SummaryRNA interference pathways use small RNAs to mediate gene silencing in eukaryotes. In addition to small interfering RNAs (siRNA) and microRNAs, several types of endogenously produced small RNAs play important roles in gene regulation, germ cell maintenance and transposon silencing 1 -4. Production of some of these RNAs requires the synthesis of aberrant RNAs (aRNAs) or pre-siRNAs, which are specifically recognized by RNA-dependent RNA polymerases (RdRPs) to make double stranded RNA (dsRNA). The mechanism for aRNA synthesis and recognition is largely unknown. Here we show that DNA damage induces the expression of the Argonaute protein QDE-2 and a novel class of small RNAs in the filamentous fungus Neurospora. This class of small RNAs, named qiRNAs for their association with QDE-2, are about 20-21 nt long (several nt shorter than Neurospora siRNAs) with a strong preference for uridine at the 5′ end and originate mostly from the ribosomal DNA locus. Production of qiRNAs requires the RdRP QDE-1, the Werner/Bloom RecQ DNA helicase homolog QDE-3 and dicers. qiRNA biogenesis also requires DNA damage-induced aRNAs as precursor, a process that is dependent on QDE-1 and QDE-3. Surprisingly, our results suggest that QDE-1 is the DNA-dependent RNA polymerase that produces aRNAs. In addition, the Neurospora RNAi mutants exhibit increased sensitivity to DNA damage, suggesting a role for qiRNAs in DNA damage response by inhibiting protein translation.In the filamentous fungus Neurospora crassa, the RNAi pathway is essential for both dsRNA and transgene-induced gene silencing (quelling) 5 . In the quelling pathway, QDE-1 and QDE-3 are thought to be involved in the generation of dsRNA 6 , 7. In addition, QDE-3 was previously shown to be involved in DNA repair 6. It has been proposed that a repetitive transgene leads to the production of transgene-specific aRNA, which is converted to dsRNA

The study of macroautophagy in mammalian cells has described induction, vesicle nucleation, and membrane elongation complexes as key signaling intermediates driving autophagosome biogenesis. How these components are recruited to nascent autophagosomes is poorly understood, and although much is known about signaling mechanisms that restrain autophagy, the nature of positive inductive signals that can promote autophagy remain cryptic. We find that the Ras-like small G-protein, RalB, is localized to nascent autophagosomes and is activated upon nutrient deprivation. RalB and its effector Exo84 are required for nutrient starvation-induced autophagocytosis, and RalB activation is sufficient to promote autophagosome formation. Through direct binding to Exo84, RalB induces the assembly of catalytically active ULK1 and Beclin1-VPS34 complexes on the exocyst, which are required for isolation membrane formation and maturation. Thus, RalB signaling is a primary adaptive response to nutrient limitation that directly engages autophagocytosis through mobilization of the core vesicle nucleation machinery.

