Synaptic neurotransmission protein UNC-13 affects RNA interference in neurons

Caenorhabditis elegans UNC-13 is an integral component of the synaptic vesicle cycle, functioning in the priming step. A recent yeast two-hybrid screen against UNC-13 identified three interacting proteins that are thought to function in pathways other than neurotransmitter release. One such protein, ERI-1, negatively regulates exogenous RNA interference in the nervous system and other tissues. This study investigates a role for UNC-13 in RNAi through analysis of RNAi penetrance in unc-13 and eri-1 mutant strains. Feeding these strains double stranded RNA corresponding to a neuronally expressed GFP reporter resulted in a significant reduction of GFP in double mutants compared to GFP expression in eri-1 mutants, indicating that UNC-13 functions in conjunction with ERI-1 in RNAi. There is no evidence for altered neurotransmission in eri-1 mutants.     

Genome-wide RNAi of C. elegans using the hypersensitive rrf-3 strain reveals novel gene functions

RNA-mediated interference (RNAi) is a method to inhibit gene function by introduction of double-stranded RNA (dsRNA). Recently, an RNAi library was constructed that consists of bacterial clones expressing dsRNA, corresponding to nearly 90% of the 19,427 predicted genes of C. elegans. Feeding of this RNAi library to the standard wild-type laboratory strain Bristol N2 detected phenotypes for approximately 10% of the corresponding genes. To increase the number of genes for which a loss-of-function phenotype can be detected, we undertook a genome-wide RNAi screen using the rrf-3 mutant strain, which we found to be hypersensitive to RNAi. Feeding of the RNAi library to rrf-3 mutants resulted in additional loss-of-function phenotypes for 393 genes, increasing the number of genes with a phenotype by 23%. These additional phenotypes are distributed over different phenotypic classes. We also studied interexperimental variability in RNAi results and found persistent levels of false negatives. In addition, we used the RNAi phenotypes obtained with the genome-wide screens to systematically clone seven existing genetic mutants with visible phenotypes. The genome-wide RNAi screen using rrf-3 significantly increased the functional data on the C. elegans genome. The resulting dataset will be valuable in conjunction with other functional genomics approaches, as well as in other model organisms.     

The ERI-6/7 helicase acts at the first stage of an siRNA amplification pathway that targets recent gene duplications

Endogenous small interfering RNAs (siRNAs) are a class of naturally occuring regulatory RNAs found in fungi, plants, and animals. Some endogenous siRNAs are required to silence transposons or function in chromosome segregation; however, the specific roles of most endogenous siRNAs are unclear. The helicase gene eri-6/7 was identified in the nematode Caenorhabditis elegans by the enhanced response to exogenous double-stranded RNAs (dsRNAs) of the null mutant. eri-6/7 encodes a helicase homologous to small RNA factors Armitage in Drosophila, SDE3 in Arabidopsis, and Mov10 in humans. Here we show that eri-6/7 mutations cause the loss of 26-nucleotide (nt) endogenous siRNAs derived from genes and pseudogenes in oocytes and embryos, as well as deficiencies in somatic 22-nucleotide secondary siRNAs corresponding to the same loci. About 80 genes are eri-6/7 targets that generate the embryonic endogenous siRNAs that silence the corresponding mRNAs. These 80 genes share extensive nucleotide sequence homology and are poorly conserved, suggesting a role for these endogenous siRNAs in silencing of and thereby directing the fate of recently acquired, duplicated genes. Unlike most endogenous siRNAs in C. elegans, eri-6/7-dependent siRNAs require Dicer. We identify that the eri-6/7-dependent siRNAs have a passenger strand that is ～19 nt and is inset by ～3-4 nts from both ends of the 26 nt guide siRNA, suggesting non-canonical Dicer processing. Mutations in the Argonaute ERGO-1, which associates with eri-6/7-dependent 26 nt siRNAs, cause passenger strand stabilization, indicating that ERGO-1 is required to separate the siRNA duplex, presumably through endonucleolytic cleavage of the passenger strand. Thus, like several other siRNA-associated Argonautes with a conserved RNaseH motif, ERGO-1 appears to be required for siRNA maturation.     

A Caenorhabditis elegans RNA-Directed RNA Polymerase in Sperm Development and Endogenous RNA Interference

