microRNAs (miRNAs) are single-stranded, 21- to 23-nucleotide cellular RNAs that control the expression of cognate target genes. Primary miRNA (pri-miRNA) transcripts are transformed to mature miRNA by the successive actions of two RNase III endonucleases. Drosha converts pri-miRNA transcripts to precursor miRNA (pre-miRNA); Dicer, in turn, converts pre-miRNA to mature miRNA. Here, we show that normal processing of Drosophila pre-miRNAs by Dicer-1 requires the double-stranded RNA-binding domain (dsRBD) protein Loquacious (Loqs), a homolog of human TRBP, a protein first identified as binding the HIV trans-activator RNA (TAR). Efficient miRNA-directed silencing of a reporter transgene, complete repression of white by a dsRNA trigger, and silencing of the endogenous Stellate locus by Suppressor of Stellate, all require Loqs. In loqs f00791 mutant ovaries, germ-line stem cells are not appropriately maintained. Loqs associates with Dcr-1, the Drosophila RNase III enzyme that processes pre-miRNA into mature miRNA. Thus, every known Drosophila RNase-III endonuclease is paired with a dsRBD protein that facilitates its function in small RNA biogenesis.

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide distinct classes of RNA-induced silencing complexes (RISCs) to repress mRNA expression in biological processes ranging from development to antiviral defense. In Drosophila, separate but conceptually similar endonucleolytic pathways produce siRNAs and miRNAs. Here, we show that despite their distinct biogenesis, double-stranded miRNAs and siRNAs participate in a common sorting step that partitions them into Ago1- or Ago2-containing effector complexes. These distinct complexes silence their target RNAs by different mechanisms. miRNA-loaded Ago2-RISC mediates RNAi, but only Ago1 is able to repress an mRNA with central mismatches in its miRNA-binding sites. Conversely, Ago1 cannot mediate RNAi, because it is an inefficient nuclease whose catalytic rate is limited by the dissociation of its reaction products. Thus, the two members of the Drosophila Ago subclade of Argonaute proteins are functionally specialized, but specific small RNA classes are not restricted to associate with Ago1 or Ago2.

Small RNAs have been implicated in numerous cellular processes, including effects on chromatin structure and the repression of transposons. We describe the generation of a small RNA response at DNA ends in Drosophila that is analogous to the recently reported double-strand break (DSB)-induced RNAs or Dicer- and Drosha-dependent small RNAs in Arabidopsis and vertebrates. Active transcription in the vicinity of the break amplifies this small RNA response, demonstrating that the normal messenger RNA contributes to the endogenous small interfering RNAs precursor. The double-stranded RNA precursor forms with an antisense transcript that initiates at the DNA break. Breaks are thus sites of transcription initiation, a novel aspect of the cellular DSB response. This response is specific to a double-strand break since nicked DNA structures do not trigger small RNA production. The small RNAs are generated independently of the exact end structure (blunt, 3′- or 5′-overhang), can repress homologous sequences in trans and may therefore—in addition to putative roles in repair—exert a quality control function by clearing potentially truncated messages from genes in the vicinity of the break.

Colonization of genomes by a new selfish genetic element is detrimental to the host species and must lead to an efficient, repressive response. In vertebrates as well as in Drosophila, piRNAs repress transposons in the germ line, whereas endogenous siRNAs take on this role in somatic cells. We show that their biogenesis depends on a new isoform of the Drosophila TRBP homologue loquacious, which arises by alternative polyadenylation and is distinct from the one that functions during the biogenesis of miRNAs. For endo-siRNAs and piRNAs, it is unclear how an efficient response can be initiated de novo. Our experiments establish that the endo-siRNA pathway will target artificially introduced sequences without the need for a pre-existing template in the genome. This response is also triggered in transiently transfected cells, thus genomic integration is not essential. Deep sequencing showed that corresponding endo-siRNAs are generated throughout the sequence, but preferentially from transcribed regions. One strand of the dsRNA precursor can come from spliced mRNA, whereas the opposite strand derives from independent transcripts in antisense orientation.

