Tabara, Hiroaki et al. (2009) International Worm Meeting "Argonaute proteins possessing mRNA cleavage activity in C. elegans."

Mochizuki, Megumi, Tabara, Hiroaki, Ogawa, Yoshito, Asanuma, Takahiro

[International Worm Meeting, 2009]

In the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) and Argonaute proteins cooperatively play important roles. Primary siRNAs bearing monophosphorylated 5'' ends are produced from trigger dsRNAs by an RNaseIII-related enzyme called Dicer. In C. elegans, secondary siRNAs bearing triphosphorylated 5'' ends are also produced by RNA-dependent RNA polymerases, which act on target mRNAs. As a result of in vitro analyses using a cell-free assay system prepared from C. elegans, we previously showed that secondary-type siRNAs induce a prominent Slicer activity to cleave target mRNAs far more effectively than primary-type siRNAs. An Argonaute protein, CSR-1, is responsible for the Slicer activity induced by secondary-type siRNAs. Monophosphorylated siRNAs also induce a weak mRNA cleavage activity in the cell-free system, but the factor responsible for the weak Slicer activity was not clear. CSR-1 in cell lysates migrates to high-molecular-weight fractions by gel filtration. Somewhat similarly, recent fluorescent immunostaining experiments have shown that P-granules in germ cells and certain granules in somatic cells are intensely stained with anti-CSR-1 antibodies; non-granular regions of cytoplasm are also moderately stained. Using the same antibodies, we are now trying to examine interactions between CSR-1 and endogenous siRNAs. On the other hand, we biochemically tested various Argonaute mutants, and found that monophosphorylated siRNAs fail to induce a proper mRNA-cleavage activity in cell lysates prepared from a mutant strain, which corresponds to a 100-kDa Argonaute protein (p100). This Argonaute-mutant strain is homozygous-viable and known to show a weak RNAi-deficient phenotype. We made a recombinant protein of p100 and further examined its activity. The recombinant p100 was able to show an mRNA cleavage activity in conjunction not only with monophosphorylated siRNAs but also with triphosphorylated siRNAs. Several experiments are in progress to investigate the position of p100 in the RNAi pathway.

Tabara, Hiroaki et al. (2015) International Worm Meeting "Homologous pairing and unpaired silencing of chromosomes are regulated by RNAi during meiosis in C. elegans."

Kohara, Yuji, Mochizuki, Megumi, Nagata, Kyosuke, Mitani, Shohei, Tabara, Hiroaki

[International Worm Meeting, 2015]

During meiosis, most chromosomes have homologous partners and undergo homologous pairing. In contrast, pairing does not occur for X-chromosomes in males of C. elegans and free-type chromosome duplications. These unpaired chromosomes are heterochromatinized by meiotic silencing of unpaired DNA, which is often abbreviated to meiotic silencing. We analyzed the differences and similarities of regulation of paired and unpaired chromosomes during meiosis. In C. elegans, unpaired DNA subjected to meiotic silencing forms facultative heterochromatin that is rich in dimethylation of lysine 9 on histone H3 (H3K9me2). Heterochromatinization of unpaired chromosome is known to be abnormal in some RNAi mutants, such as the csr-1 mutant corresponding to an Argonaute protein, which has an RNA cleavage (Slicer) activity in conjunction with triphosphorylated siRNAs produced by RNA-dependent RNA polymerases. A similar Slicer activity was detected also from C04F12.1 corresponding to another Argonaute protein. We found that C04F12.1(tm1637) mutation enhanced the meiotic silencing defect caused by csr-1(fj54) mutation. While endogenous RNAi is thought to be involved in the early phase of meiotic silencing, the nature of heterochromatin formed by meiotic silencing is not well understood. Our biochemical analysis identified a chromo-domain protein CEC-5 as a component of heterochromatin formed by meiotic silencing. Triple-mutations of cec-5 and similar genes significantly diminished H3K9me2 signals thought to correspond to unpaired chromosomes. In addition, we found that the activity of meiosis-specific cohesin containing COH-3/4 kleisin is required for meiotic silencing. Our immunostaining detected COH-3/4 on unpaired chromosomes and at lateral axes of the synaptonemal complex on paired chromosomes. Considering the distribution of COH-3/4, we next asked whether mutants corresponding to COH-3/4 and Slicer-type Argonaute proteins show any defect in homolog pairing. As results of immunofluorescence and FISH experiments, the coh-3/4 double-mutant and the Slicer-type Argonaute double-mutant showed the defects in homolog pairing, except for pairing centers. An abnormal distribution of COH-3/4 was also observed in some gonads of the Argonaute double-mutant. Slicer-type Argonaute proteins are not essential for axis formation of the synaptonemal complex, but they are necessary for formation of a synaptonemal complex with accurate homology between homologous chromosomes. Thus, mechanisms of RNAi, meiotic silencing and homologous pairing overlap partially with each other in this organism.

