Caenorhabditis elegans has been used extensively to identify the genetic requirements for proper nervous system development and function. Key to this process is the direction of vesicles to the growing axons and dendrites, which is required for growth-cone extension and synapse formation in the developing neurons. The contribution and mechanism of membrane traffic in neuronal development are not fully understood, however.The nematode C. elegans gene unc-69 is required for axon outgrowth, guidance, fasciculation and normal presynaptic organization. We identify UNC-69 as an evolutionarily conserved 108-amino-acid protein with a short coiled-coil domain. UNC-69 interacts physically with UNC-76, mutations in which produce similar defects to loss of unc-69 function. In addition, a weak reduction-of-function allele, unc-69(ju69), preferentially causes mislocalization of the synaptic vesicle marker synaptobrevin. UNC-69 and UNC-76 colocalize as puncta in neuronal processes and cooperate to regulate axon extension and synapse formation. The chicken UNC-69 homolog is highly expressed in the developing central nervous system, and its inactivation by RNA interference leads to axon guidance defects.We show that the C. elegans. As both proteins are conserved through evolution, we suggest that the mammalian homologs of UNC-69 and UNC-76  may function similarly.We have identified a novel protein complex, composed of UNC-69 and UNC-76, which promotes axonal growth and normal presynaptic organization in  At its simplest, a neuron is composed of three major structures, a central cell body and two networks of extensively branched membrane structures, the dendrite and the axon. Growing axons respond to a wide variety of extracellular attractive and repulsive signals that direct migration to a fated location. Although many guidance receptors have been identified on extending growth cones, little is known about how activation of receptors mediates coordinated neurite extension. In addition to signaling cues in the extracellular matrix, neurite elongation and growth-cone extension depend on a concerted effort of vesicular transport and regulated membrane addition. For growth cones to extend, vesicles derived from the Golgi apparatus fuse with the plasma membrane by a process of regulated exocytosis . LikewisCaenorhabditis elegans has been extensively used to study vesicular transport in neuronal development. For example, monomeric kinesin UNC-104/KIF1A, UNC-116/kinesin heavy chain (KHC), kinesin light chain KLC-2, and various cytoplasmic dynein complex components regulate various vesicle trafficking events  transcriptional reporter, which is normally stochastically activated in either the right or left AWC neuron [unc-69(e587) mutants, the axon of the AWC neuron often stopped prematurely . In contrast, the dendrite of the AWC neuron had no outgrowth defect, as 100% (136/136) of the AWC dendrites extended to their full length. In unc-69(e587) mutants, 73% (99/136) of AWC neurons had ectopic bulges or branches protruding from either the cell body or the axon , the AWB, AWC, ASG, ASI, ASK, and ASJ neurons  [To determine whether the outgrowth defects we observed in C neuron . The bily Figure , and stransgene) ,29, and unc-16 and unc-116, markers for synaptic vesicles are also mis-sorted into dendrites [unc-69 mutants also show such a general sorting defect, or whether unc-69 might be required more specifically for efficient trafficking within the axons. At the L1 larval stage, the thirteen VD neurons are not yet born, and the six DD neurons are the only D-type GABAergic motor neurons present in the VNC. At this stage, the DD neurons receive their synaptic inputs from the DNC and output onto the ventral body-wall muscles. In wild-type L1s, therefore, the SNB-1::GFP puncta can be seen only along the VNC. In unc-69(ju69) mutants, the synaptic GFP was not significantly mislocalized to the DNC  mutant L1s  mutants .In vesicle-trafficking mutants such as endrites . We wondodr-10::gfp transgene that is expressed in the AWB neurons to answer this question [unc-101 (which encodes the homolog of AP1 μ1 clathrin adaptor protein), ODR-10::GFP is not targeted to the AWB cilia [unc-69(ju69) and unc-69(e587) mutants, ODR-10::GFP was still