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I am trying to silence the tph-1 (tryptophan hydroxylase) gene in C. elegans using the pLT63 plasmid to check if that particular gene has anything to do with the pharyneal pumping or not. Am I using the right plasmid for the purpose?
"pLT63 contains 0.8 kb of the fem-1 gene inserted into the L4440 vector" (1). The gene fem-1 is involved in determining the male sexual trait in C. elegans (you can verify this on uniprot). It may be best, however, to stick to a "normal" plasmid like L4440 that'll just have your tph-1 insert ligated in.
Meiotic silencing in Caenorhabditis elegans
In many animals and some fungi, mechanisms have been described that target unpaired chromosomes and chromosomal regions for silencing during meiotic prophase. These phenomena, collectively called "meiotic silencing," target sex chromosomes in the heterogametic sex, for example, the X chromosome in male nematodes and the XY-body in male mice, and also target any other chromosomes that fail to synapse due to mutation or chromosomal rearrangement. Meiotic silencing phenomena are hypothesized to maintain genome integrity and perhaps function in setting up epigenetic control of embryogenesis. This review focuses on meiotic silencing in the nematode, Caenorhabditis elegans, including its mechanism and function(s), and its relationship to other gene silencing processes in the germ line. One hallmark of meiotic silencing in C. elegans is that unpaired/unsynapsed chromosomes and chromosomal regions become enriched for a repressive histone modification, dimethylation of histone H3 on lysine 9 (H3K9me2). Accumulation and proper targeting of H3K9me2 rely on activity of an siRNA pathway, suggesting that histone methyltransferase activity may be targeted/regulated by a small RNA-based transcriptional silencing mechanism.
Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans
Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene 1 , 2 . Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression 3 , 4 . Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny. Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process.
Clk-2 is required for normal developmental and behavioral rates
The gene clk-2 is defined by the recessive temperature-sensitive allele qm37. The phenotypes displayed by the clk-2(qm37) mutants are fully penetrant at both the permissive and the restrictive temperatures. We examined in detail the rates of development and of rhythmic behaviors of clk-2(qm37) mutants grown at 20°C. The average rates of embryonic and post-embryonic development, as well as those of the pumping, defecation and egg-laying behaviors, are dramatically slower in qm37 than in the wild type (Table 1). In addition, the self-brood size is reduced, but embryonic viability is similar to the wild type (98.4% of the embryos produced are viable and develop into fertile adults).
The developmental and behavioral phenotypes are fully rescued in homozygous mutants derived from a heterozygous mother (Table 1). However, the reproductive phenotypes are only partially rescued (Table 1). A simple interpretation of these results is that the clk-2(+) activity provided by the mother is sufficient to rescue events that occur relatively early in the life cycle and/or that require relatively small amounts of CLK-2. However, other events such as egg production might have a higher requirement and cannot be rescued by the amounts provided by the mother. Alternatively, the observed maternal effect could be based on an epigenetic setting that is erased in the germline. This interpretation is supported by the extent of the rescue considering that the animal grows more than 500-fold in volume during post-embryonic development. The dilution of any maternal product would be extreme, yet most of the somatic adult features are fully rescued.
Clk-2 is required for embryonic development
Although clk-2(qm37) embryos all develop and grow up to become long-lived adults at permissive temperatures (15-20°C), clk-2(qm37) leads to embryonic lethality at 25°C. qm37 hermaphrodites grown at a permissive temperature and transferred to 25°C before egg laying begins, produce progeny that all die during embryogenesis. Although qm37 embryos appear normal from the one-cell stage to the beginning of gastrulation, they all arrest development later on in embryogenesis (Fig. 1). Embryonic arrests occur at various stages of development, ranging from arrests of disorganized gastrulating embryos of ∼100 cells to arrests of deformed embryos at the threefold stage with differentiated tissues such as muscle, pharynx, gut and neurons. The lethal embryonic defect is irreversible, as embryos that are downshifted to 20°C at various stages of embryogenesis still die as embryos.
