Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships, but require efficient methods for cell capture and mRNA sequencing1–4. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths5, the limitations of shallow sequencing have not been directly investigated. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In developing cortex we identify diverse cell types including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells.
RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.
Cap hydrolysis by Dcp2 is a critical step in several eukaryotic mRNA decay pathways. Processing requires access to cap-proximal nucleotides and the coordinated assembly of a decapping mRNP, but the mechanism of substrate recognition and regulation by protein interactions have remained elusive. Using NMR spectroscopy and kinetic analyses, we show that yeast Dcp2 resolves interactions with the cap and RNA body using a bipartite surface that forms a channel intersecting the catalytic and regulatory Dcp1-binding domains. The interaction with cap is weak but specific and requires binding of the RNA body to a dynamic interface. The catalytic step is stimulated by Dcp1 and its interaction domain, likely through a substrate-induced conformational change. Thus, activation of the decapping mRNP is restricted by access to 5'-proximal nucleotides, a feature that could act as a checkpoint in mRNA metabolism.
Decapping by Dcp2 is an essential step in 5′-3′ mRNA decay. In yeast, decapping requires an open-to-closed transition in Dcp2, though the link between closure and catalysis remains elusive. Here we show using NMR that cap binds conserved residues on both the catalytic and regulatory domains of Dcp2. Lesions in the cap-binding site on the regulatory domain reduce the catalytic step two orders of magnitude and block formation of the closed state whereas Dcp1 enhances the catalytic step by a factor of ten and promotes closure. We conclude that closure occurs during the rate-limiting catalytic step of decapping, juxtaposing the cap-binding region of each domain to form a composite active site. This work suggests a model for regulation of decapping, where coactivators trigger decapping by stabilizing a labile composite active site.
Cap hydrolysis is a critical control point in the life of eukaryotic mRNAs and is catalyzed by the evolutionarily conserved Dcp1-Dcp2 complex. In Saccharomyces cerevisiae, decapping is modulated by several factors, including the Lsm family protein Edc3, which directly binds to Dcp2. We show that Edc3 binding to Dcp2 is mediated by a short peptide sequence located C terminal to the catalytic domain of Dcp2. This sequence is required for Edc3 to stimulate decapping activity of Dcp2 in vitro, for Dcp2 to efficiently accumulate in P-bodies, and for efficient degradation of the RPS28B mRNA, whose decay is enhanced by Edc3. In contrast, degradation of YRA1 pre-mRNA, another Edc3-regulated transcript, occurs independently from this region, suggesting that the effect of Edc3 on YRA1 is independent of its interaction with Dcp2. Deletion of the sequence also results in a subtle but significant defect in turnover of the MFA2pG reporter transcript, which is not affected by deletion of EDC3, suggesting that the region affects some other aspect of Dcp2 function in addition to binding Edc3. These results raise a model for Dcp2 recruitment to specific mRNAs where regions outside the catalytic core promote the formation of different complexes involved in mRNA decapping.
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