Nadine L Vastenhouw et al. (2001) International C. elegans Meeting "Pulling the Trigger of Transposon Silencing"

Ronald H A Plasterk, Titia Sijen, Nadine L Vastenhouw

[International C. elegans Meeting, 2001]

Transposons are mobile genetic elements that are widespread among prokaryotes and eukaryotes. C. elegans contains different types of transposons among which are Tc1, Tc3 and Tc5. These elements belong to the Tc1/mariner family. Members of this family encode a transposase that is flanked by terminal inverted repeats. The transposase binds near the inverted repeats mediating the ability to jump from one position in the genome to another through a 'cut-and-paste' mechanism. In C. elegans , the activity of these intragenomic parasites is highly regulated by the mechanism of RNA interference (RNAi), e.g. in the laboratory strain Bristol N2, transposition activity is only detectable in somatic tissue and not in the germline. The mechanism of RNAi involves the silencing of gene expression in response to double stranded RNA (dsRNA). There are at least two possible ways by which transposons can produce dsRNA. First, readthrough transcription from flanking promoters in the genome may yield both sense and antisense transcripts that can basepair and form dsRNA. Alternatively, either a sense or an antisense transcript of one transposon could snap back by basepairing between the two terminal inverted repeats, thus forming dsRNA. Here, we use Tc5 of which only four copies are present in N2. Preliminary results show that all four copies are transcribed in both the soma and germline. In addition, dsRNA of the terminal inverted repeats of Tc5 was detected in soma and germline. We are currently generating strains that contain only one element each in order to determine the origin of the dsRNA that triggers transposon silencing.

Andrews S et al. (1991) International C. elegans Meeting "NUCLEOTIDE SEQUENCE OF THE TRANSPOSON Tc5."

Rasmussen K, Collins JJ, Andrews S

[International C. elegans Meeting, 1991]

We have determined the complete nucleotide sequence of a copy of the C elegans transposon TcS. The sequence reveals an element with inverted repeat termini and several long open reading frames that could encode a protein, perhaps one involved in element activity. The ends of TcS are inverted repeats of 491 base pairs. Interestingly, the most terminal bases are nearly identical to the most terminal bases of another C elegans transposon, Tc4 (Yuan and Horvitz). The internal portion of the element contains four non- overlapping open reading frames interrupted by three regions that exhibit sequence features of C elegans introns. The spliced transcript predicted by this DNA sequence would encode a protein of 458 amino acids. Regions of this predicted protein resemble regions of similarity in the predicted products of ORFs contained in the C elegans transposons Tcl and Tc3.

Maureen Peters et al. (2007) International Worm Meeting "A gap junction-mediated calcium wave coordinates a rhythmic motor program in C. elegans."

Takayuki Teramoto, Jamie White, Maureen Peters, Kouichi Iwasaki, Erik Jorgensen

[International Worm Meeting, 2007]

A gap junction-mediated calcium wave is required for the timing and execution of a multi-tissue motor program that controls defecation in C. elegans. In feeding worms, defecation occurs approximately every 50 seconds and is controlled by a molecular timekeeper located in the intestine1. We have identified a pannexin gap junction subunit, innexin-16, that is expressed exclusively in the intestine and is localized to cell-cell junctions. In inx-16 mutants, calcium wave propagation is slowed or eliminated and wave directionality is disrupted. These results indicate that INX-16 gap junctions mediate directional and temporally precise propagation of the calcium wave. The calcium wave is apparently crucial to several aspects of the motor program. Cycle periodicity is more erratic in inx-16 mutant animals and all motor steps are abnormal or absent. The calcium wave instructs the pattern of the first muscle contraction of the program, posterior body contraction; inverted calcium waves are correlated with inverted posterior body contractions. The last two muscle contractions of the program: anterior body contraction and enteric muscle contraction rarely occur, suggesting that the signals controlling these steps are not released from the inx-16 mutant intestine. Our findings suggest that calcium waves can coordinate multiple tissues to produce an integrated set of behaviors. 1. Dal Santo et al., (1999) Cell, 98, 757-67.

Ruvolo VR et al. (1991) International C. elegans Meeting "SEQUENCE ANALYSIS OF THE TRANSPOSABLE ELEMENT Tc2."