Clk-2 acts very early in development
To establish how early clk-2 acts in development, we dissected embryos at the two- to four-cell stage from wild-type and mutant hermaphrodites, grown at either permissive or non-permissive temperature, transferred the embryos to the other temperature and examined whether they could complete development (Table 2). When development proceeds up to the two- to four-cell stages at the permissive temperature (20°C), almost all qm37 embryos carry out further embryonic and post-embryonic development at 20 or 25°C. By contrast, when qm37 embryos develop at 25°C up to the two- to four-cell stage and are then transferred to 20°C, very few hatch and succeed in completing development. Thus, clk-2 is required for embryonic viability before the two- to four-cell stages.
In addition, we examined the number of embryos present in the uteri of qm37 hermaphrodites kept at 25°C for 26 hours after adulthood. In these mutant hermaphrodites, there is an average of 9.9 embryos (which we identify by their eggshell) per worm (n=125 worms, three independent experiments). We compared this figure with the total number of dead embryos produced by mutant hermaphrodites, similarly kept at 25°C for 26 hours after adulthood, but then shifted to the permissive temperature. These animals produce an average of 10.7 dead embryos per hermaphrodite before producing only live eggs (n=133 worms, three independent experiments). These observations indicate that after transfer away from the lethal temperature only one embryo dies, on average, in addition to those that have already formed an eggshell. This suggests that clk-2(qm37) produces irreversible damage, leading to subsequent lethality, in a narrow window between the very end of oogenesis and the initiation of embryonic development. This corresponds to the time at which oocyte maturation, fertilization, completion of meiosis, pronuclear formation and eggshell formation occur.
We examined late oogenesis and early embryonic development using DIC microscopy and could not detect any obvious abnormality in the events that precede or follow fertilization (data not shown). Early embryos appear invariably normal and healthy, with cells and nuclei of normal size and shape (Fig. 1E,F). Two polar bodies are extruded upon completion of oocyte meiosis in early qm37 embryos (Fig. 1E). We also visualized chromosomes in early DAPI stained embryos and could not detect abnormal patterns of chromosome morphology or segregation, or any other defects.
Clk-2 is not required for gonad and germline development
clk-2(qm37) hermaphrodites shifted to 25°C during post-embryonic development produce reduced broods of dead embryos. The reduced brood size and the death of the embryos at 25°C does not result from a failure of the gonad or germline to develop properly, because it is fully reversible. Indeed, despite developing entirely at 25°C, the gonad and germline of these qm37 mutants is functional after downshift to the permissive temperature as they resume production of numerous live embryos. This also indicates that sperm and oocytes that have been produced at 25°C are functional after downshift. Similarly, the development and the function of male gametes do not appear to be affected in qm37 mutants: homozygous males that develop from the early L4 stage at 25°C can sire abundant progeny as adults when mated to wild-type hermaphrodites at 25°C.
Examination by DIC microscopy of the gonads and germlines of qm37 adult hermaphrodites transferred to 25°C at different stages during their larval development, and of adults that have spent ∼25 hours as adults at 25°C, revealed no obvious morphological or cellular defects. The gonads have normal shape and size, containing numerous mitotic and meiotic germ nuclei and abundant sperm of normal appearance. The oocytes develop normally and display size increase and nucleus enlargement as well as nucleolus disappearance and asymmetric nucleus location in late oogenesis (McCarter et al., 1999). Also, the DAPI stained chromosomes of the mitotic and meiotic nuclei and of the oocytes have normal morphologies.
Thus, the mutant hermaphrodites produce reduced broods in spite of containing numerous functional gametes. One possibility is that an abnormal function of the somatic gonad of qm37 mutants at the restrictive temperature contributes to the small brood size. In fact, most somatic structures of clk-2 mutants appear affected by the non-permissive temperature, as mutant hermaphrodites that are transferred to 25°C as adults take on a sick appearance, move sluggishly, and their egg-laying rate eventually drops to zero before they have exhausted all their sperm.
