The 21-nucleotide small temporal RNA (stRNA) let-7 regulates developmental timing in Caenorhabditis elegans and probably in other bilateral animals. We present in vivo and in vitro evidence that in Drosophila melanogaster a developmentally regulated precursor RNA is cleaved by an RNA interference-like mechanism to produce mature let-7 stRNA. Targeted destruction in cultured human cells of the messenger RNA encoding the enzyme Dicer, which acts in the RNA interference pathway, leads to accumulation of the let-7 precursor. Thus, the RNA interference and stRNA pathways intersect. Both pathways require the RNA-processing enzyme Dicer to produce the active small-RNA component that represses gene expression.

Puf proteins are developmental regulators that control mRNA stability and translation by binding sequences in the 3' untranslated regions of their target mRNAs. We have determined the structure of the RNA binding domain of the human Puf protein, Pumilio1, bound to a high-affinity RNA ligand. The RNA binds the concave surface of the molecule, where each of the protein's eight repeats makes contacts with a different RNA base via three amino acid side chains at conserved positions. We have mutated these three side chains in one repeat, thereby altering the sequence specificity of Pumilio1. Thus, the high affinity and specificity of the PUM-HD for RNA is achieved using multiple copies of a simple repeated motif.

bCoxiella burnetii, the etiological agent of Q fever in humans, is an intracellular pathogen that replicates in an acidified parasitophorous vacuole derived from host lysosomes. Generation of this replicative compartment requires effectors delivered into the host cell by the Dot/Icm type IVb secretion system. Several effectors crucial for C. burnetii intracellular replication have been identified, but the host pathways coopted by these essential effectors are poorly defined, and very little is known about how spacious vacuoles are formed and maintained. Here we demonstrate that the essential type IVb effector, CirA, stimulates GTPase activity of RhoA. Overexpression of CirA in mammalian cells results in cell rounding and stress fiber disruption, a phenotype that is rescued by overexpression of wild-type or constitutively active RhoA. Unlike other effector proteins that subvert Rho GTPases to modulate uptake, CirA is the first effector identified that is dispensable for uptake and instead recruits Rho GTPase to promote biogenesis of the bacterial vacuole. Collectively our results highlight the importance of CirA in coopting host Rho GTPases for establishment of Coxiella burnetii infection and virulence in mammalian cell culture and mouse models of infection.T he naturally obligate intracellular pathogen Coxiella burnetii is the causative agent of Q fever in humans. The agent's high infectivity, ease of spread by aerosols, and environmental stability have led to its classification as a category B select agent by the Centers for Disease Control and Prevention (1). The primary route of infection is through inhalation of contaminated aerosols, which typical results in acute Q fever, a debilitating flu-like illness that is generally self-limiting and readily resolves without antibiotic treatment. However, under some circumstances, persistent infection can lead to chronic Q fever that presents as endocarditis or hepatitis (1). While the number of reported cases of acute Q fever in the United States has been relatively low, a marked increase occurred in 1999 when Q fever became a reportable disease. A recent outbreak of Q fever in The Netherlands resulted in over 4,000 confirmed cases, with 20% of patients requiring hospitalization (2). The dramatic increase in reported cases suggests C. burnetii is an emerging pathogen and highlights our lack of understanding of C. burnetii virulence factors.Inhalation of C. burnetii by a mammalian host results in actindependent endocytosis and internalization in an early endosome. While numerous intracellular pathogens actively subvert the default endocytic pathway to establish a unique host-derived vacuole, C. burnetii generally follows the default trafficking pathway to establish a Coxiella-containing vacuole (CCV) derived from the host lysosomal network. Generation of this unique replicative compartment requires active bacterial protein synthesis which drives homotypic and heterotypic vesicle fusions associated with generation of a spacious CCV that occupies the majority of the hos...

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