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1.  The DEAH-box Helicase Dhr1 Dissociates U3 from the Pre-rRNA to Promote Formation of the Central Pseudoknot 
PLoS Biology  2015;13(2):e1002083.
In eukaryotes, the highly conserved U3 small nucleolar RNA (snoRNA) base-pairs to multiple sites in the pre-ribosomal RNA (pre-rRNA) to promote early cleavage and folding events. Binding of the U3 box A region to the pre-rRNA is mutually exclusive with folding of the central pseudoknot (CPK), a universally conserved rRNA structure of the small ribosomal subunit essential for protein synthesis. Here, we report that the DEAH-box helicase Dhr1 (Ecm16) is responsible for displacing U3. An active site mutant of Dhr1 blocked release of U3 from the pre-ribosome, thereby trapping a pre-40S particle. This particle had not yet achieved its mature structure because it contained U3, pre-rRNA, and a number of early-acting ribosome synthesis factors but noticeably lacked ribosomal proteins (r-proteins) that surround the CPK. Dhr1 was cross-linked in vivo to the pre-rRNA and to U3 sequences flanking regions that base-pair to the pre-rRNA including those that form the CPK. Point mutations in the box A region of U3 suppressed a cold-sensitive mutation of Dhr1, strongly indicating that U3 is an in vivo substrate of Dhr1. To support the conclusions derived from in vivo analysis we showed that Dhr1 unwinds U3-18S duplexes in vitro by using a mechanism reminiscent of DEAD box proteins.
U3 snoRNA binds to pre-rRNA, helping to orchestrate key steps in ribosome assembly. This study identifies Dhr1 as the essential RNA helicase that releases U3 snoRNA and allows ribosome maturation to continue.
Author Summary
Ribosomes are intricate assemblies of RNA and protein that are responsible for decoding a cell’s genetic information. Their assembly is a very rapid and dynamic process, requiring many ancillary factors in eukaryotic cells. One critical factor is the U3 snoRNA, which binds to the immature ribosomal RNA to direct early processing and folding of the RNA of the small subunit. Although U3 is essential to promote assembly, it must be actively removed to allow completion of RNA folding. Such RNA dynamics are often driven by RNA helicases, and here we use a broad range of experimental approaches to identify the RNA helicase Dhr1 as the enzyme responsible for removing U3 in yeast. A combination of techniques allows us to assess what goes wrong when Dhr1 is mutated, which parts of the RNA molecules the enzyme binds to, and how Dhr1 unwinds its substrates.
doi:10.1371/journal.pbio.1002083
PMCID: PMC4340053  PMID: 25710520
2.  Caprin Controls Follicle Stem Cell Fate in the Drosophila Ovary 
PLoS ONE  2012;7(4):e35365.
Adult stem cells must balance self-renewal and differentiation for tissue homeostasis. The Drosophila ovary has provided a wealth of information about the extrinsic niche signals and intrinsic molecular processes required to ensure appropriate germline stem cell renewal and differentiation. The factors controlling behavior of the more recently identified follicle stem cells of the ovary are less well-understood but equally important for fertility. Here we report that translational regulators play a critical role in controlling these cells. Specifically, the translational regulator Caprin (Capr) is required in the follicle stem cell lineage to ensure maintenance of this stem cell population and proper encapsulation of developing germ cells by follicle stem cell progeny. In addition, reduction of one copy of the gene fmr1, encoding the translational regulator Fragile X Mental Retardation Protein, exacerbates the Capr encapsulation phenotype, suggesting Capr and fmr1 are regulating a common process. Caprin was previously characterized in vertebrates as Cytoplasmic Activation/Proliferation-Associated Protein. Significantly, we find that loss of Caprin alters the dynamics of the cell cycle, and we present evidence that misregulation of CycB contributes to the disruption in behavior of follicle stem cell progeny. Our findings support the idea that translational regulators may provide a conserved mechanism for oversight of developmentally critical cell cycles such as those in stem cell populations.
doi:10.1371/journal.pone.0035365
PMCID: PMC3320888  PMID: 22493746
3.  Essential function of Drosophila Sec6 in apical exocytosis of epithelial photoreceptor cells 
The Journal of Cell Biology  2005;169(4):635-646.
Polarized exocytosis plays a major role in development and cell differentiation but the mechanisms that target exocytosis to specific membrane domains in animal cells are still poorly understood. We characterized Drosophila Sec6, a component of the exocyst complex that is believed to tether secretory vesicles to specific plasma membrane sites. sec6 mutations cause cell lethality and disrupt plasma membrane growth. In developing photoreceptor cells (PRCs), Sec6 but not Sec5 or Sec8 shows accumulation at adherens junctions. In late PRCs, Sec6, Sec5, and Sec8 colocalize at the rhabdomere, the light sensing subdomain of the apical membrane. PRCs with reduced Sec6 function accumulate secretory vesicles and fail to transport proteins to the rhabdomere, but show normal localization of proteins to the apical stalk membrane and the basolateral membrane. Furthermore, we show that Rab11 forms a complex with Sec5 and that Sec5 interacts with Sec6 suggesting that the exocyst is a Rab11 effector that facilitates protein transport to the apical rhabdomere in Drosophila PRCs.
doi:10.1083/jcb.200410081
PMCID: PMC2171699  PMID: 15897260

Results 1-3 (3)