Human nuclear RNAi-defective 2 (NRDE2) is an essential RNA splicing factor.

The accurate inheritance of genetic material is a basic necessity in all domains of life and an unexpectedly large number of RNA processing factors are required for mitotic progression and genome stability. NRDE2 (nuclear RNAi defective-2) is an evolutionarily conserved protein originally discovered for its role in nuclear RNA interference (RNAi) and heritable gene silencing in Caenorhabditis elegans (C. elegans). The function of the human NRDE2 gene remains poorly understood. Here we show that human NRDE2 is an essential protein required for suppressing intron retention in a subset of pre-mRNAs containing short, GC-rich introns with relatively weak 5' and 3' splice sites. NRDE2 preferentially interacts with components of the U5 small nuclear ribonucleoprotein (snRNP), the exon junction complex, and the RNA exosome. Interestingly, NRDE2-depleted cells exhibit greatly increased levels of genomic instability and DNA damage, as well as defects in centrosome maturation and mitotic progression. We identify the essential centriolar satellite protein, CEP131, as a direct NRDE2-regulated target. NRDE2 specifically binds to and promotes the efficient splicing of CEP131 pre-mRNA, and depleting NRDE2 dramatically reduces CEP131 protein expression, contributing to impaired recruitment of critical centrosomal proteins (e.g., γ-tubulin and Aurora Kinase A) to the spindle poles during mitosis. Our work establishes a conserved role for human NRDE2 in RNA splicing, characterizes the severe genomic instability phenotypes observed upon loss of NRDE2, and highlights the direct regulation of CEP131 splicing as one of multiple mechanisms through which such phenotypes might be explained.

Phleboviruses encapsidate their genomes by sequestering RNA bases.

Rift Valley fever and Toscana viruses are human pathogens for which no effective therapeutics exist. These and other phleboviruses have segmented negative-sense RNA genomes that are sequestered by a nucleocapsid protein (N) to form ribonucleoprotein (RNP) complexes of irregular, asymmetric structure, previously uncharacterized at high resolution. N binds nonspecifically to single-stranded RNA with nanomolar affinity. Crystal structures of Rift Valley fever virus N-RNA complexes reconstituted with defined RNAs of different length capture tetrameric, pentameric and hexameric N-RNA multimers. All N-N subunit contacts are mediated by a highly flexible α-helical arm. Arm movement gives rise to the three multimers in the crystal structures and also explains the asymmetric architecture of the RNP. Despite the flexible association of subunits, the crystal structures reveal an invariant, monomeric RNP building block, consisting of the core of one N subunit, the arm of a neighboring N, and four RNA nucleotides with the flanking phosphates. Up to three additional RNA nucleotides bind between subunits. The monomeric building block is matched in size to the repeating unit in viral RNP, as visualized by electron microscopy. N sequesters four RNA bases in a narrow hydrophobic binding slot and has polar contacts only with the sugar-phosphate backbone, which faces the solvent. All RNA bases, whether in the binding slot or in the subunit interface, face the protein in a manner that is incompatible with base pairing or with "reading" by the viral polymerase.

Structure of the Rift Valley fever virus nucleocapsid protein reveals another architecture for RNA encapsidation.

Rift Valley fever virus (RVFV) is a negative-sense RNA virus (genus Phlebovirus, family Bunyaviridae) that infects livestock and humans and is endemic to sub-Saharan Africa. Like all negative-sense viruses, the segmented RNA genome of RVFV is encapsidated by a nucleocapsid protein (N). The 1.93-A crystal structure of RVFV N and electron micrographs of ribonucleoprotein (RNP) reveal an encapsidated genome of substantially different organization than in other negative-sense RNA virus families. The RNP polymer, viewed in electron micrographs of both virus RNP and RNP reconstituted from purified N with a defined RNA, has an extended structure without helical symmetry. N-RNA species of approximately 100-kDa apparent molecular weight and heterogeneous composition were obtained by exhaustive ribonuclease treatment of virus RNP, by recombinant expression of N, and by reconstitution from purified N and an RNA oligomer. RNA-free N, obtained by denaturation and refolding, has a novel all-helical fold that is compact and well ordered at both the N and C termini. Unlike N of other negative-sense RNA viruses, RVFV N has no positively charged surface cleft for RNA binding and no protruding termini or loops to stabilize a defined N-RNA oligomer or RNP helix. A potential protein interaction site was identified in a conserved hydrophobic pocket. The nonhelical appearance of phlebovirus RNP, the heterogeneous approximately 100-kDa N-RNA multimer, and the N fold differ substantially from the RNP and N of other negative-sense RNA virus families and provide valuable insights into the structure of the encapsidated phlebovirus genome.

Organoids Model Transcriptional Hallmarks of Oncogenic KRAS Activation in Lung Epithelial Progenitor Cells.