properly targeted  pull-down assay to verify the physical interaction between UNC-69 and UNC-76. As shown in Figure in vitro translated full-length UNC-76 (UNC-76FL) was pulled down efficiently by GST-UNC-69 but only minimally by GST-CBP, a eukaryotic transcription factor used as a negative control [in vitro translated adenoviral protein E1A efficiently bound to its cognate partner GST-CBP but not to GST-UNC-69. Therefore, the interaction between UNC-76 and UNC-69 is specific and most likely direct.We used an  control . Conversin vitro . Interestingly, this 19-amino-acid region overlaps with a region predicted to form a coiled-coil structure  in unc-76(e911) mutant worms  mutants  mutants, suggesting that the amino-terminal region of UNC-76 is required for its function in vivo. Bloom and Horvitz reported that amino acids 1–197 of UNC-76 are sufficient to direct proteins into the axons in C. elegans [unc-76(e911) mutants, consistent with the hypothesis that binding to UNC-69 is critical for UNC-76 to function in vivo.To corroborate the s Figure  and assas Figure . The CFP elegans . As the in vitro  mutants. In contrast, both UNC-76(L281P) and UNC-76(L287P) mutant proteins failed to bind UNC-69 in vitro. Surprisingly, UNC-76(L287P) was still able to rescue unc-76(e911) in vivo  in vivo . In summary, amino acids 281–299 of UNC-76 probably contain or overlap with an UNC-69-binding site, and UNC-76 may require interaction with UNC-69 to function in vivo.If coiled-coil structures are important for the UNC-76-UNC-69 interaction, any mutation that abolishes the coiled-coil structure would possibly also abolish physical interaction between the two proteins. To test this idea, we mutagenized four conserved residues in UNC-76: Glu275, Leu281, Leu287, and Lys291. Both UNC-76(E275A) and UNC-76(K291A) mutant proteins still bound UNC-69 o Figure . Likewisunc-76(lf) mutants could bypass the unc-76 mutant phenotype. We overexpressed a functional unc-69::gfp transgene as an extrachromosomal array in unc-76(e911) mutants but did not see any rescue in locomotion . Likewise, overexpression of a functional unc-76::yfp transgene failed to rescue the locomotion defect of unc-69(e587) mutants (data not shown).As both UNC-69 and UNC-76 are required for axon outgrowth and fasciculation, we asked whether they function in the same genetic pathway to regulate axon extension. We first tested whether overexpression of UNC-69 in unc-69 and unc-76 act in the same pathway. In C. elegans, expression of the odorant receptor gene str-2 is randomly turned on in either the left or the right AWC sensory neuron (AWCL/R), but never in both [ON' phenotype is determined by axonal contact and calcium signaling between AWCL and AWCR. In axonal guidance mutants such as unc-76, sax-3 and vab-3, the two AWC axons often fail to meet, and Pstr-2::gfp expression is consequently silenced in both AWCs, giving rise to a '2 AWCOFF' phenotype [unc-69(lf) and unc-76(lf) mutants as well as in unc-69(lf); unc-76(lf) double mutants.We also performed a double-mutant analysis to further address the question of whether  in both . In wildhenotype . We usedunc-69(e602) and unc-69(e587), 30–34% of animals showed a 2 AWCOFF phenotype. In contrast, the hypomorphic allele unc-69(ju69) resulted in only 1% of mutant worms (n = 190) having Pstr-2::gfp expression silenced in both AWCs (Table unc-69(ju69) mutants. In agreement with previous studies [unc-76(e911) mutants (n = 101) had the 2 AWCOFF phenotype; e911 was the strongest allele among all the nine alleles that we tested. For the other unc-76 alleles, the 2 AWCOFF phenotype varied from 6% to 30%. Interestingly, the strength of the AWC expression defect (which is an indication of axon extension defects) showed an inverse colinear relationship with the position of each mutation in the open reading frame: the most 5' mutation, unc-76(n2457), showed the least defect in axon extension, whereas alleles located most carboxy-terminally showed greater defects than alleles located close to the amino terminus ; sax-3(ky123), unc-76(e911); unc-6(n102) and unc-33(e204); unc-76(e911), and slightly enhanced in unc-76(e911); vab-3(e648) and unc-119(ed3); unc-76(e911) double mutants  and unc-69(e602), we conclude that UNC-69 and UNC-76 