Hiroaki Tabara et al. (2001) International C. elegans Meeting "rde-4 encodes a double-stranded RNA-binding protein required for RNAi in C. elegans"

Susan Parrish, Erbay Yigit, Andrew Fire, Hiroaki Tabara, Craig C Mello, Darryl Conte

[International C. elegans Meeting, 2001]

In many eucaryotic species, the introduction of double-stranded RNA (dsRNA) induces potent and sequence-specific gene silencing, a phenomenon called RNA interference (RNAi). Natural functions of RNAi-related mechanisms may include antiviral immunity, as well as developmental regulation mediated by natural dsRNA encoding genes (See abstract by Grishok and Pasquanali et al.). To elucidate the mechanism of RNAi, we have isolated RNAi deficient mutants, ( rde-1 through rde-9 ). The rde-1 and rde-4 mutants are the strongest RNAi deficient strains, lacking RNAi in all tissues examined. Genetic analysis suggests that rde-1 and rde-4 function in the initiation of interference after exposure to dsRNA. The rde-1 gene is a member of a functionally novel but highly conserved eucaryotic gene family with numerous homologs in C. elegans as well as fungi, plants and animals. Here we report that the rde-4 gene encodes a 385 amino acid protein with two double-stranded RNA-binding motifs (dsRBDs). A PCR product predicted to contain this single gene rescues rde-4 and both alleles of rde-4 cause premature stop codons within this gene. The ne301 allele truncates the protein prior to the dsRBDs and behaves like a null allele when placed in trans to a chromosomal deficiency. In vitro gel shift and north-western blot analyses suggest that RDE-4 strongly binds to dsRNA. In order to ask whether RDE-4 interacts with dsRNA during RNAi in vivo , we raised RDE-4 antibodies and used them to immuno-precipitate the RDE-4 protein from worms exposed to pos-1 dsRNA. The RDE-4 precipitate contained abundant pos-1 dsRNA whose average length was about 100bp. We are currently analyzing this precipitate to determine whether it contains the trigger dsRNA, the endogenous target mRNA or both. We are also examining whether the in vivo interaction between RDE-4 and dsRNA depends on other rde(+) activities. Localization studies suggest that both RDE-1 and RDE-4 are broadly expressed cytoplasmic proteins and that they co-localize in vivo . Preliminary analysis suggests that the in vivo interaction between RDE-4 and pos-1 dsRNA depends on rde-1 activity. The genetic and biochemical data are consistent with a model in which RDE-1 and RDE-4 act directly with the trigger dsRNA in forming an interfering complex that mediates mRNA destruction.

Kohara Y et al. (1994) Worm Breeder's Gazette "The C. elegans cDNA Project: A Progress Report."

Sugimoto A, Motohashi T, Tabara H, Watanabe H, Kohara Y

[Worm Breeder's Gazette, 1994]

The C.elegans cDNA project: A progress report Yuji Kohara, Tomoko Motohashi, Akiko Sugimoto, Hisako Watanabe and Hiroaki Tabara Gene Library Lab, National Institute of Genetics, Mishima 411, Japan. e-mail: ykohara@lddbj.nig.ac.jp

Grishok A et al. (2000) Science "Genetic requirements for inheritance of RNAi in C. elegans."