Clk-2(qm37) displays maternal effects at the restrictive temperature
We examined whether the phenotypes of the qm37 mutants could be maternally rescued at 25°C. All progeny produced at 25°C from heterozygous mothers raised at 25°C are alive and develop into phenotypically wild-type adults. These maternally rescued adults appear undistinguishable from the wild type, but produce only a small brood of dead embryos. Thus, the maternal-rescue effect at 25°C is complete for development and behavior, as it is at 20°C, but fails to rescue fertility.
The maternal contribution of clk-2 to development is further revealed by the observation that the lethality of embryos produced at 25°C is strictly dependent on the maternal genotype. When clk-2 hermaphrodites are mated with wild-type males at 25°C they nonetheless produce only dead embryos. When transferred to the permissive temperature at various times after mating, these hermaphrodites produce live progeny, including ∼50% live males, indicating that the mating was successful. Thus, the presence of a wild-type allele in the embryo is insufficient for normal embryonic development to occur if the embryo is produced in a homozygous mutant mother. This strict maternal-effect lethality suggests a very early focus of action for clk-2, before activation of the zygotic genome, which is consistent with our finding of a requirement for clk-2(+) before the two- to four-cell stage (see above).
Clk-2 encodes a protein homologous to Saccharomyces cerevisiae Tel2p
clk-2 was previously mapped to LGIII between sma-4 and mab-5 (Hekimi et al., 1995). We refined this position using additional genetic markers and found clk-2 to be inseparable from lin-39 (Fig. 2A). Cosmids from the corresponding genomic region were injected into clk-2(qm37) animals to assay for rescue of the mutant phenotypes (Fig. 2B). A 3.6 kb region of the rescuing cosmid C07H6 (Fig. 2C) is sufficient to rescue the developmental and behavioral phenotypes at 20°C, as well as the lethality at 25°C.
To characterize the gene structure of clk-2, the cDNA clone yk447b4, mapped to this region by Dr Y. Kohara, was sequenced (Accession Number, AF400665) and found that it differs from the Genefinder prediction (Fig. 2D). Six bases at the 5′ end of yk447b4 correspond to transpliced sequence from SL2. RT-PCR experiments yield a single band of the expected size when amplifying with a primer internal to clk-2 and with an SL2-, but not with a SL1-specific primer (not shown), indicating that a single SL2-transpliced clk-2 transcript is produced in vivo. A single band of the expected size is detected by northern analysis. The cDNA clone yk215f6, provided by Dr Y. Kohara, which corresponds to the gene immediately upstream of clk-2, which we call cux-7 (upstream in clk-2 operon and homologous to the human gene XE7 Accession Number, I54325), was also sequenced (Accession Number, AF400666). We also amplified the ends of cux-7 in RT-PCR experiments. The 5′ end is amplified with SL1- but not with SL2-specific primers (not shown), indicating that cux-7 is SL1-transpliced. Given their proximity and transplicing pattern, clk-2 and cux-7 appear to be in an operon (Spieth et al., 1993 Zorio et al., 1994).
clk-2 encodes a predicted protein of 877 amino acids. Database searches for sequences homologous to C. elegans CLK-2 revealed that it is similar to Saccharomyces cerevisiae Tel2p, as well as to predicted proteins in vertebrates and plants (Fig. 3). S. cerevisiae TEL2 is an essential gene required for telomere length regulation (Runge and Zakian, 1996). After sporulation of a diploid heterozygote, tel2 knockout cells divide no more than two or three times and arrest with an abnormal cellular morphology. A missense temperature-sensitive mutation (tel2-1), however, leads to slow growth and shortened telomeres. Furthermore, Tel2p has been found to bind single-stranded and double-stranded yeast telomeric repeats in vitro, as well as RNA (Kota and Runge, 1998 Kota and Runge, 1999). The existence of homologs of Tel2p in multicellular organisms has not been reported before, presumably because only alignments with multiple sequences are capable of revealing their relatively weak similarity (Fig. 3). Extensive searches have uncovered only one protein of the Tel2p/CLK-2 family in every eukaryote for which a complete or almost complete genome sequence is available, suggesting that these genes are orthologs. Functional conservation between yeast and worm has also been observed in the case of mrt-2/rad1 + /RAD17, which affect telomere biology, in spite of their low sequence similarity (Ahmed and Hodgkin, 2000).