Yeast telomeres consist of approximately 300 nt of degenerate repeats with the consensus sequence G(2-3)(TG)(1-6). We developed a method for the amplification of a genetically marked telomere by PCR, allowing precise length and sequence determination of the G-rich strand including the 3' terminus. We examined wild-type cells, telomerase RNA deficient cells and a strain deleted for YKU70, which encodes for a protein involved in telomere maintenance and DNA double strand break repair. The 3' end of the G-rich strand was found to be at a variable position within the telomeric repeat. No preference for either thymine or guanine as the 3' base was detected. Comparison of telomere sequences from clonal populations revealed that telomeres consist of a centromere-proximal region of stable sequence and a distal region with differing degenerate repeats. In wild-type as well as yku70-Delta cells, variation in the degenerate telomeric repeats was detected starting 40-100 nt from the 3' end. Sequence divergence was abolished after deletion of the telomerase RNA gene. Thus, this region defines the domain where telomere shortening and telomerase-mediated extension occurs. Since this domain is much larger than the number of nucleo-tides lost per generation in the absence of telomerase, we propose that telomerase does not extend a given telomere in every cell cycle.

The ability to edit the genome is essential for many state-of-the-art experimental paradigms. Since DNA breaks stimulate repair, they can be exploited to target site-specific integration. The clustered, regularly interspaced, short palindromic repeats (CRISPR)/cas9 system from Streptococcus pyogenes has been harnessed into an efficient and programmable nuclease for eukaryotic cells. We thus combined DNA cleavage by cas9, the generation of homologous recombination donors by polymerase chain reaction (PCR) and transient depletion of the non-homologous end joining factor lig4. Using cultured Drosophila melanogaster S2-cells and the phosphoglycerate kinase gene as a model, we reached targeted integration frequencies of up to 50% in drug-selected cell populations. Homology arms as short as 29 nt appended to the PCR primer resulted in detectable integration, slightly longer extensions are beneficial. We confirmed established rules for S. pyogenes cas9 sgRNA design and demonstrate that the complementarity region allows length variation and 5′-extensions. This enables generation of U6-promoter fusion templates by overlap-extension PCR with a standardized protocol. We present a series of PCR template vectors for C-terminal protein tagging and clonal Drosophila S2 cell lines with stable expression of a myc-tagged cas9 protein. The system can be used for epitope tagging or reporter gene knock-ins in an experimental setup that can in principle be fully automated.

No abstract

Telomerase is a ribonucleoprotein enzyme that adds repetitive sequences to the ends of linear chromosomes, thereby counteracting nucleotide loss due to incomplete replication. A short region of the telomerase RNA subunit serves as template for nucleotide addition onto the telomere 3 end. Although Saccharomyces cerevisiae contains only one telomerase RNA gene, telomere repeat sequences are degenerate in this organism. Based on a detailed analysis of the telomere sequences specified by wild-type and mutant RNA templates in vivo, we show that the divergence of telomere repeats is due to abortive reverse transcription in the 3 and 5 regions of the template and due to the alignment of telomeres in multiple registers within the RNA template. Through the interpretation of wild-type telomere sequences, we identify nucleotides in the template that are not accessible for base pairing during substrate annealing. Rather, these positions become available as templates for reverse transcription only after alignment with adjacent nucleotides has occurred, indicating that a conformational change takes place upon substrate binding. We also infer that the central part of the template region is reverse transcribed processively. The inaccessibility of certain template positions for alignment and the processive polymerization of the central template portion may serve to reduce the possible repeat diversification and enhance the incorporation of binding sites for Rap1p, the telomere binding protein of budding yeast.Telomeres protect the ends of the linear eukaryotic chromosomes from end-to-end fusions and serve as buffer zones against sequence loss due to incomplete replication (1). They are maintained by the ribonucleoprotein enzyme telomerase, a cellular reverse transcriptase that uses a specific region of its RNA subunit as template for DNA synthesis (16,17,32,58). The template region of the RNA is copied repeatedly onto the 3Ј ends of the chromosomes, thus specifying the telomere repeats. The ability to maintain telomeres is a prerequisite to undergo unlimited rounds of replication (33), and reactivation of telomerase is seen in more than 80% of human tumors (23). In the yeast Saccharomyces cerevisiae, the RNA subunit of telomerase is encoded by TLC1 (49) and the catalytic protein subunit is encoded by EST2 (8, 32). EST1 and EST3 encode other telomerase-associated proteins essential for telomere maintenance in vivo but dispensable for telomerase activity in vitro (4,22,28,31,33,45).Several organisms, including some protozoa, fungi, slime molds, and plants, have irregular telomere repeat sequences (56). The degeneracy is most pronounced in S. cerevisiae and Schizosaccharomyces pombe with the telomere consensus sequences (TG) 1-4 G 2-3 (5, 48, 52, 54) and GGTTACA(G) [1][2][3][4]