Unfolding of a Temperature-Sensitive Domain Controls Voltage-Gated Channel Activation.

Voltage-gated ion channels (VGICs) are outfitted with diverse cytoplasmic domains that impact function. To examine how such elements may affect VGIC behavior, we addressed how the bacterial voltage-gated sodium channel (BacNa(V)) C-terminal cytoplasmic domain (CTD) affects function. Our studies show that the BacNa(V) CTD exerts a profound influence on gating through a temperature-dependent unfolding transition in a discrete cytoplasmic domain, the neck domain, proximal to the pore. Structural and functional studies establish that the BacNa(V) CTD comprises a bi-partite four-helix bundle that bears an unusual hydrophilic core whose integrity is central to the unfolding mechanism and that couples directly to the channel activation gate. Together, our findings define a general principle for how the widespread four-helix bundle cytoplasmic domain architecture can control VGIC responses, uncover a mechanism underlying the diverse BacNa(V) voltage dependencies, and demonstrate that a discrete domain can encode the temperature-dependent response of a channel.

A Distinct Type of Pilus from the Human Microbiome.

Pili are proteinaceous polymers of linked pilins that protrude from the cell surface of many bacteria and often mediate adherence and virulence. We investigated a set of 20 Bacteroidia pilins from the human microbiome whose structures and mechanism of assembly were unknown. Crystal structures and biochemical data revealed a diverse protein superfamily with a common Greek-key ß sandwich fold with two transthyretin-like repeats that polymerize into a pilus through a strand-exchange mechanism. The assembly mechanism of the central, structural pilins involves proteinase-assisted removal of their N-terminal ß strand, creating an extended hydrophobic groove that binds the C-terminal donor strands of the incoming pilin. Accessory pilins at the tip and base have unique structural features specific to their location, allowing initiation or termination of the assembly. The Bacteroidia pilus, therefore, has a biogenesis mechanism that is distinct from other known pili and likely represents a different type of bacterial pilus.

The RNase PARN-1 Trims piRNA 3' Ends to Promote Transcriptome Surveillance in C. elegans.

Piwi-interacting RNAs (piRNAs) engage Piwi proteins to suppress transposons and are essential for fertility in diverse organisms. An interesting feature of piRNAs is that, while piRNA lengths are stereotypical within a species, they can differ widely between species. For example, piRNAs are mainly 29 and 30 nucleotides in humans, 24 to 30 nucleotides in D. melanogaster, and uniformly 21 nucleotides in C. elegans. However, how piRNA length is determined and whether length impacts function remains unknown. Here, we show that C. elegans deficient for PARN-1, a conserved RNase, accumulate untrimmed piRNAs with 3' extensions. Surprisingly, these longer piRNAs are stable and associate with the Piwi protein PRG-1 but fail to robustly recruit downstream silencing factors. Our findings identify PARN-1 as a key regulator of piRNA length in C. elegans and suggest that length is regulated to promote efficient transcriptome surveillance.

Maturation Pathway from Germline to Broad HIV-1 Neutralizer of a CD4-Mimic Antibody.

Antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. Here, we define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level of somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. We integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages.

Post-translational Control of the Temporal Dynamics of Transcription Factor Activity Regulates Neurogenesis.

Neurogenesis is initiated by the transient expression of the highly conserved proneural proteins, bHLH transcriptional regulators. Here, we discover a conserved post-translational switch governing the duration of proneural protein activity that is required for proper neuronal development. Phosphorylation of a single Serine at the same position in Scute and Atonal proneural proteins governs the transition from active to inactive forms by regulating DNA binding. The equivalent Neurogenin2 Threonine also regulates DNA binding and proneural activity in the developing mammalian neocortex. Using genome editing in Drosophila, we show that Atonal outlives its mRNA but is inactivated by phosphorylation. Inhibiting the phosphorylation of the conserved proneural Serine causes quantitative changes in expression dynamics and target gene expression resulting in neuronal number and fate defects. Strikingly, even a subtle change from Serine to Threonine appears to shift the duration of Atonal activity in vivo, resulting in neuronal fate defects.

Oncogenic Role of Fusion-circRNAs Derived from Cancer-Associated Chromosomal Translocations.

Chromosomal translocations encode oncogenic fusion proteins that have been proven to be causally involved in tumorigenesis. Our understanding of whether such genomic alterations also affect non-coding RNAs is limited, and their impact on circular RNAs (circRNAs) has not been explored. Here, we show that well-established cancer-associated chromosomal translocations give rise to fusion circRNAs (f-circRNA) that are produced from transcribed exons of distinct genes affected by the translocations. F-circRNAs contribute to cellular transformation, promote cell viability and resistance upon therapy, and have tumor-promoting properties in in vivo models. Our work expands the current knowledge regarding molecular mechanisms involved in cancer onset and progression, with potential diagnostic and therapeutic implications.