Key Points• CXCL13 and CXCL12 mediate chemotaxis of CNS lymphoma cells, and CXCL13 concentration in CSF is prognostic.• CXCL13 plus IL-10 is highly specific for the diagnosis of CNS lymphoma.Establishing the diagnosis of focal brain lesions in patients with unexplained neurologic symptoms represents a challenge. The goal of this study is to provide evidence supporting functional roles for CXC chemokine ligand (CXCL)13 and interleukin (IL)-10 in central nervous system (CNS) lymphomas and to evaluate the utility of each as prognostic and diagnostic biomarkers. We demonstrate for the first time that elevated CXCL13 concentration in cerebrospinal fluid (CSF) is prognostic and that CXCL13 and CXCL12 mediate chemotaxis of lymphoma cells isolated from CNS lymphoma lesions. Expression of the activated form of Janus kinase 1 supported a role for IL-10 in prosurvival signaling. We determined the concentration of CXCL13 and IL-10 in CSF of CNS lymphoma patients and control cohorts including inflammatory and degenerative neurologic disease in a multicenter study involving 220 patients. Bivariate elevated CXCL13 plus IL-10 was 99.3% specific for primary and secondary CNS lymphoma, with sensitivity significantly greater than reference standard CSF tests. These results identify CXCL13 and IL-10 as potentially important biomarkers of CNS lymphoma that merit further evaluation and support incorporation of CXCL13 and IL-10 into diagnostic algorithms for the workup of focal brain lesions in which lymphoma is a consideration. (Blood. 2013;121(23):4740-4748) IntroductionDetermination of the pathological basis of focal brain lesions in patients with unexplained neurologic symptoms is a major clinical challenge. Persistent symptoms or rapid neurologic decline often mandates stereotactic brain biopsy, a highly invasive procedure with a 10% to 35% rate of diagnostic failure.1-3 Moreover, many lesions are not amenable to biopsy because of small size, location in deep brain structures, risk of hemorrhage, and other comorbidities.The diagnosis of central nervous system (CNS) involvement of non-Hodgkin lymphoma is a particular challenge because of lesional response to glucocorticoids and features on magnetic resonance imaging (MRI) that are shared with other pathologies including astrocytic neoplasms, demyelination, neurosarcoid, vasculitis, infections, and leptomeningeal dissemination of systemic cancer. Although flowcytometric and cytological analysis of cerebrospinal fluid (CSF) is useful in the evaluation of leptomeningeal disease, these tests are usually insensitive to pathological processes based in deep brain structures and rarely provide information that eliminates the need for brain biopsy; the sensitivity of CSF cytological analysis in the evaluation of primary CNS lymphoma (PCNSL) is ;15%. 4Advances that facilitate diagnosis and early treatment of CNS lymphoma would likely be cost-effective, minimize repeat diagnostic CSF and MRI evaluations and brain biopsies, and also lead to improved outcomes. [5][6][7] The molecular const...

Single-stranded small interfering RNA (siRNA) guides the cleavage of homologous mRNA by Argonaute proteins, the catalytic core of the RNA-induced silencing complex (RISC), in the conserved RNA interference (RNAi) pathway. The separation of the siRNA duplex into single strands is essential for the activation of RISC. Previous biochemical studies have suggested that Argonaute proteins cleave and remove the passenger strand of siRNA duplex from RISC, but the in vivo importance of this process and the mechanism for the removal of the nicked passenger strand are not known. Here, we show that in the filamentous fungus Neurospora, the Argonaute homolog QDE-2 and its slicer function are required for the generation of single-stranded siRNA and gene silencing in vivo. Biochemical purification of QDE-2 led to the identification of QIP, a QDE-2-interacting protein, with an exonuclease domain. The disruption of qip in Neurospora impaired gene silencing and siRNA accumulated, mostly in nicked duplex form. Furthermore, our results suggest that QIP acts as an exonuclease that cleaves and removes the nicked passenger strand from siRNA duplex in a QDE-2-dependent manner. Together, these results suggest that both the cleavage and removal of the passenger strand from the siRNA duplex are important steps in RNAi pathways.[Keywords: RNAi; Neurospora; Argonaute; siRNA; exonuclease; RISC] Supplemental material is available at http://www.genesdev.org.

When recognized by the RNA interference (RNAi) pathway, double-stranded RNA (dsRNA) produced in eukaryotic cells results in posttranscriptional gene silencing. In addition, dsRNA can trigger the interferon response as part of the immune response in vertebrates. In this study, we show that dsRNA, but not short interfering RNA (siRNA), induces the expression of qde-2 (an Argonaute gene) and dcl-2 (a Dicer gene), two central components of the RNAi pathway in the filamentous fungus Neurospora crassa. The induction of QDE-2 by dsRNA is required for normal gene silencing, indicating that this is a regulatory mechanism that allows the optimal function of the RNAi pathway. In addition, we demonstrate that Dicer proteins (DCLs) regulate QDE-2 posttranscriptionally, suggesting a role for DCLs or siRNA in QDE-2 accumulation. Finally, a genome-wide search revealed that additional RNAi components and homologs of antiviral and interferon-stimulated genes are also dsRNA-activated genes in Neurospora. Together, our results suggest that the activation of the RNAi components is part of a broad ancient host defense response against viral and transposon infections.