Transcriptomic analyses have identified tens of thousands of intergenic, intronic, and cis-antisense long noncoding RNAs (lncRNAs) that are expressed from mammalian genomes. Despite progress in ...functional characterization, little is known about the post-transcriptional regulation of lncRNAs and their half-lives. Although many are easily detectable by a variety of techniques, it has been assumed that lncRNAs are generally unstable, but this has not been examined genome-wide. Utilizing a custom noncoding RNA array, we determined the half-lives of ∼800 lncRNAs and ∼12,000 mRNAs in the mouse Neuro-2a cell line. We find only a minority of lncRNAs are unstable. LncRNA half-lives vary over a wide range, comparable to, although on average less than, that of mRNAs, suggestive of complex metabolism and widespread functionality. Combining half-lives with comprehensive lncRNA annotations identified hundreds of unstable (half-life < 2 h) intergenic, cis-antisense, and intronic lncRNAs, as well as lncRNAs showing extreme stability (half-life > 16 h). Analysis of lncRNA features revealed that intergenic and cis-antisense RNAs are more stable than those derived from introns, as are spliced lncRNAs compared to unspliced (single exon) transcripts. Subcellular localization of lncRNAs indicated widespread trafficking to different cellular locations, with nuclear-localized lncRNAs more likely to be unstable. Surprisingly, one of the least stable lncRNAs is the well-characterized paraspeckle RNA Neat1, suggesting Neat1 instability contributes to the dynamic nature of this subnuclear domain. We have created an online interactive resource (http://stability.matticklab.com) that allows easy navigation of lncRNA and mRNA stability profiles and provides a comprehensive annotation of ~7200 mouse lncRNAs.
We identified the neurons comprising the Drosophila mushroom body (MB), an associative center in invertebrate brains, and provide a comprehensive map describing their potential connections. Each of ...the 21 MB output neuron (MBON) types elaborates segregated dendritic arbors along the parallel axons of ∼2000 Kenyon cells, forming 15 compartments that collectively tile the MB lobes. MBON axons project to five discrete neuropils outside of the MB and three MBON types form a feedforward network in the lobes. Each of the 20 dopaminergic neuron (DAN) types projects axons to one, or at most two, of the MBON compartments. Convergence of DAN axons on compartmentalized Kenyon cell-MBON synapses creates a highly ordered unit that can support learning to impose valence on sensory representations. The elucidation of the complement of neurons of the MB provides a comprehensive anatomical substrate from which one can infer a functional logic of associative olfactory learning and memory.
Animals discriminate stimuli, learn their predictive value and use this knowledge to modify their behavior. In Drosophila, the mushroom body (MB) plays a key role in these processes. Sensory stimuli ...are sparsely represented by ∼2000 Kenyon cells, which converge onto 34 output neurons (MBONs) of 21 types. We studied the role of MBONs in several associative learning tasks and in sleep regulation, revealing the extent to which information flow is segregated into distinct channels and suggesting possible roles for the multi-layered MBON network. We also show that optogenetic activation of MBONs can, depending on cell type, induce repulsion or attraction in flies. The behavioral effects of MBON perturbation are combinatorial, suggesting that the MBON ensemble collectively represents valence. We propose that local, stimulus-specific dopaminergic modulation selectively alters the balance within the MBON network for those stimuli. Our results suggest that valence encoded by the MBON ensemble biases memory-based action selection.
The use of genetically encoded 'self-labeling tags' with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we ...synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved Drosophila melanogaster transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.
Ernest Hemingway was known for writing with the “Iceberg Theory” in mind. Thus, there are deeper meanings and contexts moving beneath the surface of his works. His war novel A Farewell to Arms takes ...place along the Soča/Isonzo Front both before and after the Battle of Kobarid/Caporetto and in this setting, consistent with his “Iceberg Theory,” Hemingway has placed both characters and settings that deserve a reconsideration below the surface. While the Italians in the novel are on the surface of the story and thus more easily recognizable, it is the Slovenes and Friuli who run under the surface and carry a deeper meaning. Slovenes and Friuli are not named directly, but as Hemingway was historically accurate in the novel, both ethnic groups are placed along the Front and collectively they represent the “other” in Hemingway’s novels, both unseen and integral to the storyline.
