaction networks on long RNAs in living cells. Networks of proteins bound to single RNAs are identified by correlated chemical crosslinking.

The amylome is the universe of proteins that are capable of forming amyloid-like fibrils. Here we investigate the factors that enable a protein to belong to the amylome. A major factor is the presence in the protein of a segment that can form a tightly complementary interface with an identical segment, which permits the formation of a steric zipper--two self-complementary beta sheets that form the spine of an amyloid fibril. Another factor is sufficient conformational freedom of the self-complementary segment to interact with other molecules. Using RNase A as a model system, we validate our fibrillogenic predictions by the 3D profile method based on the crystal structure of NNQQNY and demonstrate that a specific residue order is required for fiber formation. Our genome-wide analysis revealed that self-complementary segments are found in almost all proteins, yet not all proteins form amyloids. The implication is that chaperoning effects have evolved to constrain self-complementary segments from interaction with each other.