probably act in the same pathway to control axon extension, at least in the case of the AWC sensory neurons.In both strong loss-of-function mutants,  studies , 47% of unc-76 alleles have severe axonal outgrowth defects, making interpretations of defect in synaptic vesicle localization difficult. To bypass this problem and to reveal possible genetic interactions between unc-69 and unc-76, we looked at the localization of the synaptobrevin SNB-1::GFP puncta in unc-69(lf)/+; unc-76(lf)/+ double heterozygotes /+ heterozygotes /+; unc-76(e911)/+ and unc-69(e587)/+; unc-76(n2457)/+ double heterozygous hermaphrodites, SNB-1::GFP puncta were occasionally more diffused, larger, or completely absent within a stretch of DNC /+; unc-76(e911)/+ and unc-69(e587)/+; unc-76(n2457)/+ double heterozygotes occasionally had a slight Unc phenotype in locomotion, resembling weak synaptic transmission mutants. The weak locomotion defect could be a direct or indirect effect of the synaptic vesicle mislocalization defect.We showed above that UNC-69 is required for localization of synaptic vesicles in axons. Does UNC-76 also have a role in this process, and if so, does UNC-76 control presynaptic organization together with UNC-69? Unfortunately, all existing s Figure . In wilds Figure . The diss Figure  as compaC Figure ; the absunc-69/+; unc-76/+ double heterozygotes show phenotypes that are similar, albeit significantly weaker, to those observed in unc-69(ju69) homozygotes. Haplo-insufficient genetic interactions of this type, commonly known as nonallelic (or unlinked) noncomplementation, are often observed with proteins that form heterodimers or function in a common protein complex . Severatubulin; ). Thus, Punc-69::cfp::unc-69 and Punc-76::unc-76::yfp constructs at low concentration (5 ng/μl) into unc-76(e911) mutant hermaphrodites, and selected rescued transgenic animals for examination. At low concentration, both CFP::UNC-69 and UNC-76::YFP often appeared as puncta along the DNC, in CAN neurons, as well as in other neuronal processes that run along the subdorsal and sub-ventral tracts  mutants, UNC-76::YFP puncta were occasionally diffuse and sometimes failed to be accompanied by CFP::UNC-69 puncta in a stretch of axon  mutants  mutants, perinuclear UNC-76::YFP either split into several smaller dots  mutants. The unc-116(rh24) mutants carry two missense mutations (I304M and E338K) at the end of the motor domain of KHC (amino acids 1–358) [In n Figure . In addis Figure . Whereass 1–358) . Thus, tunc-104(e1265), the C. elegans KIF1A homolog [unc-116(rh24) mutants, overexpression pattern of UNC-69::GFP and UNC-76::YFP were both significantly different from wild-type animals. The CAN neuron accumulated UNC-69::GFP in the vicinity of its cell body, which was swollen and deformed. In addition, there were ectopic branches near the cell body, and UNC-69::GFP also accumulated in these processes  mutant, as if UNC-76::YFP was removed from the cytoplasm and concentrated in certain subcellular compartments  mutants  mutants. It is striking that the nearly perfect co-localization of UNC-69 and UNC-76 is disrupted in unc-116 mutants. We are still at a loss to explain the molecular basis of this unexpected finding. What is clear, however, is that axonal transport of UNC-69 and UNC-76 is still occurring in unc-116(rh24) mutants. Thus, other kinesin motors and/or additional factors probably contribute to transport of UNC-69 and UNC-76 along the axons.In summary, our data show that the subcellular distribution of both UNC-69 and UNC-76 is altered in S. cerevisiae and mammals have been reported to interact physically with members of the family of ARF-like small GTPases. To investigate whether a similar interaction occurs in C. elegans, we first used yeast two-hybrid assays to study protein-protein interactions between UNC-69 and three closely related but distinct ARF-like small GTPases, ARL-1 (F54C9.10), ARL-3 (F19H8.3), and ARFRP (Y54E10BR.2) [UNC-69 homologs in E10BR.2) . WhereasE10BR.2) , we alsoE10BR.2)  also hadE10BR.2) , a homolE10BR.2) -43  [To address directly the question of whether UNC-69 is a Golgi-associating protein, we coexpressed CFP::UNC-69 and a YFP-tagged fragment of the II::YFP) . Unlike Taken together, our results suggest