RNAi, a gene-silencing pathway triggered by double-stranded RNA, is conserved in diverse eukaryotic species but has been lost in the model budding yeast, Saccharomyces cerevisiae. Here, we show that RNAi is present in other budding-yeast species, including Saccharomyces castellii and Candida albicans. These species use noncanonical Dicer proteins to generate siRNAs, which mostly correspond to transposable elements and Y’ subtelomeric repeats. In S. castellii, RNAi mutants are viable but have excess Y’ mRNA levels. In S. cerevisiae, introducing Dicer and Argonaute of S. castellii restores RNAi, and the reconstituted pathway silences endogenous retrotransposons. These results identify a novel class of Dicer proteins, bring the tool of RNAi to the study of budding yeasts, and bring the tools of budding yeast to the study of RNAi.

Live cell imaging is a powerful method for studying protein dynamics at the cell surface, but conventional probes, such as antibodies and fluorescent ligands, are bulky, interfere with protein function 1,2 , or dissociate after internalization 3,4 . To overcome these limitations, we developed a method to covalently tag any cell surface protein with any chemical probe with remarkable specificity. Through rational design, we re-directed a microbial lipoic acid ligase (LplA) 5 to specifically ligate an alkyl azide to an engineered LplA acceptor peptide (LAP) tag. The alkyl azide is then selectively derivatized with a cyclooctyne 6 conjugated to any probe of interest. We demonstrate the utility of this method by first labeling LAP fusion proteins expressed on the surface of living mammalian cells with Cy3, Alexa Fluor 568, and biotin. Next, we combined LAP-tagging with our previously reported tagging method 7,8 to simultaneously monitor the dynamics of two receptors, co-expressed in the same cell, with different fluorophores. Using a wound-healing assay, we found that while the LDL receptor maintains a uniform distribution on the cell surface, the ephrin receptor EphA3 is polarized to the leading edge. Our methodology should provide general access to biochemical and imaging studies of cell surface proteins, using small fluorophores introduced via a short peptide tag.Fluorescent labeling of cell surface proteins enables imaging of the trafficking and function of receptors, channels, and transporters. Many protein labeling methods have been developed in recent years 9 , but none currently allows the covalent attachment of small fluorophores of any structure onto cell surface proteins modified only by a small peptide tag, with short labeling times and with extremely high specificity over a wide range of expression levels and labeling conditions. To address this shortcoming, we developed a new protein labeling method based on the E. coli enzyme lipoic acid ligase (LplA) 5 . In E. coli, LplA catalyzes the ATP-dependent covalent ligation of lipoic acid to one of three proteins involved in oxidative metabolism (E2p, E2o, and H-protein 5 ) (Fig. 1a, top). LplA naturally exhibits extremely high sequence specificity, but previous work showing that the enzyme accepts octanoic acid, 6-thio-octanoic acid, and selenolipoic acid in place of lipoic acid 5 suggest that the small-molecule binding site has considerable plasticity. To harness LplA for fluorescent labeling, we re-engineered the system in three stages. First, through synthesis and testing of ten different substrate analogs, we discovered an alkyl azide substrate that can be efficiently used by LplA in place of lipoic acid. Once ligated to the target protein, the azide functional group can be selectively derivatized with any fluorescent probe conjugated to a cyclooctyne reaction partner 6 (Figure 1a). Second, to create a minimally invasive tag to direct the ligation of the alkyl azide, we engineered, through iterative cycles of rational design, a 22-amino acid replacement...

Successful ex vivo expansion of hematopoietic stem cells (HSCs) would greatly benefit the treatment of disease and the understanding of crucial questions of stem cell biology. Here we show, using microarray studies, that the HSC-supportive mouse fetal liver CD3 + cells specifically express the proteins angiopoietin-like 2 (Angptl2) and angiopoietin-like 3 (Angptl3). We observed a 24-or 30-fold net expansion of long-term HSCs by reconstitution analysis when we cultured highly enriched HSCs for 10 days in the presence of Angptl2 or Angptl3 together with saturating levels of other growth factors. The coiled-coil domain of Angptl2 was capable of stimulating expansion of HSCs. Furthermore, angiopoietin-like 5, angiopoietin-like 7 and microfibril-associated glycoprotein 4 also supported expansion of HSCs in culture.HSCs, defined by their ability to self-renew and to differentiate into all blood cell types, form the basis of bone marrow transplantation for treatment of cancers and hematopoietic disorders 1 , and are also a promising cell target for gene therapies that can potentially treat a broad variety of human diseases 2 . Development of these important clinical applications of HSCs is greatly hampered by the lack of understanding of the extracellular and intracellular signals that govern their fates as well as by the difficulty in carrying out ex vivo expansion of these cells.Numerous attempts have been made to increase the number of long-term HSCs in culture 3,4 . The use of stromal cell lines or combinations of cytokines have resulted in considerable selfrenewal of mouse HSCs assayed 4-6 weeks after transplant, and have led to as much as a sixfold increase in mouse long-term HSC activity in culture [5][6][7][8][9] . The introduction of exogenous transcription factors can expand HSCs more substantially [10][11][12] 13 . We subsequently developed a simple culture system using low but saturating levels of stem cell factor (SCF), thrombopoietin (TPO), IGF-2 and fibroblast growth factor 1 (FGF-1) in serum-free medium. As measured by competitive repopulation analyses, there was a greater than eightfold increase in numbers of long-term repopulating HSCs (LT-HSCs) after 10 d of culture of highly enriched bone marrow HSCs 14 .Here we further analyzed gene expression of E15 mouse liver CD3 + cells using Affymetrix U74Bv2 and U74Cv2 mouse microarrays, and identified the proteins angiopoietin-like 2 (Angptl2) and angiopoietin-like 3 (Angptl3) as well as several other members of the family of angiopoietin-like proteins as potent stimulators of ex vivo expansion of HSCs. RESULTS Fetal liver CD3 + cells specifically express Angptl2 and Angptl3In Supplementary Fig. 1 online). These proteins are also expressed in adult bone marrow cells, including the side population (SP) CD45 + Sca-1 + highly enriched HSC population 17 ( Supplementary Fig. 1 online). We therefore hypothesized that these two proteins, not previously thought to be involved in HSC biology, might support expansion of HSCs. Angptl2 and Angptl3 stimulate ex ...