Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (6 results)

  • Article
  • Peer Reviewed
UC Berkeley Previously Published Works (2014)
In a recent paper Siegfried et al. published a new sequence-based structural RNA assay that utilizes mutational profiling to detect base pairing (MaP). Output from MaP provides information about both pairing (via reactivities) and contact (via correlations). Reactivities can be coupled to partition function folding models for structural inference, while correlations can reveal pairs of sites that may be in structural proximity. The possibility for inference of 3D contacts via MaP suggests a novel approach to structural prediction for RNA analogous to covariance structural prediction for proteins. We explore this approach and show that partial correlation analysis outperforms na\"ive correlation analysis. Our results should be applicable to a wide range of high-throughput sequencing based RNA structural assays that are under development.
    Cover page: Identifying RNA contacts from SHAPE-MaP by partial correlation analysis
    • Thesis
    • Peer Reviewed
    UC Berkeley Electronic Theses and Dissertations (2017)

    Genomic data has revolutionized biology. The related development of high-throughput DNA sequencers has had a major impact on this, as the DNA sequencing platforms are widely adaptable to a number of assays that measure aspects of biology that are increasingly unrelated to the problem for which they were built: to understand the content of a genome. Such experiments, termed sequence-census assays, leverage the power of these machines to yield high throughput digital information about a molecular sample. Although there is large variability within these assays, there are main themes to the structure of these experiments: they encode molecular information into a sequencable format by performing a limited set of molecular biology operations on a pool of nucleic acids, then they read out this information by sequencing, and finally decode the sequenced message computationally. Importantly, during this process the computational decoding and experimental encoding of this information are coupled, and as such both ’wet’ and ’dry’ parts of an assay must be designed and treated together. In this thesis I explore these aspects of sequence-census assays, and their application to biologically unrelated, but computationally similar problems, ranging from CRISPR biology to single-cell sequencing.

      Cover page: Developing experimental and computational tools for sequence-census assays
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2017)
      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.
        Cover page: PROBer Provides a General Toolkit for Analyzing Sequencing-Based Toeprinting Assays
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2014)
        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.
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2018)
          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.
            • Article
            • Peer Reviewed
            UC Berkeley Previously Published Works (2015)
            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.
              Top