Qm37 is a partial loss-of-function allele at 20°c
In order to further confirm the identity of the gene and determine the null phenotype of clk-2, we depleted clk-2 transcripts by RNA interference (Fire et al., 1998). While injection of wild-type worms with cux-7 dsRNA produced no phenotype, injection of clk-2 dsRNA elicited a robust phenocopy of the clk-2 phenotypes in the F1 progeny of injected hermaphrodites. At first, all wild-type and clk-2(qm37) animals injected with clk-2 dsRNA produce embryos that hatch and develop into slow growing larvae that become slow behaving and sterile adults. Approximately 24 hours after dsRNA injection, the injected animals lay only dead embryos at all temperatures. These results indicate that clk-2 is required for embryonic development at all temperatures and that qm37 is a partial loss-of-function mutation that, at 25°C, displays a much stronger loss-of-function phenotype, which might be the null phenotype. Sequencing of the clk-2 genomic region in qm37 reveals the mutation to be a G to A transition at base 32069 of C07H6 (Fig. 2C), resulting in a cysteine to tyrosine substitution at residue 772 of the predicted protein.
The level of the CLK-2 protein is similar at all temperatures (Fig. 4C), in both the wild type and in qm37 mutants. The qm37 mutation greatly reduces the level of CLK-2 at all temperatures (Fig. 4C). This indicates that the stronger phenotype of qm37 at 25°C is not caused by increased instability of the mutant CLK-2 protein at this temperature. It is unclear therefore, whether CLK-2 is required at a high level at 25°C or whether the mutant protein is less functional at 25°C in a way that does not affect stability.
Abundant clk-2 transcript is stored in the hermaphrodite germline
We examined the abundance of the clk-2 transcript throughout development in wild-type worm populations at different developmental stages, by northern analysis (see Materials and Methods). While a modest level of the clk-2 transcript is detected from the embryonic stage through the larval stages, the highest level is detected in young adults (Fig. 4A). Wild-type young adults contain a large germline with mitotic and meiotic germ nuclei, sperm, and developing oocytes. Given the maternal contribution of clk-2 to development and the high transcript level in young adults, we investigated the clk-2 transcript levels in the germline by comparing wild-type young adults with adults carrying mutations that affect the germline. The clk-2 transcript level is highly reduced in glp-4(bn2ts) mutants, which fail to develop a germline altogether at 25°C (Beanan and Strome, 1992), indicating that most of the clk-2 mRNA in young adults is in the germline. However, the clk-2 mRNA level is low in mid-L4 larvae compared with young adults, even though these larvae possess an already large germline. While the L4 germline contains numerous mitotic and early meiotic germ nuclei, it contains few sperm and lacks oocytes. We interpret this result to indicate that most of the clk-2 mRNA in wild-type young adults is likely to be localized to oocytes.
That clk-2 mRNA is abundant in the oocytes is consistent with our finding that the level of clk-2 transcript is high in fem-2(b245ts) mutants that make only oocytes at 25°C (Fig. 4B). clk-2 mRNA appears even more abundant in fem-2(b245ts) than in wild-type young adults, which is probably due to the abnormal accumulation of oocytes in these mutants. We also find that the level of clk-2 transcripts in fem-3(q20ts) adults that make only sperm at 25°C is no different from that of wild-type young adults (Fig. 4B), indicating that sperm also contains clk-2 mRNA. Note that the similarity of the levels of clk-2 transcript in fem-3(q20ts) mutants and the wild type does not indicate that in young adults much of the clk-2 mRNA comes from sperm, as these mutants contain a quantity of sperm that largely exceeds the amount normally present in wild-type adult hermaphrodites. Taken together, these results indicate that gametes, in particular oocytes, accumulate high levels of clk-2 mRNA, presumably as a store to be used by the embryo, which is consistent with the maternal-rescue effect. However, as no paternal rescue is observed with clk-2 mutants, the amount present in sperm appears insufficient to the requirement of the developing embryo.
