Cross-Species Single-Cell Analysis Reveals Divergence of the Primate Microglia Program

Summary Microglia, the brain-resident immune cells, are critically involved in many physiological and pathological brain processes, including neurodegeneration. Here we characterize microglia morphology and transcriptional programs across ten species spanning more than 450 million years of evolution. We find that microglia express a conserved core gene program of orthologous genes from rodents to humans, including ligands and receptors associated with interactions between glia and neurons. In most species, microglia show a single dominant transcriptional state, whereas human microglia display significant heterogeneity. In addition, we observed notable differences in several gene modules of rodents compared with primate microglia, including complement, phagocytic, and susceptibility genes to neurodegeneration, such as Alzheimer’s and Parkinson’s disease. Our study provides an essential resource of conserved and divergent microglia pathways across evolution, with important implications for future development of microglia-based therapies in humans

Single cell dissection of plasma cell heterogeneity in symptomatic and asymptomatic myeloma

Multiple myeloma, a plasma cell malignancy, is the second most common blood cancer. Despite extensive research, disease heterogeneity is poorly characterized, hampering efforts for early diagnosis and improved treatments. Here, we apply single cell RNA sequencing to study the heterogeneity of 40 individuals along the multiple myeloma progression spectrum, including 11 healthy controls, demonstrating high interindividual variability that can be explained by expression of known multiple myeloma drivers and additional putative factors. We identify extensive subclonal structures for 10 of 29 individuals with multiple myeloma. In asymptomatic individuals with early disease and in those with minimal residual disease post-treatment, we detect rare tumor plasma cells with molecular characteristics similar to those of active myeloma, with possible implications for personalized therapies. Single cell analysis of rare circulating tumor cells allows for accurate liquid biopsy and detection of malignant plasma cells, which reflect bone marrow disease. Our work establishes single cell RNA sequencing for dissecting blood malignancies and devising detailed molecular characterization of tumor cells in symptomatic and asymptomatic patients

Pre-mRNA splicing is a determinant of nucleosome organization.

Chromatin organization affects alternative splicing and previous studies have shown that exons have increased nucleosome occupancy compared with their flanking introns. To determine whether alternative splicing affects chromatin organization we developed a system in which the alternative splicing pattern switched from inclusion to skipping as a function of time. Changes in nucleosome occupancy were correlated with the change in the splicing pattern. Surprisingly, strengthening of the 5' splice site or strengthening the base pairing of U1 snRNA with an internal exon abrogated the skipping of the internal exons and also affected chromatin organization. Over-expression of splicing regulatory proteins also affected the splicing pattern and changed nucleosome occupancy. A specific splicing inhibitor was used to show that splicing impacts nucleosome organization endogenously. The effect of splicing on the chromatin required a functional U1 snRNA base pairing with the 5' splice site, but U1 pairing was not essential for U1 snRNA enhancement of transcription. Overall, these results suggest that splicing can affect chromatin organization

Alternative splicing affects nucleosome occupancy in endogenous genes.

<p>(<b>A</b>) HeLa cells were treated with 10 nM meayamycin and RNA was extracted 24 hr later. Splicing products were separated on a 1.5% agarose gel after RT-PCR reaction using appropriate primers for the flanking the alternative exon (marked with an arrow). Ten endogenous genes were analyzed: BCL2-like 11 (apoptosis facilitator; BCL2L11); calcium channel, voltage-dependent, T type, alpha 1G subunit (CACNA1G); vacuolar protein sorting 26 homolog A (<i>S. pombe</i>) (VPS26A); calcium channel, voltage-dependent, T type, alpha 1H subunit (CACNA1H); cathepsin A (CTSA); polymerase delta interacting protein 3 (POLDIP3); KH domain containing, RNA binding, signal transduction associated 1 (KHDR); bromodomain containing 8 (BRD8); deoxyguanosine kinase, nuclear gene encoding mitochondrial protein (DGUOK); and proline-rich coiled coil 2B (PRRC2B). (<b>B</b>) DNA was extracted from HeLa nuclei 24 hr after meayamycin treatments. An MNase assay was then performed and the mononucleosomal DNA was subjected to absolute QPCR analysis on the alternative exon. Data are presented as DNA copy number.</p

Alternative splicing affects nucleosome occupancy.