Mello CC, Grishok A, Tabara H

[Science, 2000]

In Caenorhabditis elegans, the introduction of double-stranded RNA triggers sequence-specific genetic interference (RNAi) that is transmitted to offspring. The inheritance properties associated with this phenomenon were examined. Transmission of the interference effect occurred through a dominant extragenic agent. The wild-type activities of the RNAi pathway genes rde-1 and rde-4 were required for the formation of this interfering agent but were not needed for interference thereafter. In contrast, the rde-2 and mut-7 genes were required downstream for interference. These findings provide evidence for germ line transmission of an extragenic sequence-specific silencing factor and implicate rde-1 and rde-4 in the formation of the inherited agent.AD - Program in Molecular Medicine, Department of Cell Biology, University of Massachusetts Cancer Center, Two Biotech Suite 213, 373 Plantation Street, Worcester, MA 01605, USA.FAU - Grishok, AAU - Grishok AFAU - Tabara, HAU - Tabara HFAU - Mello, C CAU - Mello CCLA - engID - GM58800/GM/NIGMSPT - Journal ArticleCY - UNITED STATESTA - ScienceJID - 0404511RN - 0 (DNA Transposable Elements)RN - 0 (Helminth Proteins)RN - 0 (RNA, Double-Stranded)RN - 0 (RNA, Helminth)RN - 0 (rde1 protein)SB - IM

Lin R et al. (1999) Nature "RNA interference. Policing rogue genes."

Lin R, Avery L

[Nature, 1999]

Life is based on social contract, genes work for the good of the organism, and they are reproduced. But certain rogue genes, called transposons, wantonly reproduce at the expense of the organism, inserting new copies of themselves all over the genome. Reporting in Cell, Tabara et al., and Ketting et al. now show that organisms have systems to hold transposons in check. They suggest that one of the clues that organisms use to detect illicit activity is double-stranded RNA, and their results could explain the reason for the mysterious phenomenon of RNA interference.

Wang J et al. (2005) Methods Enzymol "RNA interference in Caenorhabditis elegans."

Barr MM, Wang J

[Methods Enzymol, 2005]

RNA interference (RNAi) was first discovered in the nematode Caenorhabditis elegans (Fire et al., 1998; Guo and Kemphues, 1995). The completion of the C. elegans genome in 1998 coupled with the advent of RNAi techniques to knock down gene function ushered in a new age in the field of functional genomics. There are four methods for double-stranded RNA (dsRNA) delivery in C. elegans: (1) injection of dsRNA into any region of the animal (Fire et al., 1998), (2) feeding with bacteria producing dsRNA (Timmons et al., 2001), (3) soaking in dsRNA (Tabara et al., 1998), and (4) in vivo production of dsRNA from transgenic promoters (Tavernarakis et al., 2000). In this chapter, we discuss the molecular genetic mechanisms, techniques, and applications of RNAi in C. elegans.

Scott W Knight et al. (2003) International Worm Meeting "Biochemical analysis of Dicer activity in C. elegans extracts"

Brenda L Bass, Scott W Knight

[International Worm Meeting, 2003]

Dicer is an RNaseIII-like enzyme that is required for RNA interference (RNAi) and essential for proper development. dcr-1 (-/-) animals are RNAi defective, sterile, and exhibit heterochronic phenotypes. In an early step in the RNAi pathway, Dicer cleaves the initiating double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that are approximately 23 nucleotides (nts) in length. Dicer also cleaves endogenous dsRNAs that form intramolecular hairpins into single-stranded micro RNAs (miRNAs) that, like siRNAs, are 23 nts long. Biochemical and genetic data has indicated that RDE-4, a dsRNA-binding protein, associates with Dicer and is required for cleavage of long dsRNA into siRNAs (Tabara et al., 2002; Parrish and Fire, 2001). Unlike siRNA production, however, genetic evidence suggests that cleavage of at least some miRNAs occurs in the absence of RDE-4. rde-4 mutants, while being RNAi defective, do not exhibit any of the additional phenotypes associated with a dcr-1 knockout (Tabara et al., 1999). Using biochemical extracts made from C. elegans wild-type embryos and rde-4 embryos we investigated the role of RDE-4 in dsRNA cleavage and the production of mature miRNAs. We demonstrate that wild-type extracts efficiently cleave dsRNA into siRNAs and also process miRNA precursors into mature miRNAs. In agreement with previously described molecular data, we find that extracts made from rde-4 mutants fail to process dsRNA into siRNAs. We are currently investigating the role of RDE-4 in processing miRNAs and will present data on how variations in the dsRNA structure affect substrate recognition and cleavage by Dicer.