We further characterized the expression pattern of clk-2 by determining the levels of protein throughout development and in germline mutants by western analysis (see Materials and Methods). Surprisingly, the content of CLK-2 protein is similar through all developmental stages including in young adults (Fig. 4A). In addition, the concentration of CLK-2 in glp-4(bn2ts), fem-3(q20ts) and fem-2(b245ts) mutants is no different from the wild type (Fig. 4B). These observations suggest that the transcript levels differences are mostly due to stores of presumably untranslated transcripts in the germline. However, the constant levels of CLK-2 protein throughout development are consistent with a continual requirement for CLK-2 in somatic tissues, as reflected by the temperature-sensitive period that extends throughout life (see above).
A functional CLK-2::GFP fusion is cytoplasmic
We constructed transcriptional and translational fusions of clk-2 with the gene that encodes green fluorescent protein, and examined transgenic worms carrying these reporter genes (see Materials and Methods). Expression from the transgene is ubiquitous in somatic tissues including hypodermis, muscles, neurons, pharynx, gut, excretory canal, somatic gonad, vulva and presumably all cells (Fig. 5 data not shown). However, even using complex arrays to help prevent transgene silencing in the germline (Kelly et al., 1997), no CLK-2::GFP expression could be detected in the germline.
A full-length CLK-2::GFP fusion protein that complements the mutant phenotype for development, behavior and viability at 25°C, is localized virtually exclusively to the cytoplasm (Fig. 5), which is consistent with the absence of an obvious nuclear localization signal in the predicted protein. The pattern we observe is unlikely to be a consequence of overexpression as we have used very small transgene concentrations in complex arrays (Kelly et al., 1997). However, although the nucleus appears dark in the fluorescent images, we cannot exclude the possibility that it contains very small amounts of the fusion protein. Whether the CLK-2::GFP fusion exactly reflects the distribution of native CLK-2 will need to be addressed in further studies.
Clk-2 is required for the regulation of telomere length in C. elegans
We examined the length of telomeres in clk-2(qm37) worms raised for numerous generations at 20°C and 25°C by Southern blotting (see Materials and Methods). In C. elegans, tracks of numerous TTAGGC telomeric repeats are present at the ends of the six chromosomes (Wicky et al., 1996). In addition, numerous interstitial blocks of perfect and degenerate telomeric repeats are located more internally to the chromosomes (Riddle, 1997). Analysis of genomic DNA after restriction digestion with a frequent cutter that does not cleave within the telomeric repeats (HinfI), electrophoresis, and hybridization to telomeric probes, reveals the telomere-carrying end fragments of the chromosomes (Wicky et al., 1996). Telomeres, and thus the restriction fragments containing them, are heterogeneous in size and appear as smears. However, restriction fragments carrying tracts of internal telomeric repeats are of fixed size and appear as discrete bands in the 0.5-3 kb range (Ahmed and Hodgkin, 2000 Wicky et al., 1996).
In qm37 mutants, telomeres are up to two times longer on average than in the wild type (Fig. 6A-C). We detect the smear corresponding to the terminal telomeric fragments in the range of 2.5-6 kb in clk-2(qm37), which in the wild-type animals is in the range of 2-4 kb (Fig. 6A-C). The increased telomeric length of qm37 mutants is a stable feature of the phenotype that we have repeatedly observed in more than four independent worm cultures for each genotype, at a variety of temperatures, and in at least two independent DNA preparations and analyses for each of the worm cultures. Lengthening of telomeric repeats in qm37 mutants appears to occur only at the terminal telomeric fragments and not at internal sites, as the bands that correspond to them are at identical positions in clk-2(qm37) and the wild type (Fig. 6A-C).