that any interaction between UNC-69 and Golgi is at best transient, and that the UNC-69 puncta probably represent a structure distinct from the Golgi.C. elegans, two lines of evidence do suggest that the molecular function of UNC-69/SCOCO is conserved through evolution. First, the level of conservation between family members is extremely high in all the metazoans analyzed  [unc-69/SCOCO RNAi treated embryos (n = 26) showed defects, versus 10% of control embryos (n = 20); Figure in ovo RNAi approach selectively knocks down UNC-69/SCOCO expression in the spinal cord neurons, we conclude that the chick homolog of UNC-69/SCOCO is likely to function autonomously in epaxial nerve cells to control axon pathfinding, consistent with our observations in worms.Expression of the chick homolog of 6 Figure . In chic6 Figure . Blockine (RNAi)  resultedFrom the above analysis, we conclude that the function of UNC-69/SCOCO in axon guidance and nervous-system development is probably conserved through evolution. On the basis of its high degree of sequence conservation and its expression pattern, we predict that SCOCO is also required for nervous system development in mammals, including humans.unc-69 results in a selective defect in synaptic vesicle localization, strong unc-69 mutants have extensive axonal outgrowth, fasciculation and guidance defects. Both of the strong unc-69(lf) alleles, unc-69(e602) and unc-69(e587), truncate the coiled-coil domain of UNC-69. In contrast, the hypomorphic allele unc-69(ju69) results in a missense mutation of the start codon and presumably interferes with translation initiation. The lack of extensive axonal outgrowth defects in the unc-69(ju69) mutants suggests that UNC-69 protein translation might not be totally abolished and enough UNC-69 protein is still being produced to meet the requirement for growth-cone extension. In contrast, the process of proper localization of synaptic vesicles appears to be more sensitive to reduction in levels of UNC-69 protein. It is possible that unc-69 mediates different cellular processes in parallel: axonal outgrowth, fasciculation and guidance on one hand, synaptic vesicle localization on the other.In this work we show that mutations that affect the small 108-amino-acid protein UNC-69 abrogated a spectrum of processes, including synaptic-vesicle targeting, axonal outgrowth, pathfinding, and fasciculation. Although a weak reduction-of-function allele of unc-69(lf) mutants could be secondary to a more general transport defect, as loss of UNC-69 function might interfere not only with the transport of synaptic cargos but also with the transport of axonogenic vesicles to growth cones.UNC-69 is clearly different from most other molecules that have been implicated in axon outgrowth and guidance, which are either guidance cues, membrane receptors, or cell-adhesion proteins ,47. It iDrosophila is an axonal transport protein [in vivo function. Our genetic experiments suggest that both UNC-69 and UNC-76 act in the same pathway to regulate axon extension. In addition, we found that UNC-76 and UNC-69 cooperate to regulate the size and position of SNB-1::GFP puncta, a marker of presynaptic regions. Finally, we showed that UNC-69 and UNC-76 colocalize in neurons as puncta and that their normal subcellular distribution requires UNC-116/KHC. The physical interaction, subcellular colocalization, and similar mutant phenotypes all suggest that the UNC-69-UNC-76 protein complex acts as a functional unit that promotes transport of vesicles along axons.We identified UNC-76 as an UNC-69-interacting protein. UNC-76 is required for axonal outgrowth and fasciculation in worms and its homolog in  protein ,15. We iC. elegans requires many proteins, including UNC-116/KHC, KLC-2, UNC-14, UNC-51, as well as UNC-16, the worm homolog of Drosophila Sunday Driver, which serves as a scaffold protein receiving regulatory signals from the JNK pathway [unc-69(ju69) mutants, suggesting that UNC-69 is intrinsically different from these proteins. UNC-69 may function in a distinct step of axonal transport, and is possibly not involved in polarized sorting. Moreover, UNC-69 is unlikely to control general protein trafficking in neurons, as it was not required for dendritic transport of the