Ashe A et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Small RNAs With Characteristics Of 22Gs Are Present Multiple Generations After dsRNA Induced Silencing"

Miska E A, Goldstein L D, Ashe A, Lehrbach N J

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Since the discovery of RNAi in C. elegans in 1998, our knowledge of the complexity of small RNA pathways in this organism has grown rapidly. We now know that there are many exogenous and endogenous small RNA pathways interacting in complex ways within the organism. Much of this knowledge has come about through advances in sequencing technology that allow us to see in great detail the numerous small RNAs present in cells. Despite this abundance of data, there are still many aspects of small RNA biology that remain unclear. From the first reports of gene silencing induced by exogenous dsRNA, it has been observed that the silencing can be heritable (1). For RNAi of most genes, silencing of the target gene is limited to the F1 generation, with subsequent generations reverting back to normal levels of gene expression. However, there are a small number of reported cases where silencing of a specific gene can be transmitted across multiple generations (2, 3, 4). The mechanism of inheritance is unknown, but studies have suggested the involvement of a diffusible element (4), or chromatin modifiers (3). Here we show that silencing of a single-copy GFP transgene can be inherited for multiple generations, and that this silencing correlates with the presence of small RNAs targeted to the transgene. 1. H. Tabara, A. Grishok, C. Mello, Science 282, 430-431 (1998) 2. A. Grishok, H. Tabara, C. Mello, Science 287, 2494-2497 (2000) 3. N. Vastenhouw, K. Brunschwig, K. Okihara, F. Muller, M. Tijsterman, R. Plasterk, Nature, 442, 882 (2006) 4. R. Alcazar, R. Lin, A. Fire, Genetics, 180, 1275-1288 (2008)

Kohara Y et al. (1995) Worm Breeder's Gazette "The C. elegans cDNA Project: A Progress Report."

Kohara Y, Motohashi T, Watanabe H, Sugimoto A, Tabara H

[Worm Breeder's Gazette, 1995]

The C.elegans cDNA project: A progress report Yuji Kohara, Tomoko Motohashi, Akiko Sugimoto, Hisako Watanabe and Hiroaki Tabara Gene Library Lab, National Institute of Genetics, Mishima 411, Japan e-mail: ykohara/*ddbj.nig.ac.jp Tag sequencing is now on the third set of cDNA dones. After analysis of the first set of cDNA clones (some 4,400 clones), each 10,000 clones were picked up randomly from 3 different cDNA libraries (an embryonic cDNA library and libraries of >2kb cDNA and unfractionated cDNA made from mixed stage population). The total 30,000 clones were gridded and probed with the cDNA clones belonging to the species which had been represented by more than 4 clones in the analysis of the first set. A set of some 4,800 cDNA clones (the second set) were selected out of the unhybridized clones (from rare or not analyzed cDNA species) and has been subjected to the tag sequencing. This analysis produced 3,667 clean 3'-tags which gave 1,532 more unique cDNA species (see Fig.). As the next step, the grids were further screened with the cDNA probes the groups containing more than 4 clones at the point. A set of some 4,000 cDNA clones (the third set) was selected out of the unhybridized clones and tag sequencing has been continued on this set. The current status of our progress is that we have identified 3,324 unique cDNA species out of 7,647 clones (clean 3'-tags). The unique cDNA species were assigned serial numbers from CELK00001 to CELK03324. These analyses have also detected many pairs of clones which appeared to be generated by alternative splicing. In some cases, two groups were turned out that they were derived from the same gene but had different 3'-end sequences due to alternative splicing or differential poly-A addition. We are going to make a list of such differential splicings. BLASTX search showed that 653 groups out of the 1,816 groups identified through the analysis of the second and the third sets gave significant similarities (blastx score > 100), which are listed below. (Note; "-" in the column of "Frame" means BLASTX search was made using only 3'-tag sequences so far.) Mapping and in situ analysis are in progress.