Retinitis pigmentosa (RP) is a genetically heterogeneous heritable disease characterized by apoptotic death of photoreceptor cells. We used exome sequencing to identify a homozygous Alu insertion in ...exon 9 of male germ cell-associated kinase (MAK) as the cause of disease in an isolated individual with RP. Screening of 1,798 unrelated RP patients identified 20 additional probands homozygous for this insertion (1.2%). All 21 affected probands are of Jewish ancestry. MAK encodes a kinase involved in the regulation of photoreceptor-connecting cilium length. Immunohistochemistry of human donor tissue revealed that MAK is expressed in the inner segments, cell bodies, and axons of rod and cone photoreceptors. Several isoforms of MAK that result from alternative splicing were identified. Induced pluripotent stem cells were derived from the skin of the proband and a patient with non-MAK–associated RP (RP control). In the RP control individual, we found that a transcript lacking exon 9 was predominant in undifferentiated cells, whereas a transcript bearing exon 9 and a previously unrecognized exon 12 predominated in cells that were differentiated into retinal precursors. However, in the proband with the Alu insertion, the developmental switch to the MAK transcript bearing exons 9 and 12 did not occur. In addition to showing the use of induced pluripotent stem cells to efficiently evaluate the pathogenicity of specific mutations in relatively inaccessible tissues like retina, this study reveals algorithmic and molecular obstacles to the discovery of pathogenic insertions and suggests specific changes in strategy that can be implemented to more fully harness the power of sequencing technologies.
Age-related macular degeneration (AMD) is a common disease that can result in severe visual impairment. Abnormal regulation of the complement system has been implicated in its pathogenesis, and CFH ...polymorphisms contribute substantially to risk. How these polymorphisms exert their effects is poorly understood. We performed enzyme-linked immunosorbent assay (ELISA) analysis on young, aged, and AMD choroids to determine the abundance of the membrane attack complex (MAC) and performed immunofluorescence studies on eyes from 117 donors to evaluate the MAC in aging, early AMD, and advanced AMD. Morphometric studies were performed on eyes with high- or low-risk CFH genotypes. ELISA confirmed that MAC increases significantly with aging and with AMD. MAC was localized to Bruch's membrane and the choriocapillaris and was detectable at low levels as early as 5 years of age. Hard drusen were labeled with anti-MAC antibody, but large or confluent drusen and basal deposits were generally unlabeled. Labeling of retinal pigment epithelium was observed in some cases of advanced AMD, but not in early disease. Eyes homozygous for the high-risk CFH genotype had thinner choroids than low-risk homozygotes (P < 0.05). These findings suggest that increased complement activation in AMD and in high-risk genotypes can lead to loss of endothelial cells in early AMD. Treatments to protect the choriocapillaris in early AMD are needed.
T-cell-engaging bispecific antibody (T-BsAb, also known as BiTE) therapy has emerged as a powerful therapeutic modality against multiple myeloma. Given that T-BsAb therapy redirects endogenous T ...cells to eliminate tumor cells, reinvigorating dysfunctional T cells may be a potential approach to improve the efficacy of T-BsAb. While various immunostimulatory cytokines can potentiate effector T-cell functions, the optimal cytokine treatment for T-BsAb therapy is yet to be established, partly due to a concern of cytokine release syndrome driven by aberrant interferon (IFN)-γ production. Here, we functionally screen immunostimulatory cytokines to determine an ideal combination partner for T-BsAb therapy. This approach reveals IL-21 as a potential immunostimulatory cytokine with the ability to augment T-BsAb-mediated release of granzyme B and perforin, without increasing IFN-γ release. Transcriptome profiling and functional characterization strongly support that IL-21 selectively targets the cytotoxic granule exocytosis pathway, but not pro-inflammatory responses. Notably, IL-21 modulates multiple steps of cytotoxic effector functions including upregulation of co-activating CD226 receptor, increasing cytotoxic granules, and promoting cytotoxic granule delivery at the immunological synapse. Indeed, T-BsAb-mediated myeloma-killing is cytotoxic granule-dependent, and IL-21 priming significantly augments cytotoxic activities. Furthermore, in vivo IL-21 treatment induces cytotoxic effector reprogramming in bone marrow T cells, showing synergistic anti-myeloma effects in combination with T-BsAb therapy. Together, harnessing the cytotoxic granule exocytosis pathway by IL-21 may be a potential approach to achieve better responses by T-BsAb therapy.
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