Levitt A, Ruvolo VR

[International C. elegans Meeting, 1991]

We have sequenced a transposed copy of the transposable element Tc2. The Tc2 element is 2074 base pairs in length and has perfect inverted terminal repeats of 24 base pairs. m e structure of the transposon suggests that it may have the capacity to code for a transposase protein and/or for regulatory functions. Tc2 contains nine potential open reading frames of 75 codons or longer, six on one strand and three on the other. Three open reading frames on one strand show non- random codon usage an,d may be exons in a single message. The first of these coding regions is preceded by a potential CAAT box, TATA box, and consensus heat shock sequence. In addition to its inverted terminal repeats, TC2 has an unusual structural feature: subterminal degenerate direct repeats that are arranged in an irregular overlapping pattern. The Spm elements of maize also have a subtermunal repetitive region, which is believed to be required for transpcsition (Masson et al., 1987. Genetics 177: 117- 137). However, unlike the subterminal repeats of Tc2, the Spm repeats are present in discrete units and are oriented in both directions. We found that the 12 base pair motif of the Spm repeats is similar at seven positions to a 10 base pair motif in Tc2. Using GoG profile analysis to ccmpare amino acid sequences, we determined that TC2 is not a member of the Tcl-like y.-oup of transpc60ns, which includes some elements of nematodes and fruit flies (Brezinsky et al., 1990. NAR 18: 20s3-2059). m e Tcl-like elements are shorter than Tc2 by about 500 base pairs, and they have one large open reading frame which specifies a protein of about 270 amino acids. The multi-codon structure of Tc2 is more similar to the Ac and Spm elements of maize and the P elements of Drosophila. What some of the Tcl-like elements do have in ccmmon with Tc2 is the generation of a target site duplication of the dinucleotide TA. We examined the insertion sites of two transposed elements which were identified as the cause of restriction fragment length polymorphisms. Except for the TA dinucleotide, the two sites were not similar to each other. One element was located in a highly repetitive chromosomal region. The other had inaertel inside the inverted terminal repeat of a Tcl element, splitting it into two unequal parts.

Remi Sonneville et al. (2006) Development & Evolution Meeting "Using zyg-11 mutants to identify novel components required for A-P polarity"

Pierre Gonczy, Remi Sonneville

[Development & Evolution Meeting, 2006]

In response to a signal from the centrosome, A-P polarity is established shortly after fertilization in the one cell stage embryo. We reported previously that embryos lacking zyg-11 function appear to use a signal from oocyte derived chromosomes to establish an inverted polarity during meiosis II. How zyg-11 normally acts to prevent precocious polarity establishment is not understood. ZYG-11 appears to function as a substrate recruitment subunit of a CUL-2 based E3 ligase to target substrates to proteasome destruction. This E3 ligase may have several substrates, based on the phenotypes observed after zyg-11/cul-2 inactivation. First, such embryos have a delayed metaphase to anaphase transition and M phase exit, due in part to an increased levels in B-type cyclins. Second, such embryos establish an inverted polarity during meiosis II, a phenotype that can be dissociated from the cell cycle delays, indicating that zyg-11 may have a distinct function and substrate for A-P polarity establishment. Third, such embryos fail to degrade OMA-1/OMA-2. Destabilization of these two proteins also requires phosphorylation by the two kinases MBK-2 and GSK-3 and is necessary for proper localization of P-granules. To identify novel components that function together with the ZYG-11/CUL-2 E3 ligase, we conducted a genome-wide RNAi-based synthetic lethal screen with the temperature-sensitive mutant allele zyg-11(b2). We identified 294 candidate enhancers in this manner. Next, we sought to focus specifically on those genes that may participate in polarity establishment by assaying the synthetic lethality of these candidates with other conditional mutants. We retained those genes that also enhanced par-2(it5) or mbk-2(ne992) and excluded those genes that also enhanced two other conditional mutants not related to zyg-11/cul-2 function. Conducting these positive and negative selections, we identified 5 enhancers of zyg-11(b2ts) and par-2(it5), 1 enhancer of zyg-11(b2ts) and mbk-2(ne992), 2 enhancers of zyg-11(b2ts), par-2(it5) and mbk-2(ne992). We are in the process of examining polarity markers and OMA-1 levels after inactivation of these candidates in the wild-type, as well as in sensitized background conditions, to better understand their mode of action. We expect that analysis of these genes will help us decipher how inverted polarity is established following zyg-11/cul-2 inactivation, which may shed light on the mechanism by which A-P polarity is set up in the wild-type.