We analyzed the length of terminal telomeric fragments in the animals of the strain MQ691, which carries an extrachromosomal array containing functional wild-type CLK-2 that rescues development and behavior at 25°C in a clk-2(qm37) chromosomal background. In these animals, the length of terminal telomeric fragments appear very similar to the wild type, and even slightly shorter, indicating that the lengthened telomere phenotype of qm37 mutants is rescued by the expression of clk-2(+) (Fig. 6). We further examined the telomere length of non-transgenic animals of the strain MQ931, derived from MQ691, which have lost the extrachromosomal array and thus again lack clk-2(+). The terminal telomeric repeats in this strain are long again (data not shown). Thus, the lengthened telomere phenotype of clk-2(qm37) can be rescued by clk-2(+) and reverses back to mutant length after the loss of the transgene.
The quality of visualization of the length of telomeres in C. elegans with a hybridization probe that detects telomeric repeats is marred by the numerous internal repeats that also hybridize to the probe. In particular, they can mask the detection of the telomeres of chromosomes that have small HinfI terminal telomeric fragments. To further describe the telomere phenotype of clk-2(qm37) mutants, we have characterized the length of individual telomeres. The subtelomeric regions just adjacent to the terminal telomeric repeats share no sequence homology among telomeres (Wicky et al., 1996). Taking advantage of this sequence diversity, we designed probes specific to particular telomeres. The size of a given HinfI terminal fragment is related to the fixed distance between the most exterior HinfI site of the chromosome and the beginning of the telomeric repeats, and by the variable number of terminal telomeric repeats. Upon genomic DNA digestion with HinfI and Southern blotting with a probe specific to a particular telomere, the terminal fragments, which are heterogeneous in size, again appear as a smear. We present the results obtained for two individual telomeres in Fig. 6D,E.
The length of the terminal fragment of the left telomere of chromosome X is ∼1 kb longer in qm37 than in the wild type, which ranging from 2.4 to 4.2 kb and from 1.7 to 2.8 kb, respectively (Fig. 6D). This telomere is of wild-type length in MQ691, which carries the rescuing transgene, and lengthens again to the clk-2(qm37) values in the non-rescued MQ931 strain (Fig. 6D). The length of another terminal fragment (left telomere of chromome IV) is also ∼1 kb longer in qm37 than in the wild type, ranging from 2.2 to 3.9 kb and from 1.8 to 2.8 kb, respectively (Fig. 6E). This telomere becomes shorter than the wild type in MQ691, ranging from 1.3 to 2 kb only (Fig. 6E). This telomere acquires the mutant length again after loss of the transgene in MQ931 (Fig. 6E). Thus, the overexpression of clk-2 can shorten the tracks of telomeric repeats, but not at each telomere.
Evidence indicates that short telomeres are sensed as double-stranded breaks in the cell and elicit a DNA repair response that can lead to end-to-end chromosome fusions and genomic instability (Gasser, 2000). However, in contrast to what happens in S. cerevisiae tel2-1 mutants, telomeres are lengthened in qm37 mutants and not shortened. We nonetheless examined a number of markers of chromosome instability. However, we found no high frequency of males, no embryonic lethality and no decrease in the reproductive capacity over time in qm37 mutants at 20°C (data not shown). In addition, DAPI stained chromosomes of oocytes arrested in diakinesis of meiosis I in qm37 hermaphrodites after ∼25 hours of adulthood at 25°C, and at 20°C, display normal morphology and relative disposition (n>300 diakinesis oocytes in ∼60 gonad arms at each temperature). Finally, while γ-radiation, a double-stranded break generator, of qm37 L4 hermaphrodites grown at 20°C significantly affects the mutants’ brood size, the survival of the progeny produced is not affected (data not shown).