transmembrane receptor ODR-10.Proper SNB-1::GFP localization in  pathway ,9,48. Bounc-116(rh24) mutants, but not in the unc-104(e1268) or unc-104(rh43) mutants (our unpublished observations), our data imply that UNC-69 and UNC-76 are possibly required for events other than transporting synaptic vesicles.Kinesin-1 transports various cargos, including Golgi, endoplasmic reticulum, mitochondria and synaptic membrane proteins, but not synaptic vesicles, in the nervous system ,49,50. IDrosophila UNC-76 is a KHC-associating protein further suggests that the UNC-69-UNC-76 protein complex might constitute an alternative pathway for Kinesin-1-mediated transport. Association of the UNC-69-UNC-76 protein complex with the tail of KHC could either provide additional levels of regulation, or allow intracellular trafficking of a different repertoire of cargos. So far, however, we could not verify a direct physical interaction between C. elegans UNC-76 and the tail region (amino acids 696–815) of UNC-116 in vitro . Further analysis will be required to definitively address this issue.Indeed, the observation that unc-69 mutants suggests that a similar interaction occurs in C. elegans. We failed to detect any physical interaction between UNC-69 and either ARL-1 or ARFRP, however. Moreover, UNC-69 often appears to occupy subcellular regions distinct from those of the Golgi marker mansII. Thus, the exact step at which UNC-69 acts in vesicular transport  is still obscure.UNC-69 is not predicted to have enzymatic activity and possibly functions only by interacting with other coiled-coil domain-containing proteins. The budding yeast and mammalian homologs of UNC-69, Slo1p and SCOCO, have been shown to interact, respectively, with Arl3p and ARL1 - two related, Golgi-associated, GTP-binding ADP-ribosylation factor (ARF)-like proteins. Mammalian ARL1 is involved in post-Golgi transport . Yeast Aunc-69 and unc-76 mutants, the UNC-69-UNC-76 protein complex might also help restrict membrane addition to growth cones during neuronal development, thereby preventing unwanted membrane extension elsewhere along the axons. Similar biochemical properties of the UNC-69-UNC-76 protein complex might be used to regulate synaptic vesicle clustering or maintenance in the presynaptic regions in the adult nervous system. UNC-69-UNC-76 puncta could reflect certain post-Golgi compartments that shuttle between their budding sites on the trans-Golgi and their docking sites near the plasma membrane. As such, an UNC-69-UNC-76 protein complex might function at an intermediate step before vesicle maturation. Indeed, UNC-69-UNC-76 puncta were present along the commissures in addition to axons. As commissures do not form synapses [We propose that UNC-69 and UNC-76 participate in a protein complex that is localized to certain subcellular compartments in the cytoplasm to control vesicle transport between the Golgi and the plasma membrane. The whole UNC-69-UNC-76 protein complex might be recruited and targeted by Kinesin-1 to its final destination in axons. As multiple ectopic branches were consistently observed in both synapses , these pC. elegans unc-69 mutants  and lin-15(n765ts) mutants, which were grown at 15°C before injection to improve viability and at 25°C following injection to enhance selection of transgenic F1 animals. Wild-type worms were of the Bristol N2 strain.escribed . All strunc-69 gene is tightly linked to RFLP nP55, which is recognized by cosmid C15B3. The three overlapping cosmids C15B3, C41B4 and F11D2, but not the flanking cosmids C30B11 and F46H1, rescued the Unc phenotype of unc-69(e587) mutant. Subsequent subclonings identified a 1.2-kb EcoRI-SacI rescuing genomic fragment, which contained a single gene composed of three exons. A frameshift mutation was introduced into the unc-69 open reading frame of the rescuing EcoRI-SacI fragment by cutting and filling the unique MluI restriction site, followed by re-ligation of the blunt ends. The frameshifted construct failed to rescue unc-69 mutant worms. To identify the molecular lesion(s) present in unc-69 mutants, the unc-69 locus from wild-type and unc-69 mutants was amplified using primers flanking the gene (5'-GCTCCGCAGTACGTCTTCTAAGCCC-3' and 5'-GCGAGAATGGAACAATCAATGGACG-3') and sequenced. In