Baere IJD et al. (1995) International C. elegans Meeting "IN VITRO ACTIVITIES OF Tc3A TRANSPOSASE"

Plasterk RHA, Baere IJD

[International C. elegans Meeting, 1995]

C. elegans Tc1 and Tc3 are transposons that belong to the Tc1/mariner family. For the biochemical analysis of transposition, we are characterizing the properties of recombinant transposase. Tc3A transposase fused to the carboxy-terminus of Schistosoma japonicum glutathione S-transferase was overexpressed in and purified from E. coli and shows improved solubility. This fusion protein binds specifically to the ends of Tc3 transposon inverted repeats in gel retardation assays. We also observed production of single stranded nicks near the 5' ends of the transposon. This activity is not seen at near the 3' ends. The binding and nuclease activity are analogous to those noticed with recombinant Tc1A transposase [1]. Unfused Tc3A from E. coli is not well soluble, and therefore we now also use alternative sources as yeast, and transgenic C. elegans strains containing a heat shock promoter - Tc3A transposase construct [2].

Shroff, Hari et al. (2013) International Worm Meeting "DiSPIM: time to leave your confocal behind."

Winter, Peter, Christensen, Ryan, Colon-Ramos, Daniel, Shroff, Hari, Wawrzusin, Peter, Santella, Anthony, Bao, Zhirong, Senseney, Justin, Fischer, Robert, McAuliffe, Matthew, Waterman, Clare, York, Andrew, Wu, Yicong

[International Worm Meeting, 2013]

Optimal 3D time-lapse imaging requires microscopy that provides high resolution in all spatial dimensions, high speed, and minimal photobleaching and photodamage. By creating algorithms that register and appropriately combine two perpendicular specimen views, we developed a dual-view inverted selective plane illumination microscope (diSPIM) with 330 nm isotropic resolution and 200 Hz acquisition, enabling rapid 3D imaging at 2 volumes/second. Unlike spinning-disk confocal or Bessel beam illumination methods that dose the sample outside the focal plane, leading to significant photobleaching, we maintain this spatiotemporal resolution over hundreds of volumes (hundreds of thousands of images) with negligible photobleaching. DiSPIM enables the study of biological systems that are intractable using other optical microscopes, including 4D microtubule tracking in live cells, improved nuclear imaging over 14 hours of nematode embryogenesis, and visualization of neural wiring during C. elegans brain development.

Garett M Padilla et al. (2004) Mid-west Worm Meeting "Investigating the Role of a Putative STAR Domain Protein in Caenorhabditis elegans"

Garett M Padilla, Elizabeth B Goodwin

[Mid-west Worm Meeting, 2004]

TRA-2 protein is necessary to promote female sexual development. Male development, at least in part, requires translational repression of tra-2 by two elements called TGEs ( tra-2 and GLI elements) located in the 3! (inverted exclamation mark)- (macron) UTR. tra-2 gain of function mutants that disrupt the TGEs feminize hermaphrodites. These mutations also feminize male animals, resulting in yolk production in the intestines and oocyte development in the germline. Therefore, TGE control regulates tra-2 activity in the germline and soma. GLD-1 is a germline specific protein that contains an RNA binding Maxi-KH/STAR domain that regulates tra-2 translation; however, a tra-2 somatic regulator has yet to be identified. We have identified a STAR domain containing protein in C. elegans , T21G5.5. This gene has two putative transcripts which differ in a 3! (inverted exclamation mark)- (macron) exon and is predicted to bind the same mRNAs as GLD-1 (Ryder et al , 2004). T21G5.5 are 63% identical throughout the STAR domain and 74% identical in the KH RNA binding domain. Both transcripts have been cloned and we are currently investigating their biochemical functions. Western analysis of a deletion strain of T21G5.5 demonstrates increased levels of TRA-2 protein relative to wildtype worms. In addition, RNA interference against T21G5.5a results in some in embryonic lethality and a decrease in fecundity. However, unlike GLD-1 gross germline phenotypes are not observed. Together these results indicate that T21G5.5 may function in the soma as a translational repressor in a role equivalent to GLD-1. Given slight germline defects observed, T21G5.5 may also function in the germline as well. Our future experiments include investigating the tissue specific and subcellular localization of T21G5.5, phenotypic analysis of the deletion strain, and determination of biochemical function. Ryder SP, Frater LA, Abramovitz DL, Goodwin EB, and Williamson JR. RNA target specificity of the STAR/GSG domain post-transcriptional regulatory protein GLD- 1. Nature Structural and Molecular Biology 11 , 20-28 (2004).