Short interfering RNAs (siRNAs) are a class of regulatory effectors that enforce gene silencing through formation of RNA duplexes. Although progress has been made in identifying the capabilities of siRNAs in silencing foreign RNA and transposable elements, siRNA functions in endogenous gene regulation have remained mysterious. In certain organisms, siRNA biosynthesis involves novel enzymes that act as RNA-directed RNA polymerases (RdRPs). Here we analyze the function of a Caenorhabditis elegans RdRP, RRF-3, during spermatogenesis. We found that loss of RRF-3 function resulted in pleiotropic defects in sperm development and that sperm defects led to embryonic lethality. Notably, sperm nuclei in mutants of either rrf-3 or another component of the siRNA pathway, eri-1, were frequently surrounded by ectopic microtubule structures, with spindle abnormalities in a subset of the resulting embryos. Through high-throughput small RNA sequencing, we identified a population of cellular mRNAs from spermatogenic cells that appear to serve as templates for antisense siRNA synthesis. This set of genes includes the majority of genes known to have enriched expression during spermatogenesis, as well as many genes not previously known to be expressed during spermatogenesis. In a subset of these genes, we found that RRF-3 was required for effective siRNA accumulation. These and other data suggest a working model in which a major role of the RRF-3/ERI pathway is to generate siRNAs that set patterns of gene expression through feedback repression of a set of critical targets during spermatogenesis.REPRESSION of gene expression by small RNAs of ∼20–30 nt in length is important for many aspects of multicellular eukaryotic development. A variety of classes of small RNA with distinct structural features, modes of biogenesis, and biological functions have been identified (reviewed in Hutvagner and Simard 2008). We are particularly interested in a class of small RNAs, called endogenous short interfering RNAs (siRNAs), that are similar to intermediates in exogenously triggered RNA interference (RNAi) in their perfect complementarity to mRNA targets. High-throughput sequencing technology has provided a valuable tool for characterization of endogenous siRNA populations from many diverse sources, including mouse embryonic stem cells (Babiarz et al. 2008), Drosophila heads (Ghildiyal et al. 2008), and Arabidopsis pollen (Slotkin et al. 2009). These siRNAs have been proposed to function in the regulation of both cellular processes and genome defense through downregulation of gene expression. Caenorhabditis elegans, like plants and fungi, utilizes RNA-copying enzymes called RNA-directed RNA polymerases (RdRPs) as part of the RNAi machinery (Smardon et al. 2000; Sijen et al. 2001). While two of the C. elegans RdRPs are nonessential (RRF-1 and RRF-2), mutations in either of the remaining two (EGO-1 or RRF-3) lead to fertility defects (Smardon et al. 2000; Simmer et al. 2002). RRF-3 is functionally distinct from EGO-1 in that the RRF-3 requirement in fertility is temperature dependent. In addition, RRF-3 activity has an inhibitory effect on exogenously triggered RNAi (resulting in an ERI, or enhanced RNAi, mutant phenotype in rrf-3 mutants). Mutants lacking either RRF-3 or another ERI factor, ERI-1, have been used as experimental tools because of their enhanced sensitivity in RNAi-based screens. One proposed mechanism for the enhancement in RNAi in rrf-3 and eri mutants has been a competition for cofactors between the exogenously triggered RNAi pathway and an endogenous RNAi pathway. Consistent with this hypothesis, siRNAs corresponding to several genes have been shown by Northern analysis to depend upon RRF-3 and other ERI factors for their accumulation (Duchaine et al. 2006; Lee et al. 2006; Yigit et al. 2006). Global microarray analyses have also been undertaken to identify messenger RNAs whose expression is affected by RRF-3 and ERI-1 (Lee et al. 2006; Asikainen et al. 2007).A functional significance of the RRF-3/ERI pathway has been inferred by the inability of rrf-3, eri-1, eri-3, and eri-5 mutant strains to propagate at a high growth temperature (Simmer et al. 2002; Duchaine et al. 2006). Rather than producing temperature-sensitive mutant protein effects, RRF-3 and other ERI proteins are thought to act in a temperature-sensitive process, as evidenced by the predicted truncated and presumed nonfunctional protein fragments that would result from the available deletion alleles and by their shared temperature-sensitive phenotypes. rrf-3 mutant animals have been observed to exhibit X-chromosome missegregation (Simmer et al. 2002) and an unusual persistence of a chromatin mark on the X chromosome during male spermatogenesis (Maine et al. 2005). X-chromosome missegregation and defective spermatogenesis have been referred to in previous studies of eri-1 (Kennedy et al. 2004) and eri-3 and eri-5 (Duchaine et al. 2006). Furthermore, eri-3 mutant sterility can be rescued by insemination with wild-type sperm (Duchaine et al. 2006).Here we investigated the role of RRF-3 during spermatogenesis. We found defects evident at multiple stages, including after fertilization, where defects in rrf-3 mutant sperm can produce subsequent nonviable embryos. By using high-throughput sequencing, we characterized a large population of siRNAs present in spermatogenic cells and found a strong enrichment for antisense siRNAs from genes with known mRNA expression during spermatogenesis. While the majority of siRNA production during spermatogenesis does not require RRF-3, we found a set of genes for which siRNA production was dependent upon RRF-3. Existing data indicate increased expression for these genes in rrf-3 and/or eri-1 mutants. Taken together, our analyses suggest a working model in which the RRF-3/ERI pathway generates siRNAs that downregulate specific genes during spermatogenesis, with this regulation playing a key role in generating functional sperm.     

A riboprinting scheme for identification of unknown Acanthamoeba isolates at species level

We describe a riboprinting scheme for identification of unknown Acanthamoeba isolates at the species level. It involved the use of PCR-RFLP of small subunit ribosomal RNA gene (riboprint) of 24 reference strains by 4 kinds of restriction enzymes. Seven strains in morphological group I and III were identified at species level with their unique sizes of PCR product and riboprint type by Rsa I. Unique RFCP of 17 strains in group II by Dde I, Taq I and Hae III were classified into: (1) four taxa that were identifiable at the species level, (2) a subgroup of 4 taxa and a pair of 2 taxa that were identical with each other, and (3) a species complex of 7 taxa assigned to A. castellanii complex that were closely related. These results were consistent with those obtained by 18s rDNA sequence analysis. This approach provides an alternative to the rDNA sequencing for rapid identification of a new clinical isolate or a large number of environmental isolates of Acanthamoeba.