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Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2014)

Proteins that harbor double stranded RNA-binding domains (dsRBDs) play

functional roles in processes as diverse as RNA localization, RNA splicing, RNA editing,

nuclear export of RNA and translation, yet the mechanistic basis and functional

significance of dsRBDs remain unclear. To unravel this enigma, we investigated

transactivation response (TAR) RNA-binding protein (TRBP), comprising three dsRBDs,

which has functions in HIV replication, PKR-mediated immune response and RNA

silencing. In collaborative studies using single-molecule methods, I found that TRBP

exhibits an ATP-independent diffusion activity exclusively on double-stranded RNA

(dsRNA) in a length-dependent manner. The first two dsRBDs of TRBP are essential for

diffusion, whereas the third dsRBD is dispensable. Two homologs of TRBP, PKR

activator (PACT) and R3D1-L, displayed the same behavior, implying that the ability to

diffuse along dsRNA is a universal property of this protein family. Furthermore, a Dicer-

TRBP complex on dsRNA exhibited dynamic diffusion, which was correlated with

Dicer's catalytic activity, suggesting that the dsRNA-specific diffusion activity of TRBP

contributes to enhancing small interfering RNA (siRNA) and microRNA (miRNA)

processing by Dicer.

The enzyme Dicer generates 21-25 nucleotide-long dsRNAs that target specific

mRNAs for silencing during RNA interference and related pathways. Although the active

site and RNA-binding regions of Dicer are functionally conserved within eukaryotes, the

helicase domain has distinct activities in the context of different Dicer enzymes. To

examine the evolutionary origins of Dicer helicase functions, we investigated two related

Dicer enzymes from the thermophilic fungus Sporotrichum thermophile. RNA cleavage

assays showed that S. thermophile Dicer-1 (StDicer-1) can process hairpin precursors

to miRNAs, whereas StDicer-2 can only cleave linear dsRNAs. Furthermore, only

StDicer-2 possesses robust ATP hydrolytic activity in the presence of dsRNA. Deletion

of the StDicer-2 helicase domain increases both StDicer-2 cleavage activity and affinity

for hairpin RNA. Notably, the full-length forms (but not truncated versions lacking their

helicase domain) of both StDicer-1 and StDicer-2 could complement the phenotype of a

mutant of a distantly related yeast Schizosaccharomyces pombe lacking its endogenous

Dicer gene, underscoring the importance of the helicase domain for Dicer function.

These results further suggest that an in vivo regulatory function for the helicase domain

may be conserved from fungi to humans.

    Cover page: Mechanisms of RNA Recognition and Cleavage in RNA Interference
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2014)
    The enzyme Dicer generates 21-25 nucleotide RNAs that target specific mRNAs for silencing during RNA interference and related pathways. Although their active sites and RNA binding regions are functionally conserved, the helicase domains have distinct activities in the context of different Dicer enzymes. To examine the evolutionary origins of Dicer helicase functions, we investigated two related Dicer enzymes from the thermophilic fungus Sporotrichum thermophile. RNA cleavage assays showed that S. thermophile Dicer-1 (StDicer-1) can process hairpin precursor microRNAs, whereas StDicer-2 can only cleave linear double-stranded RNAs. Furthermore, only StDicer-2 possesses robust ATP hydrolytic activity in the presence of double-stranded RNA. Deletion of the StDicer-2 helicase domain increases both StDicer-2 cleavage activity and affinity for hairpin RNA. Notably, both StDicer-1 and StDicer-2 could complement the distantly related yeast Schizosaccharomyces pombe lacking its endogenous Dicer gene but only in their full-length forms, underscoring the importance of the helicase domain. These results suggest an in vivo regulatory function for the helicase domain that may be conserved from fungi to humans.
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2017)
      Advanced single-molecule techniques have enabled tracking of nanometer-scale movements of DNA and RNA motor proteins in real time. Previously, we reported an ATP-independent diffusion of transactivation response RNA binding protein (TRBP) on dsRNA, yet the mechanistic details remain elusive. Using single-molecule fluorescence assays, we demonstrate that the diffusion activity of TRBP is coordinated by an independent movement of two subdomains, dsRBD1 and dsRBD2, in which the diffusion distance is determined by the length of a flexible linker domain that connects the two dsRBDs. When the linker is shortened, the diffusion distance is reduced proportionally, suggesting a ruler-like function of the linker domain. Diffusion stalls upon encountering a physical barrier in the form of an RNA:DNA hybrid segment or bulky secondary structures, indicating a dsRNA scanning mode of TRBP. The results display a plausible mechanism of TRBP in scanning for pre-miRNA or pre-siRNA as proper substrates for the RNAi pathway.
        Cover page: RNA Scanning of a Molecular Machine with a Built-in Ruler
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2012)
        The conserved ribonuclease Dicer generates microRNAs and short-interfering RNAs that guide gene silencing in eukaryotes. The specific contributions of human Dicer's structural domains to RNA product length and substrate preference are incompletely understood, due in part to the difficulties of Dicer purification. Here, we show that active forms of human Dicer can be assembled from recombinant polypeptides expressed in bacteria. Using this system, we find that three distinct modes of RNA recognition give rise to Dicer's fidelity and product length specificity. The first involves anchoring one end of a double-stranded RNA helix within the PAZ domain, which can assemble in trans with Dicer's catalytic domains to reconstitute an accurate but non-substrate-selective dicing activity. The second entails nonspecific RNA binding by the double-stranded RNA binding domain, an interaction that is essential for substrate recruitment in the absence of the PAZ domain. The third mode of recognition involves hairpin RNA loop recognition by the helicase domain, which ensures efficient processing of specific substrates. These results reveal distinct interactions of each Dicer domain with different RNA structural features and provide a facile system for investigating the molecular mechanisms of human microRNA biogenesis.
          Cover page: Coordinated Activities of Human Dicer Domains in Regulatory RNA ProcessingCreative Commons 'BY' version 4.0 license
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2013)
          The proteins harboring double-stranded RNA binding domains (dsRBDs) play diverse functional roles such as RNA localization, splicing, editing, export, and translation, yet mechanistic basis and functional significance of dsRBDs remain unclear. To unravel this enigma, we investigated transactivation response RNA binding protein (TRBP) consisting of three dsRBDs, which functions in HIV replication, protein kinase R(PKR)-mediated immune response, and RNA silencing. Here we report an ATP-independent diffusion activity of TRBP exclusively on dsRNA in a length-dependent manner. The first two dsRBDs of TRBP are essential for diffusion, whereas the third dsRBD is dispensable. Two homologs of TRBP, PKR activator and R3D1-L, displayed the same diffusion, implying a universality of the diffusion activity among this protein family. Furthermore, a Dicer-TRBP complex on dsRNA exhibited dynamic diffusion, which was correlated with Dicer's catalytic activity. These results implicate the dsRNA-specific diffusion activity of TRBP that contributes to enhancing siRNA and miRNA processing by Dicer.
            Cover page: ATP-independent diffusion of double-stranded RNA binding proteinsCreative Commons 'BY' version 4.0 license
            • 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.
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