De Novo Evolutionary Emergence of a Symmetrical Protein Is Shaped by Folding Constraints.

Molecular evolution has focused on the divergence of molecular functions, yet we know little about how structurally distinct protein folds emerge de novo. We characterized the evolutionary trajectories and selection forces underlying emergence of ß-propeller proteins, a globular and symmetric fold group with diverse functions. The identification of short propeller-like motifs (<50 amino acids) in natural genomes indicated that they expanded via tandem duplications to form extant propellers. We phylogenetically reconstructed 47-residue ancestral motifs that form five-bladed lectin propellers via oligomeric assembly. We demonstrate a functional trajectory of tandem duplications of these motifs leading to monomeric lectins. Foldability, i.e., higher efficiency of folding, was the main parameter leading to improved functionality along the entire evolutionary trajectory. However, folding constraints changed along the trajectory: initially, conflicts between monomer folding and oligomer assembly dominated, whereas subsequently, upon tandem duplication, tradeoffs between monomer stability and foldability took precedence.

Kv3.3 Channels Bind Hax-1 and Arp2/3 to Assemble a Stable Local Actin Network that Regulates Channel Gating.

Mutations in the Kv3.3 potassium channel (KCNC3) cause cerebellar neurodegeneration and impair auditory processing. The cytoplasmic C terminus of Kv3.3 contains a proline-rich domain conserved in proteins that activate actin nucleation through Arp2/3. We found that Kv3.3 recruits Arp2/3 to the plasma membrane, resulting in formation of a relatively stable cortical actin filament network resistant to cytochalasin D that inhibits fast barbed end actin assembly. These Kv3.3-associated actin structures are required to prevent very rapid N-type channel inactivation during short depolarizations of the plasma membrane. The effects of Kv3.3 on the actin cytoskeleton are mediated by the binding of the cytoplasmic C terminus of Kv3.3 to Hax-1, an anti-apoptotic protein that regulates actin nucleation through Arp2/3. A human Kv3.3 mutation within a conserved proline-rich domain produces channels that bind Hax-1 but are impaired in recruiting Arp2/3 to the plasma membrane, resulting in growth cones with deficient actin veils in stem cell-derived neurons.

New Brain Tumor Entities Emerge from Molecular Classification of CNS-PNETs.

Primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) are highly aggressive, poorly differentiated embryonal tumors occurring predominantly in young children but also affecting adolescents and adults. Herein, we demonstrate that a significant proportion of institutionally diagnosed CNS-PNETs display molecular profiles indistinguishable from those of various other well-defined CNS tumor entities, facilitating diagnosis and appropriate therapy for patients with these tumors. From the remaining fraction of CNS-PNETs, we identify four new CNS tumor entities, each associated with a recurrent genetic alteration and distinct histopathological and clinical features. These new molecular entities, designated "CNS neuroblastoma with FOXR2 activation (CNS NB-FOXR2)," "CNS Ewing sarcoma family tumor with CIC alteration (CNS EFT-CIC)," "CNS high-grade neuroepithelial tumor with MN1 alteration (CNS HGNET-MN1)," and "CNS high-grade neuroepithelial tumor with BCOR alteration (CNS HGNET-BCOR)," will enable meaningful clinical trials and the development of therapeutic strategies for patients affected by poorly differentiated CNS tumors.

Structure of Human DROSHA.

MicroRNA maturation is initiated by RNase III DROSHA that cleaves the stem loop of primary microRNA. DROSHA functions together with its cofactor DGCR8 in a heterotrimeric complex known as Microprocessor. Here, we report the X-ray structure of DROSHA in complex with the C-terminal helix of DGCR8. We find that DROSHA contains two DGCR8-binding sites, one on each RNase III domain (RIIID), which mediate the assembly of Microprocessor. The overall structure of DROSHA is surprisingly similar to that of Dicer despite no sequence homology apart from the C-terminal part, suggesting that DROSHA may have evolved from a Dicer homolog. DROSHA exhibits unique features, including non-canonical zinc-finger motifs, a long insertion in the first RIIID, and the kinked link between Connector helix and RIIID, which explains the 11-bp-measuring "ruler" activity of DROSHA. Our study implicates the evolutionary origin of DROSHA and elucidates the molecular basis of Microprocessor assembly and primary microRNA processing.

Ebola Viral Glycoprotein Bound to Its Endosomal Receptor Niemann-Pick C1.