Inducible Systemic RNA Silencing in Caenorhabditis elegans
Introduction of double-stranded RNA (dsRNA) can elicit a gene-specific RNA interference response in a variety of organisms and cell types. In many cases, this response has a systemic character in that silencing of gene expression is observed in cells distal from the site of dsRNA delivery. The molecular mechanisms underlying the mobile nature of RNA silencing are unknown. For example, although cellular entry of dsRNA is possible, cellular exit of dsRNA from normal animal cells has not been directly observed. We provide evidence that transgenic strains of Caenorhabditis elegans transcribing dsRNA from a tissue-specific promoter do not exhibit comprehensive systemic RNA interference phenotypes. In these same animals, modifications of environmental conditions can result in more robust systemic RNA silencing. Additionally, we find that genetic mutations can influence the systemic character of RNA silencing in C. elegans and can separate mechanisms underlying systemic RNA silencing into tissue-specific components. These data suggest that trafficking of RNA silencing signals in C. elegans is regulated by specific physiological and genetic factors.
Jose, A.M. & Hunter, C.P. Transport of sequence-specific RNA interference information between cells. Annu. Rev. Genet. 41, 305–330 (2007).
Molnar, A. et al. Small silencing RNAs in plants are mobile and direct epigenetic modification in recipient cells. Science 328, 872–875 (2010).
Dunoyer, P. et al. Small RNA duplexes function as mobile silencing signals between plant cells. Science 328, 912–916 (2010).
Dunoyer, P. et al. An endogenous, systemic RNAi pathway in plants. EMBO J. 29, 1699–1712 (2010).
Winston, W.M., Molodowitch, C. & Hunter, C.P. Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1. Science 295, 2456–2459 (2002).
Feinberg, E.H. & Hunter, C.P. Transport of dsRNA into cells by the transmembrane protein SID-1. Science 301, 1545–1547 (2003).
Wolfrum, C. et al. Mechanisms and optimization of in vivo delivery of lipophlic siRNAs. Nat. Biotechnol. 25, 1149–1157 (2007).
Jose, A.M., Smith, J.J. & Hunter, C.P. Export of RNA silencing from C. elegans tissues does not require the RNA channel SID-1. Proc. Natl. Acad. Sci. USA 106, 2283–2288 (2009).
Faghihi, M.A. & Wahlestedt, C. Regulatory roles of natural antisense transcripts. Nat. Rev. Mol. Cell Biol. 10, 637–643 (2009).
Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998).
Grishok, A. RNAi mechanisms in Caenorhabditis elegans. FEBS Lett. 579, 5932–5939 (2005).
Aoki, K., Moriguchi, H., Yoshioka, T., Okawa, K. & Tabara, H. In vitro analyses of the production and activity of secondary small interfering RNAs in C. elegans. EMBO J. 26, 5007–5019 (2007).
Steiner, F.A., Okihara, K.L., Hoogstrate, S.W., Sijen, T. & Ketting, R.F. RDE-1 slicer activity is required only for passenger-strand cleavage during RNAi in Caenorhabditis elegans. Nat. Struct. Mol. Biol. 16, 207–211 (2009).
Chen, C.C. et al. A member of the polymerase beta nucleotidyltransferase superfamily is required for RNA interference in C. elegans. Curr. Biol. 15, 378–383 (2005).
Moazed, D. et al. Studies on the mechanism of RNAi-dependent heterochromatin assembly. Cold Spring Harb. Symp. Quant. Biol. 71, 461–471 (2006).
van Wolfswinkel, J.C. et al. CDE-1 affects chromosome segregation through uridylation of CSR-1-bound siRNAs. Cell 139, 135–148 (2009).
Tabara, H. et al. The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99, 123–132 (1999).
Grishok, A., Tabara, H. & Mello, C.C. Genetic requirements for inheritance of RNAi in C. elegans. Science 287, 2494–2497 (2000).
Parker, G.S., Eckert, D.M. & Bass, B.L. RDE-4 preferentially binds long dsRNA and its dimerization is necessary for cleavage of dsRNA to siRNA. RNA 12, 807–818 (2006).
Habig, J.W., Aruscavage, P.J. & Bass, B.L. In C. elegans, high levels of dsRNA allow RNAi in the absence of RDE-4. PLoS ONE 3, e4052 (2008).
Timmons, L. & Fire, A. Specific interference by ingested dsRNA. Nature 395, 854 (1998).