Mutant KRAS is a common driver in epithelial cancers. Nevertheless, molecular changes occurring early after activation of oncogenic KRAS in epithelial cells remain poorly understood. We compared transcriptional changes at single-cell resolution after KRAS activation in four sample sets. In addition to patient samples and genetically engineered mouse models, we developed organoid systems from primary mouse and human induced pluripotent stem cell-derived lung epithelial cells to model early-stage lung adenocarcinoma. In all four settings, alveolar epithelial progenitor (AT2) cells expressing oncogenic KRAS had reduced expression of mature lineage identity genes. These findings demonstrate the utility of our in vitro organoid approaches for uncovering the early consequences of oncogenic KRAS expression. This resource provides an extensive collection of datasets and describes organoid tools to study the transcriptional and proteomic changes that distinguish normal epithelial progenitor cells from early-stage lung cancer, facilitating the search for targets for KRAS-driven tumors.

The Adrenal Clock Prevents Aberrant Light-Induced Alterations in Circadian Glucocorticoid Rhythms.

The glucocorticoid (GC) rhythm is entrained to light-dark (LD) cycles via a molecular clock in the suprachiasmatic nucleus (SCN) and is maintained by an adrenal clock synchronized by SCN-dependent signals. Targeted deletion of the core clock gene Bmal1 can disrupt adrenal clock function. The requirement of the adrenal clock to stabilize the circadian GC rhythm during exposure to aberrant LD cycles was determined using novel aldosterone synthase (AS)Cre/+::Bmal1Fl/Fl mice in which Bmal1 deletion occurred during postnatal adrenal transdifferentiation. To examine whether adrenal Bmal1 deletion results in loss of the adrenal clock, mice were crossed with mPER2::Luciferase (mPER2Luc/+) mice. Adrenals from ASCre/+::Bmal1+/+::PER2Luc/+ [control (CTRL)] mice show mPER2Luc rhythms ex vivo, whereas slices from ASCre/+::Bmal1Fl/Fl::PER2Luc/+ [knockout (KO)] mice show dampened rhythms. To monitor corticosterone rhythmicity, mice were implanted with subcutaneous microdialysis probes and sampled at 60-minute intervals for up to 3 days under 12:12-hour [τ (T) 24] LD or 3.5:3.5-hour (T7) LD cycles. Corticosterone rhythms were entrained to T24 LD in CTRL and KO mice. Under T7 LD, circadian corticosterone rhythms persisted in most CTRL mice but not KO mice. Hyperadrenocorticism also was observed in KO mice under T7 LD, reflected by increased corticosterone peak amplitude, total daily corticosterone, and responses to ACTH. Analysis of dysregulated adrenal genes in KO mice exposed to aberrant light identified candidates involved in cholesterol metabolism and trafficking, including steroidogenic acute regulatory protein, which could increase steroidogenesis. Our results show that the adrenal clock functions to buffer steroidogenic responses to aberrant light and stabilize circadian GC rhythmicity.

Efficient cellular release of Rift Valley fever virus requires genomic RNA.

The Rift Valley fever virus is responsible for periodic, explosive epizootics throughout sub-Saharan Africa. The development of therapeutics targeting this virus is difficult due to a limited understanding of the viral replicative cycle. Utilizing a virus-like particle system, we have established roles for each of the viral structural components in assembly, release, and virus infectivity. The envelope glycoprotein, Gn, was discovered to be necessary and sufficient for packaging of the genome, nucleocapsid protein and the RNA-dependent RNA polymerase into virus particles. Additionally, packaging of the genome was found to be necessary for the efficient release of particles, revealing a novel mechanism for the efficient generation of infectious virus. Our results identify possible conserved targets for development of anti-phlebovirus therapies.

A novel system for identification of inhibitors of rift valley Fever virus replication.

Rift Valley fever virus (RVFV) is a human and livestock pathogen endemic to sub-Saharan Africa. We have developed a T7-dependent system for the efficient production of RVFV-like particles (RVF-VLPs) based on the virulent ZH-501 strain of RVFV. The RVF-VLPs are capable of performing a single round of infection, allowing for the study of viral replication, assembly, and infectivity. We demonstrate that these RVF-VLPs are antigenically indistinguishable from authentic RVFV and respond similarly to a wide array of known and previously unknown chemical inhibitors. This system should be useful for screening for small molecule inhibitors of RVFV replication.

Characterization of the branching patterns of glycogen branching enzyme truncated on the N-terminus.

Truncation of 112 amino acids at the N-terminus (Nd(1-112)) changes the chain transfer pattern of the Escherichia coli glycogen branching enzyme (GBE) [Arch. Biochem. Biophys. 397 (2002) 279]. We investigated further the role of the N-terminus by engineering other truncated GBEs and analyzing the branching pattern by high-performance anion-exchange chromatography. The wild type GBE transfers mainly chains with a degree of polymerization (d.p.) of 8-14, the Nd(1-112) enzyme transfers a greater proportion of chains with higher d.p. 15-20, whereas the 63- and 83-amino acid deleted enzymes had an intermediate pattern of transferred chains (d.p. 10-20). These data showed that a progressive shortening of the N-terminus leads to a gradual increase in the length of the transferred chains, suggesting that the N-terminus provides a support for the glucan substrate during the processes of cleavage and transfer of the alpha-(1-4) glucan chains.