Filoviruses, including Ebola and Marburg, cause fatal hemorrhagic fever in humans and primates. Understanding how these viruses enter host cells could help to develop effective therapeutics. An endosomal protein, Niemann-Pick C1 (NPC1), has been identified as a necessary entry receptor for this process, and priming of the viral glycoprotein (GP) to a fusion-competent state is a prerequisite for NPC1 binding. Here, we have determined the crystal structure of the primed GP (GPcl) of Ebola virus bound to domain C of NPC1 (NPC1-C) at a resolution of 2.3 Å. NPC1-C utilizes two protruding loops to engage a hydrophobic cavity on head of GPcl. Upon enzymatic cleavage and NPC1-C binding, conformational change in the GPcl further affects the state of the internal fusion loop, triggering membrane fusion. Our data therefore provide structural insights into filovirus entry in the late endosome and the molecular basis for design of therapeutic inhibitors of viral entry.

Insertion of the Biogenesis Factor Rei1 Probes the Ribosomal Tunnel during 60S Maturation.

Eukaryotic ribosome biogenesis depends on several hundred assembly factors to produce functional 40S and 60S ribosomal subunits. The final phase of 60S subunit biogenesis is cytoplasmic maturation, which includes the proofreading of functional centers of the 60S subunit and the release of several ribosome biogenesis factors. We report the cryo-electron microscopy (cryo-EM) structure of the yeast 60S subunit in complex with the biogenesis factors Rei1, Arx1, and Alb1 at 3.4 Å resolution. In addition to the network of interactions formed by Alb1, the structure reveals a mechanism for ensuring the integrity of the ribosomal polypeptide exit tunnel. Arx1 probes the entire set of inner-ring proteins surrounding the tunnel exit, and the C terminus of Rei1 is deeply inserted into the ribosomal tunnel, where it forms specific contacts along almost its entire length. We provide genetic and biochemical evidence that failure to insert the C terminus of Rei1 precludes subsequent steps of 60S maturation.

A Small RNA-Catalytic Argonaute Pathway Tunes Germline Transcript Levels to Ensure Embryonic Divisions.

Multiple division cycles without growth are a characteristic feature of early embryogenesis. The female germline loads proteins and RNAs into oocytes to support these divisions, which lack many quality control mechanisms operating in somatic cells undergoing growth. Here, we describe a small RNA-Argonaute pathway that ensures early embryonic divisions in C. elegans by employing catalytic slicing activity to broadly tune, instead of silence, germline gene expression. Misregulation of one target, a kinesin-13 microtubule depolymerase, underlies a major phenotype associated with pathway loss. Tuning of target transcript levels is guided by the density of homologous small RNAs, whose generation must ultimately be related to target sequence. Thus, the tuning action of a small RNA-catalytic Argonaute pathway generates oocytes capable of supporting embryogenesis. We speculate that the specialized nature of germline chromatin led to the emergence of small RNA-catalytic Argonaute pathways in the female germline as a post-transcriptional control layer to optimize oocyte composition.

Axon Degeneration Gated by Retrograde Activation of Somatic Pro-apoptotic Signaling.

During development, sensory axons compete for limiting neurotrophic support, and local neurotrophin insufficiency triggers caspase-dependent axon degeneration. The signaling driving axon degeneration upon local deprivation is proposed to reside within axons. Our results instead support a model in which, despite the apoptotic machinery being present in axons, the cell body is an active participant in gating axonal caspase activation and axon degeneration. Loss of trophic support in axons initiates retrograde activation of a somatic pro-apoptotic pathway, which, in turn, is required for distal axon degeneration via an anterograde pro-degenerative factor. At a molecular level, the cell body is the convergence point of two signaling pathways whose integrated action drives upregulation of pro-apoptotic Puma, which, unexpectedly, is confined to the cell body. Puma then overcomes inhibition by pro-survival Bcl-xL and Bcl-w and initiates the anterograde pro-degenerative program, highlighting the role of the cell body as an arbiter of large-scale axon removal.

Asprosin, a Fasting-Induced Glucogenic Protein Hormone.

Hepatic glucose release into the circulation is vital for brain function and survival during periods of fasting and is modulated by an array of hormones that precisely regulate plasma glucose levels. We have identified a fasting-induced protein hormone that modulates hepatic glucose release. It is the C-terminal cleavage product of profibrillin, and we name it Asprosin. Asprosin is secreted by white adipose, circulates at nanomolar levels, and is recruited to the liver, where it activates the G protein-cAMP-PKA pathway, resulting in rapid glucose release into the circulation. Humans and mice with insulin resistance show pathologically elevated plasma asprosin, and its loss of function via immunologic or genetic means has a profound glucose- and insulin-lowering effect secondary to reduced hepatic glucose release. Asprosin represents a glucogenic protein hormone, and therapeutically targeting it may be beneficial in type II diabetes and metabolic syndrome.