Qadota, H. et al. Establishment of a tissue-specific RNAi system in C. elegans. Gene 400, 166–173 (2007).
Kwak, J.E. & Wickens, M. A family of poly(U) polymerases. RNA 13, 860–867 (2007).
Ren, H. & Zhang, H. Wnt signaling controls temporal identities of seam cells in Caenorhabditis elegans. Dev. Biol. 345, 144–155 (2010).
Kennedy, S., Wang, D. & Ruvkun, G. A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans. Nature 427, 645–649 (2004).
Hyun, T.K., Uddin, M.N., Rim, Y. & Kim, J.Y. Cell-to-cell trafficking of RNA and RNA silencing through plasmodesmata. Protoplasma 248, 101–116 (2011).
Shih, J.D. & Hunter, C.P. SID-1 is a ds-RNA selective ds-RNA gated channel. RNA 17, 1057–1065 (2011).
Elbashir, S.M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).
Kok, K.H., Ng, M.-H.J., Ching, Y.-P. & Jin, D.-Y. Human TRBP and PACT directly interact with each other and associate with Dicer to facilitate the production of small interfering RNA. J. Biol. Chem. 282, 17649–17657 (2007).
Timmons, L., Tabara, H., Mello, C.C. & Fire, A.Z. Inducible systemic RNA silencing in Caenorhabditis elegans. Mol. Biol. Cell 14, 2972–2983 (2003).
Tournier, B., Tabler, M. & Kalantidis, K. Phloem flow strongly influences the systemic spread of silencing in GFP Nicotiana benthamiana plants. Plant J. 47, 383–394 (2006).
Calixto, A., Chelur, D., Topalidou, I., Chen, X. & Chalfie, M. Enhanced neuronal RNAi in C. elegans using SID-1. Nat. Methods 7, 554–559 (2010).
Hobert, O. PCR fusion-based approach to create reporter gene constructs for expression analysis in transgenic C. elegans. Biotechniques 32, 728–730 (2002).
Mello, C.C., Kramer, J.M., Stinchcomb, D. & Ambros, V. Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, 3959–3970 (1991).
Newcombe, R.G. Two-sided confidence intervals for the single proportion: comparison of seven methods. Stat. Med. 17, 857–872 (1998).
RNAI AS A COMPLEMENT TO MUTATIONAL ANALYSIS
Caenorhabditis elegans researchers use RNAi to complement mutational analysis in many contexts. As described above in the context of RNAi-based screens, focused analysis of a regulatory pathway or developmental process can benefit from combining RNAi with traditional genetic analysis. For example, RNAi can silence gene expression at a specific developmental stage (helpful in the absence of an available temperature-sensitive allele) or be used in combination with genetic mutations to simultaneously silence multiple gene products. In many cases, gene-x(-) gene-y(RNAi) animals may be far easier to generate than gene-x(-) gene-y(-) double mutants [ 86]. Another common use of RNAi is in gene cloning, particularly in tissues where DNA-mediated transformation rescue is problematic (e.g. the germ line). Once a gene of interest is mapped to a discrete chromosomal interval, candidate genes located within the interval can be assayed by RNAi to identify any of those whose knockdown mimics the mutant phenotype of the gene of interest [ 87].
Tissue-specific RNAi can also function as a sort of poor man's genetic mosaic analysis. In particular, rrf-1 ( R NA-dependent R NA polymerase f amily) mutants disrupt RNAi in the soma but not the germ line, allowing one to distinguish between germ line and somatic gene expression [ 88]. If dsRNA treatment of wild-type animals produces a defect that does not arise when rrf-1 mutants are treated with the same dsRNA, then the standard interpretation is that the defect depends on gene silencing in the soma. This approach has been used extensively to distinguish germ line versus soma as the tissue site of action for many genes that promote germ line development [e.g. 87, 89–94, among others]. Similarly, comparative RNAi in wild-type versus Rb pathway mutants may be useful for distinguishing the tissue site of action for genes that regulate development of specific neurons.
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