Barcode identification for single cell genomics.

BACKGROUND: Single-cell sequencing experiments use short DNA barcode 'tags' to identify reads that originate from the same cell. In order to recover single-cell information from such experiments, reads must be grouped based on their barcode tag, a crucial processing step that precedes other computations. However, this step can be difficult due to high rates of mismatch and deletion errors that can afflict barcodes. RESULTS: Here we present an approach to identify and error-correct barcodes by traversing the de Bruijn graph of circularized barcode k-mers. Our approach is based on the observation that circularizing a barcode sequence can yield error-free k-mers even when the size of k is large relative to the length of the barcode sequence, a regime which is typical single-cell barcoding applications. This allows for assignment of reads to consensus fingerprints constructed from k-mers. CONCLUSION: We show that for single-cell RNA-Seq circularization improves the recovery of accurate single-cell transcriptome estimates, especially when there are a high number of errors per read. This approach is robust to the type of error (mismatch, insertion, deletion), as well as to the relative abundances of the cells. Sircel, a software package that implements this approach is described and publically available.

RNA Binding and HEPN-Nuclease Activation Are Decoupled in CRISPR-Cas13a.

CRISPR-Cas13a enzymes are RNA-guided, RNA-activated RNases. Their properties have been exploited as powerful tools for RNA detection, RNA imaging, and RNA regulation. However, the relationship between target RNA binding and HEPN (higher eukaryotes and prokaryotes nucleotide binding) domain nuclease activation is poorly understood. Using sequencing experiments coupled with in vitro biochemistry, we find that Cas13a target RNA binding affinity and HEPN-nuclease activity are differentially affected by the number and the position of mismatches between the guide and the target. We identify a central binding seed for which perfect base pairing is required for target binding and a separate nuclease switch for which imperfect base pairing results in tight binding, but not HEPN-nuclease activation. These results demonstrate that the binding and cleavage activities of Cas13a are decoupled, highlighting a complex specificity landscape. Our findings underscore a need to consider the range of effects off-target recognition has on Cas13a RNA binding and cleavage behavior for RNA-targeting tool development.

PROBer Provides a General Toolkit for Analyzing Sequencing-Based Toeprinting Assays.

A number of sequencing-based transcriptase drop-off assays have recently been developed to probe post-transcriptional dynamics of RNA-protein interaction, RNA structure, and RNA modification. Although these assays survey a diverse set of epitranscriptomic marks, we use the term toeprinting assays since they share methodological similarities. Their interpretation is predicated on addressing a similar computational challenge: how to learn isoform-specific chemical modification profiles in the face of complex read multi-mapping. We introduce PROBer, a statistical model and associated software, that addresses this challenge for the analysis of toeprinting assays. PROBer takes sequencing data as input and outputs estimated transcript abundances and isoform-specific modification profiles. Results on both simulated and biological data demonstrate that PROBer significantly outperforms individual methods tailored for specific toeprinting assays. Since the space of toeprinting assays is ever expanding and these assays are likely to be performed and analyzed together, we believe PROBer's unified data analysis solution will be valuable to the RNA community.

Dicer-TRBP complex formation ensures accurate mammalian microRNA biogenesis.

RNA-mediated gene silencing in human cells requires the accurate generation of ∼22 nt microRNAs (miRNAs) from double-stranded RNA substrates by the endonuclease Dicer. Although the phylogenetically conserved RNA-binding proteins TRBP and PACT are known to contribute to this process, their mode of Dicer binding and their genome-wide effects on miRNA processing have not been determined. We solved the crystal structure of the human Dicer-TRBP interface, revealing the structural basis of the interaction. Interface residues conserved between TRBP and PACT show that the proteins bind to Dicer in a similar manner and by mutual exclusion. Based on the structure, a catalytically active Dicer that cannot bind TRBP or PACT was designed and introduced into Dicer-deficient mammalian cells, revealing selective defects in guide strand selection. These results demonstrate the role of Dicer-associated RNA binding proteins in maintenance of gene silencing fidelity.

RNA-guided assembly of Rev-RRE nuclear export complexes.

HIV replication requires nuclear export of unspliced and singly spliced viral transcripts. Although a unique RNA structure has been proposed for the Rev-response element (RRE) responsible for viral mRNA export, how it recruits multiple HIV Rev proteins to form an export complex has been unclear. We show here that initial binding of Rev to the RRE triggers RNA tertiary structural changes, enabling further Rev binding and the rapid formation of a viral export complex. Analysis of the Rev-RRE assembly pathway using SHAPE-Seq and small-angle X-ray scattering (SAXS) reveals two major steps of Rev-RRE complex formation, beginning with rapid Rev binding to a pre-organized region presenting multiple Rev binding sites. This step induces long-range remodeling of the RNA to expose a cryptic Rev binding site, enabling rapid assembly of additional Rev proteins into the RNA export complex. This kinetic pathway may help maintain the balance between viral replication and maturation.DOI: http://dx.doi.org/10.7554/eLife.03656.001.