A Small Molecule RAS-Mimetic Disrupts RAS Association with Effector Proteins to Block Signaling.

Oncogenic activation of RAS genes via point mutations occurs in 20%-30% of human cancers. The development of effective RAS inhibitors has been challenging, necessitating new approaches to inhibit this oncogenic protein. Functional studies have shown that the switch region of RAS interacts with a large number of effector proteins containing a common RAS-binding domain (RBD). Because RBD-mediated interactions are essential for RAS signaling, blocking RBD association with small molecules constitutes an attractive therapeutic approach. Here, we present evidence that rigosertib, a styryl-benzyl sulfone, acts as a RAS-mimetic and interacts with the RBDs of RAF kinases, resulting in their inability to bind to RAS, disruption of RAF activation, and inhibition of the RAS-RAF-MEK pathway. We also find that ribosertib binds to the RBDs of Ral-GDS and PI3Ks. These results suggest that targeting of RBDs across multiple signaling pathways by rigosertib may represent an effective strategy for inactivation of RAS signaling.

Insights into Sex Chromosome Evolution and Aging from the Genome of a Short-Lived Fish.

The killifish Nothobranchius furzeri is the shortest-lived vertebrate that can be bred in the laboratory. Its rapid growth, early sexual maturation, fast aging, and arrested embryonic development (diapause) make it an attractive model organism in biomedical research. Here, we report a draft sequence of its genome that allowed us to uncover an intra-species Y chromosome polymorphism representing-in real time-different stages of sex chromosome formation that display features of early mammalian XY evolution "in action." Our data suggest that gdf6Y, encoding a TGF-ß family growth factor, is the master sex-determining gene in N. furzeri. Moreover, we observed genomic clustering of aging-related genes, identified genes under positive selection, and revealed significant similarities of gene expression profiles between diapause and aging, particularly for genes controlling cell cycle and translation. The annotated genome sequence is provided as an online resource (http://www.nothobranchius.info/NFINgb).

The African Turquoise Killifish Genome Provides Insights into Evolution and Genetic Architecture of Lifespan.

Lifespan is a remarkably diverse trait ranging from a few days to several hundred years in nature, but the mechanisms underlying the evolution of lifespan differences remain elusive. Here we de novo assemble a reference genome for the naturally short-lived African turquoise killifish, providing a unique resource for comparative and experimental genomics. The identification of genes under positive selection in this fish reveals potential candidates to explain its compressed lifespan. Several aging genes are under positive selection in this short-lived fish and long-lived species, raising the intriguing possibility that the same gene could underlie evolution of both compressed and extended lifespans. Comparative genomics and linkage analysis identify candidate genes associated with lifespan differences between various turquoise killifish strains. Remarkably, these genes are clustered on the sex chromosome, suggesting that short lifespan might have co-evolved with sex determination. Our study provides insights into the evolutionary forces that shape lifespan in nature.

Learning the sequence determinants of alternative splicing from millions of random sequences.

Most human transcripts are alternatively spliced, and many disease-causing mutations affect RNA splicing. Toward better modeling the sequence determinants of alternative splicing, we measured the splicing patterns of over two million (M) synthetic mini-genes, which include degenerate subsequences totaling over 100 M bases of variation. The massive size of these training data allowed us to improve upon current models of splicing, as well as to gain new mechanistic insights. Our results show that the vast majority of hexamer sequence motifs measurably influence splice site selection when positioned within alternative exons, with multiple motifs acting additively rather than cooperatively. Intriguingly, motifs that enhance (suppress) exon inclusion in alternative 5' splicing also enhance (suppress) exon inclusion in alternative 3' or cassette exon splicing, suggesting a universal mechanism for alternative exon recognition. Finally, our empirically trained models are highly predictive of the effects of naturally occurring variants on alternative splicing in vivo.

Evolution of the Grain Dispersal System in Barley.

About 12,000 years ago in the Near East, humans began the transition from hunter-gathering to agriculture-based societies. Barley was a founder crop in this process, and the most important steps in its domestication were mutations in two adjacent, dominant, and complementary genes, through which grains were retained on the inflorescence at maturity, enabling effective harvesting. Independent recessive mutations in each of these genes caused cell wall thickening in a highly specific grain "disarticulation zone," converting the brittle floral axis (the rachis) of the wild-type into a tough, non-brittle form that promoted grain retention. By tracing the evolutionary history of allelic variation in both genes, we conclude that spatially and temporally independent selections of germplasm with a non-brittle rachis were made during the domestication of barley by farmers in the southern and northern regions of the Levant, actions that made a major contribution to the emergence of early agrarian societies.