<p>(<b>A</b>) IKAP19–23 minigenes with different mutations that strengthened the 5′ss of each internal exon were transfected into 293 cells and splicing was evaluated. “Strong-ss” indicates a mutation that strengthened the splice site score <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053506#pone.0053506-Nilsen1" target="_blank">[1]</a>. The positions of the 5′ss mutations for each minigene are indicated with an arrow in the lower part of the panel. (<b>B</b>) The IKAP19–23 minigene with a strong exon 20 5′ss was transfected into 293 cells, and nuclei were extracted 24 and 72 hr following transfection. Half of each sample was treated with MNase and half was untreated. Mononucleosomal DNA was extracted from an agarose gel and subjected to absolute QPCR analysis with primers that cover most of the minigene. Data are presented as DNA copy number and were normalized to transfection efficiency using primers for the GFP area of the plasmid using untreated samples. (<b>C</b>) Co-transfection of the IKAP19–23 minigene into 293 cells was performed with plasmids that express U1 snRNAs. Three different U1 snRNA plasmids were used (as shown in the lower part of the panel): wt U1, U1 with a mutation that strengthens its base pairing with exon 20 5′ss (U1 strong), and U1 with a mutation that weakens the base pairing with exon 20 5′ss (U1 weak). Watson-Crick base pairing is marked by dashed line. The position of the relevant mutation in U1 snRNA is indicated with an arrow. RNA samples were extracted 24, 48, or 72 hr following transfection. The splicing products were separated on a 2% agarose gel after RT-PCR. The PCR products were eluted and sequenced. (<b>D</b>) The IKAP19–23 minigene was co-transfected into 293 cells with either U1 wt, U1 strong or U1 weak plasmid. 72 hr after the transfection, nucleosome occupancy was determined using an MNase assay as above. All experiments were repeated independently three times, and the results shown are representative of an average experiment. QPCR experiments were performed in triplicate; results shown are mean values ± SD.</p

Splicing affects RNAPII occupancy.

<p>(<b>A</b>) At 72 hr following transfection with either the wt or strong exon 20 5′ss minigene, cells were collected and used for an RNAPII-ChIP analysis. The precipitated DNA fragments were subjected to QPCR. Enrichment values were normalized to the unbound fraction, to a non-specific IgG antibody, and to the GFP area of the plasmid. Results are presented as RNAPII fold change between strong exon 20 5′ss and the wt plasmid. (<b>B</b>) At 72 h following co-transfection of IKAP19–23 and U1 plasmids, RNAPII-ChIP was performed. All experiments were repeated independently three times, and the results shown are representative of an average experiment. QPCR experiments were amplified in triplicate; results shown are mean values ± SD.</p

Exploring differential exon usage via short- and long-read RNA sequencing strategies

Alternative splicing produces various mRNAs, and thereby various protein products, from one gene, impacting a wide range of cellular activities. However, accurate reconstruction and quantification of full-length transcripts using short-reads is limited, due to their length. Long-reads sequencing technologies may provide a solution by sequencing full-length transcripts. We explored the use of both Illumina short-reads and two long Oxford Nanopore Technology (cDNA and Direct RNA) RNA-Seq reads for detecting global differential splicing during mouse embryonic stem cell differentiation, applying several bioinformatics strategies: gene-based, isoform-based and exon-based. We detected the strongest similarity among the sequencing platforms at the gene level compared to exon-based and isoform-based. Furthermore, the exon-based strategy discovered many differential exon usage (DEU) events, mostly in a platform-dependent manner and in non-differentially expressed genes. Thus, the platforms complemented each other in the ability to detect DEUs (i.e. long-reads exhibited an advantage in detecting DEUs at the UTRs, and short-reads detected more DEUs). Exons within 20 genes, detected in one or more platforms, were here validated by PCR, including key differentiation genes, such as Mdb3 and Aplp1. We provide an important analysis resource for discovering transcriptome changes during stem cell differentiation and insights for analysing such data

Transneuronal Dpr12/DIP-delta interactions facilitate compartmentalized dopaminergic innervation of Drosophila mushroom body axons

The mechanisms controlling wiring of neuronal networks are not completely understood. The stereotypic architecture of the Drosophila mushroom body (MB) offers a unique system to study circuit assembly. The adult medial MB gamma-lobe is comprised of a long bundle of axons that wire with specific modulatory and output neurons in a tiled manner, defining five distinct zones. We found that the immunoglobulin superfamily protein Dpr12 is cell-autonomously required in gamma-neurons for their developmental regrowth into the distal gamma 4/5 zones, where both Dpr12 and its interacting protein, DIP-delta, are enriched. DIP-delta functions in a subset of dopaminergic neurons that wire with gamma-neurons within the gamma 4/5 zone. During metamorphosis, these dopaminergic projections arrive to the gamma 4/5 zone prior to gamma-axons, suggesting that gamma-axons extend through a prepatterned region. Thus, Dpr12/DIP-delta transneuronal interaction is required for gamma 4/5 zone formation. Our study sheds light onto molecular and cellular mechanisms underlying circuit formation within subcellular resolution

Dissecting Immune Circuits by Linking CRISPR-Pooled Screens with Single-Cell RNA-Seq

In multicellular organisms, dedicated regulatory circuits control cell type diversity and responses. The crosstalk and redundancies within these circuits and substantial cellular heterogeneity pose a major research challenge. Here, we present CRISP-seq, an integrated method for massively parallel single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-pooled screens. We show that profiling the genomic perturbation and transcriptome in the same cell enables us to simultaneously elucidate the function of multiple factors and their interactions. We applied CRISP-seq to probe regulatory circuits of innate immunity. By sampling tens of thousands of perturbed cells in vitro and in mice, we identified interactions and redundancies between developmental and signaling-dependent factors. These include opposing effects of Cebpb and Irf8 in regulating the monocyte/macrophage versus dendritic cell lineages and differential functions for Rela and Stat1/2 in monocyte versus dendritic cell responses to pathogens. This study establishes CRISP-seq as a broadly applicable, comprehensive, and unbiased approach for elucidating mammalian regulatory circuits