Hfq, a protein required for small RNA (sRNA)-mediated regulation in bacteria, binds RNA with low-nanomolar K d values and long half-lives of complexes (>100 min). This cannot be reconciled with the 1-2-min response time of regulation in vivo. We show that RNAs displace each other on Hfq on a short time scale by RNA concentrationdriven (active) cycling. Already at submicromolar concentrations of competitor RNA, half-lives of RNA-Hfq complexes are »1 min. We propose that competitor RNA associates transiently with RNA-Hfq complexes, RNAs exchange binding sites, and one of the RNAs eventually dissociates. This solves the ''strong binding-high turnover'' paradox and permits efficient use of the Hfq pool. The homohexameric Hfq ring displays two faces: proximal and distal. Hfq-RNA interactions show a preference of U-rich for proximal and A-rich RNA sequences for distal face binding (de Haseth and Uhlenbeck 1980a;Mikulecky et al. 2004). Simultaneous binding may occur on both sides as well, which could facilitate intermolecular base-pairing and regulation (Rajkowitsch and Schroeder 2007).Structures of Hfq from E. coli, Staphylococcus aureus, and Pseudomonas aeroginosa have been determined by X-ray crystallography (Schumacher et al. 2002;Sauter et al. 2003;Nikulin et al. 2005). Two cocrystal structures support two distinct binding surfaces: In S. aureus Hfq, AU 5 G RNA is bound around the inner rim of the proximal face (Schumacher et al. 2002), and E. coli Hfq has oligo-A bound on the distal face (Link et al. 2009 Holmqvist et al. 2010). Thus, if binding-competent RNAs were in molar excess, almost all Hfq would be bound to RNAs. Hfq-RNA dissociation rate constants in vitro are too low to be compatible with a biologically relevant time scale; half-lives of complexes are in the range of a generation time. If newly induced sRNAs only could access free Hfq after its dissociation from bound RNAs, their activity should be severely delayed. Yet, the time frame from induction of an sRNA to a significant regulatory effect is short (1-2 min) (Massé et al. 2003), and hence sRNAs can acquire Hfq rapidly. This highlights a paradox, with Hfq being tightly sequestered by the intracellular pool of RNAs, contrasted by the need of new sRNAs to rapidly access Hfq. We considered here a conventional cycling model (dissociative/passive) (Fig. 1A) and associative/active cycling (Fig. 1B). In model A, newly synthesized RNA (Fig. 1A, in red) can only bind Hfq after the resident RNA (Fig. 1A, in blue) has dissociated; i.e., the rate of binding of the incoming RNA is limited by the Hfq-RNA dissociation rate constant and is not affected by the concentration of the free RNA. In model B, free RNA transiently binds the Hfq-RNA complex, whereupon one of the RNAs eventually dissociates. Thus, the dissociation rate of the bound RNA is a function of the concentration of the free RNA (Fig. 1B). This would render cycling much more rapidly, and the intracellular pool of binder RNAs would rapidly equilibrate on Hfq. The two models are distinguishable, since th...

The human mitochondrion possesses a translational machinery devoted to the synthesis of 13 proteins. While the required tRNAs and rRNAs are produced by transcription of the mitochondrial genome, all other factors needed for protein synthesis are synthesized in the cytosol and imported. This is the case for aminoacyl-tRNA synthetases, the enzymes which esterify their cognate tRNA with the specific amino acid. The genes for the full set of cytosolic aaRSs are well defined, but only nine genes for mitochondrial synthetases are known. Here we describe the genes for human mitochondrial aspartyl- and tyrosyl-tRNA synthetases and the initial characterization of the enzymes. Both belong to the expected class of synthetases, have a dimeric organization, and aminoacylate Escherichia coli tRNAs as well as in vitro transcribed human mitochondrial tRNAs. Genes for the remaining missing synthetases were also found with the exception of glutaminyl-tRNA synthetase. Their sequence analysis confirms and further extends the view that, except for lysyl- and glycyl-tRNA synthetases, human mitochondrial and cytosolic enzymes are coded by two different sets of genes.

The formation of heterochromatin at the centromeres in fission yeast depends on transcription of the outer repeats. These transcripts are processed into siRNAs that target homologous loci for heterochromatin formation. Here, high throughput sequencing of small RNA provides a comprehensive analysis of centromere-derived small RNAs. We found that the centromeric small RNAs are Dcr1 dependent, carry 5 0 -monophosphates and are associated with Ago1. The majority of centromeric small RNAs originate from two remarkably wellconserved sequences that are present in all centromeres. The high degree of similarity suggests that this non-coding sequence in itself may be of importance. Consistent with this, secondary structure-probing experiments indicate that this centromeric RNA is partially double-stranded and is processed by Dicer in vitro. We further demonstrate the existence of small centromeric RNA in rdp1D cells. Our data suggest a pathway for siRNA generation that is distinct from the well-documented model involving RITS/RDRC. We propose that primary transcripts fold into hairpin-like structures that may be processed by Dcr1 into siRNAs, and that these siRNAs may initiate heterochromatin formation independent of RDRC activity.

Kaposi's sarcoma herpesvirus (KSHV) encodes a cluster of twelve micro (mi)RNAs, which are abundantly expressed during both latent and lytic infection. Previous studies reported that KSHV is able to inhibit apoptosis during latent infection; we thus tested the involvement of viral miRNAs in this process. We found that both HEK293 epithelial cells and DG75 cells stably expressing KSHV miRNAs were protected from apoptosis. Potential cellular targets that were significantly down-regulated upon KSHV miRNAs expression were identified by microarray profiling. Among them, we validated by luciferase reporter assays, quantitative PCR and western blotting caspase 3 (Casp3), a critical factor for the control of apoptosis. Using site-directed mutagenesis, we found that three KSHV miRNAs, miR-K12-1, 3 and 4-3p, were responsible for the targeting of Casp3. Specific inhibition of these miRNAs in KSHV-infected cells resulted in increased expression levels of endogenous Casp3 and enhanced apoptosis. Altogether, our results suggest that KSHV miRNAs directly participate in the previously reported inhibition of apoptosis by the virus, and are thus likely to play a role in KSHV-induced oncogenesis.