Tracing the origin of CRISPR-Cas CRISPR-Cas systems have transformed genome editing and other biotechnologies; however, the broader origins and diversity of RNA-guided nucleases have largely remained unexplored. Altae-Tran et al . show that three distinct transposon-encoded proteins, IscB, IsrB, and TnpB, are naturally occurring, reprogrammable RNA-guided DNA nucleases (see the Perspective by Rousset and Sorek). In addition to identifying diverse guide-encoding mechanisms, the authors elucidate the evolutionary relationship between IsrB, IscB, and CRISPR-Cas9. Overall, these newly characterized systems, called OMEGA (for obligate mobile element–guided activity) systems, are found in all domains of life and may be harnessed for biotechnology development. —DJ

The Actinobacillus actinomycetemcomitans cytolethal distending toxin (Cdt) is a potent immunotoxin that induces G2 arrest in human lymphocytes. We now show that the CdtB subunit exhibits phosphatidylinositol (PI)-3,4,5-triphosphate phosphatase activity. Breakdown product analysis indicates that CdtB hydrolyzes PI-3,4,5-P3 to PI-3,4-P2 and therefore functions in a manner similar to phosphatidylinositol 5-phosphatases. Conserved amino acids critical to catalysis in this family of enzymes were mutated in the cdtB gene. The mutant proteins exhibit reduced phosphatase activity along with decreased ability to induce G2 arrest. Consistent with this activity, Cdt induces time-dependent reduction of PI-3,4,5-P3 in Jurkat cells. Lymphoid cells with defects in SHIP1 and/or ptase and tensin homolog deleted on chromosome 10 (PTEN) (such as Jurkat, CEM, Molt) and, concomitantly, elevated PI-3,4,5-P3 levels were more sensitive to the toxin than HUT78 cells which contain functional levels of both enzymes and low levels of PI-3,4,5-P3. Finally, reduction of Jurkat cell PI-3,4,5-P3 synthesis using the PI3K inhibitors, wortmannin and LY290004, protects cells from toxin-induced cell cycle arrest. Collectively, these studies show that the CdtB not only exhibits PI-3,4,5-P3 phosphatase activity, but also that toxicity in lymphocytes is related to this activity.

Ssf1p and Ssf2p are two nearly identical and functionally redundant nucleolar proteins. In the absence of Ssf1p and Ssf2p, the 27SA(2) pre-rRNA was prematurely cleaved, inhibiting synthesis of the 27SB and 7S pre-rRNAs and the 5.8S and 25S rRNA components of the large ribosomal subunit. On sucrose gradients, Ssf1p sedimented with pre-60S ribosomal particles. The 27SA(2), 27SA(3), and 27SB pre-rRNAs were copurified with tagged Ssf1p, as were 23 large subunit ribosomal proteins and 21 other proteins implicated in ribosome biogenesis. These included four Brix family proteins, Ssf1p, Rpf1p, Rpf2p, and Brx1p, indicating that the entire family functions in ribosome synthesis. This complex is distinct from recently reported pre-60S complexes in RNA and protein composition. We describe a multistep pathway of 60S preribosome maturation.

We report the characterization of a novel factor, Nob1p (Yor056c), which is essential for the synthesis of 40S ribosome subunits. Genetic depletion of Nob1p strongly inhibits the processing of the 20S pre-rRNA to the mature 18S rRNA, leading to the accumulation of high levels of the 20S pre-rRNA together with novel degradation intermediates. 20S processing occurs within a pre-40S particle after its export from the nucleus to the cytoplasm. Consistent with a direct role in this cleavage, Nob1p was shown to be associated with the pre-40S particle and to be present in both the nucleus and the cytoplasm. This suggests that Nob1p accompanies the pre-40S ribosomes during nuclear export. Pre-40S export is not, however, inhibited by depletion of Nob1p.Eukaryotic ribosome synthesis takes place largely within the nucleolus, but the late steps in both pre-40S and pre-60S maturation occur after export of the subunits to the cytoplasm. In Saccharomyces cerevisiae, this cytoplasmic maturation includes an endonuclease cleavage at site D, the 3Ј end of the 18S rRNA that generates the mature rRNA from the 20S prerRNA ( Fig. 1) (27,37). Around 150 proteins and 80 small nucleolar RNAs are known to be required for the posttranscriptional steps in yeast ribosome synthesis. Despite the identification of this large number of factors, there are a great many gaps in our knowledge. Among these are the identities of the endonucleases that perform several of the pre-rRNA cleavages including that at the 3Ј end of the mature 18S rRNA (Fig. 1). The pathway of ribosome subunit synthesis in yeast was initially studied by sucrose gradient centrifugation (16,36,38), leading to the identification of a 90S particle that contains the 35S pre-rRNA, a 66S particle that contains the 27S prerRNAs, and a 43S particle containing the 20S pre-rRNA. Recent analyses have shown that the precursors to the 60S particle are substantially more complex than was initially proposed, with multiple pre-60S particles containing different complements of the precursors to the 25S and 5.8S rRNAs and different protein compositions (6, 13, 28, 31; reviewed in references 7 and 41). Equivalent analyses of the pre-40S ribosomes have not yet been reported, but a high-throughput proteomic analysis of yeast protein interactions identified three complexes that potentially represented such particles (8; reviewed in reference 7), one of which was associated with tandem affinity purification (TAP)-tagged Nob1p (Yor056c).Each of these complexes contained Tsr1p, which is required for 20S-to-18S processing (9), and one also contained the dimethylase Dim1p, which modifies the 20S pre-rRNA, probably in the cytoplasm, and is additionally required for nucleolar pre-rRNA processing (4,19,22). Rrp10p (Rio1p) is also required for 20S-to-18S processing (39) but was not identified in these putative pre-40S particles.Nob1p (for "Nin one binding protein") was initially reported to interact in a two-hybrid screen with Nin1p/Rpn12p, a subunit of the 19S regulatory particle of the yeast 26S proteosom...

The fission yeast centromeric repeats are transcribed and ultimately processed into small interfering RNAs (siRNAs) that are required for heterochromatin formation. siRNA generation requires dsRNA synthesis by the RNA-directed RNA polymerase complex (RDRC) and processing by the Dicer ribonuclease. Here we show that Dcr1, the fission yeast Dicer, is physically associated with RDRC. Dcr1 generates siRNAs in an ATP-dependent manner that requires its conserved N-terminal helicase domain. Furthermore, C-terminal truncations of Dcr1 that abolish its interaction with RDRC, but can generate siRNA in vitro, abolish siRNA generation and heterochromatic gene silencing in vivo. Finally, reconstitution experiments show that the association of Dcr1 with RDRC strongly stimulates the dsRNA synthesis activity of RDRC. Our results suggest that heterochromatic dsRNA synthesis and siRNA generation are physically coupled processes. This coupling has implications for cis-restriction of siRNA-mediated heterochromatin assembly and for mechanisms that give rise to siRNA strand polarity.