Ketting RF et al. (1995) International C. elegans Meeting "REGULATION OF TRANSPOSITION IN C.elegans."

Vos JC, Plasterk RHA, Ketting RF, Smits MT

[International C. elegans Meeting, 1995]

We study the process of transposition of the Tc1 and Tc3 elements of C.elegans. The elements encode a protein, a transposase, that is necessary for transposition. These transposases are called Tc1A and Tc3A, respectively. The first 63 and 65 amino acids of the respective transposases are able to bind specifically to the inverted repeats of their elements [Colloms S.D. et al. (1994) Nucl. Acids Res. 22(25), 5548-5554, Vos J.C. et al. (1993) Genes Dev. 7, 1244-1253.]. The binding characteristics of the Tc3A protein have been determined in further detail. A 192 amino acid fragment appears to be more restricted in the DNA it can bind than the first 65 amino acids: both proteins will bind to an oligo of 38 basepairs inverted repeat sequence, but only the first 65 amino acids will bind to a smaller fragment of 22 basepairs, which is similar to what is found for Tc1A, where a GG to CC mutation only abolishes binding of the bigger DNA binding fragment, which is 143 amino acids long [Vos J.C. and Plasterk R.H.A. (1994) EMBO J. 13(24), 6125-6132]. We are currently trying to obtain crystalls of the first 65 amino acids of Tc3A together with its binding site, in order to study the interactions of the transposase with the transposon ends. We are also looking for additional mutator loci in a Bristol background. In wild- type Bristol strains there is no detectable transposition in the germline such as found in Bergerac strains. John Collins has shown that it is possible to increase germline transposition activity in a Bergerac background [Collins J. et al. (1987) Nature 328, 726-728]. One spontaneous Tc1 mutator line has been obtained from a Bistol N2 background [Babity J.M. et al. (1990) Mol. Gen. Genet. 222, 65-70]. Is it also possible to raise the transposition frequency in the Bristol germline to a detectable level by EMS mutagenesis? We are now looking for new mutator lines in Bristol N2 backgrounds, using genetic screens. Progress will be reported.

van Luenen HGAM et al. (1997) International C. elegans Meeting "REQUIREMENTS FOR Tc1/Tc3 TRANSPOSITION AND THE USE OF Tc1/Tc3 FOR THE TRANSGENESIS OF C. elegans AND OTHER SPECIES"

van Luenen HGAM, Fischer SE, Rezsohazy R, Raz E, Vos JC, Plasterk RHA, Driever W

[International C. elegans Meeting, 1997]

Members of the Tc1 transposon family have been found in many different organisms: animals, fungi and protozoa. We have previously shown that Tc1 and Tc3 transpose via a cut-and-paste mechanism. We determined the minimal requirements for transposition. The only protein required for the excision and transposition reaction is the element-encoded transposase. Using purified recombinant transposase we can perform the excision and transposition reaction in vitro (Genes & Dev. (1996) 10: 755- 761). In cis, Tc1 and Tc3 only need the ends of the inverted repeats. These ends contain the transposase binding site and a short sequence at the extreme ends of the transposon which is conserved among members of the Tc1 family. In addition, Tc3 contains a region located approximately 100 bp from the ends which enhances transposition. The region between the ends of the inverted repeat can be replaced by other sequences. We attempted to use Tc1/Tc3 for the transgenesis of C. elegans. We used a sensitive positive/negative selection to select transposition off a transgenic array into the genome. We found less than one in a million survivors, and none of those resulted from genuine transposition. Apparently something precluded jumping off an array in the germ-line; we had no problems detecting somatic jumps of the transgenic transposons nor germ-line jumps of endogenous elements. The limited requirements for transposition may explain why Tc1-like elements have invaded so many different species. We recently attempted to use Tc1/Tc3 as vehicles for transgenesis of several species. In collaboration with the lab of W. Driever we co-injected zebrafish oocytes with Tc3 and Tc3 transposase mRNA , and showed transposase mediated transposition to occur. We sequenced the genomic insertion site of one Tc3 element and found that it was a genuine integration event into a TA target sequence. Into this zebrafish-line we again injected Tc3 transposase mRNA and detected excision of the Tc3 element. We are currently using Tc1 for the transgenesis of mice and human cell lines.