addition to the stop codon, e602 also contains a silent (third base) G-to-A transition in Lys107.All genetic mapping data were deposited into WormBase . The uncAssays of egg-laying behavior were performed either in M9 buffer  or on plIndirect immunofluorescence staining for serotonin and GABA were performed as previously described -59. Anti4 and 0.1 M cacodylate buffer for 1 h on ice. Subsequently, samples were cut and postfixed in 2% OsO4 and 0.1 M cacodylate buffer, mounted into an agar block, dehydrated in series of alcohols, and embedded in a mixture of epon-araldite. Thin sections (50 nm) were cut on an Ultracut E and pictures were taken with a JEOL 1200X at 80 kV.Adult hermaphrodites were fixed in 0.8% glutaraldehyde, 0.7% OsOAdult worms were soaked in 10 μg/ml DiI  in M9, and incubated in the dark for 2 h at 20°C, followed by several washes in M9 buffer. Staining was analyzed using appropriate filters.EcoRI genomic fragment was used to probe a C. elegans lambdaZAP cDNA library (gift of R. Barstead). From approximately 300,000 plaques we isolated four cDNAs. The sequences present at the ends of the inserts were determined for all four clones, and the DNA sequence of the longest intact clone was determined (one strand only). A random-primed, 32P-labeled unc-69 cDNA was used to probe a northern blot of wild-type poly(A)+ RNA. The blot was visualized and band intensities quantified using a Fuji Film phosphor imager. Poly(A)+RNA was isolated from purified embryos or mixed stages of wild-type strain N2 as described by Sambrook et al. [SCOCO cDNA.A rescuing 2.8 kb k et al. , except unc-69 genomic fragment containing 700 bp upstream and 300 bp downstream of the coding region was engineered through site-directed mutagenesis to construct various amino- and carboxy-terminal CFP or GFP fusions . The rescuing ability of the constructs was tested for both amino- and carboxy-terminal GFP fusions. A full-length unc-69 cDNA was cloned into pGEX-4T1 to generate a prokaryotic expression unc-69-gst construct. To generate a full-length unc-76 major splicing form cDNA (pSU001), the EcoRI-XbaI fragment of p76-c4 [EcoRI-BamHI fragment of yk784h09 and the XbaI-BamHI fragment of p76-c7, using three-way ligation. Progressive unc-76 deletions were made either by a PCR-based method, or using ExoIII and S1 nucleases following Erase-a-Base  and ExoIII/S1 Deletion Kit  protocols. To generate genomic unc-76 constructs, an AscI site was engineered 5' to the start ATG and was used to insert a 1 kb KpnI-AscI unc-76 promoter region up to the start codon. Subsequently a BglII-SphI genomic fragment encompassing exons 1 to 3 was used to replace the corresponding region of the cDNA. An AscI CFP cassette was inserted in-frame to create the amino-terminal CFP fusion plasmid (pSU065). Likewise, a NotI site was engineered immediately 5' to the stop codon to allow creation of the carboxy-terminal YFP fusion plasmid (pSU026). A genomic fragment containing the first 82 amino acids of mannosidase II was placed 3' to the unc-69 promoter and was carboxy-terminally fused with YFP to create pSU137. Full-length and truncated cDNAs of unc-69, scoco, unc-76, arl-1, arl-3, arfrp, and unc-119 were amplified from mixed-stage N2 mRNA pool or existing cDNA clones and subsequently subcloned into pRE192 and pRH143 to create LexA-bait and GAD-prey plasmids. pRE192 and pRH143 are derivatives of pBTM116 and pGAD424, respectively, and are gifts of K. Basler (University of Zurich).All manipulations were done following standard protocols . A 1.5 kf p76-c4  was replSingle-amino-acid changes were made following QuikChange Site-Directed Mutagenesis Kit protocols . Sequences for each of the mutations are as follows: UNC-76(E275A): GAG→GCG; UNC-76(L281P): CTG→CCA; UNC-76(L287P): CTG→CCG; UNC-76(K291A): AAA→GCC; ARL-1(Q70L): CAA→CTA; ARFRP(Q79L): CAG→CTG. Mutations were confirmed by sequencing and swapped back into the original plasmids before further subcloning. All plasmids and construct sequences are available upon request.Prediction of coiled-coil structures was carried out using the COILS version 2.1 program . The coiunc-69 cDNA was subcloned into pDBTrp and pDBLeu vectors  and cotransformed with the AD plasmids. Transformed yeast reporter strain Mav203 (GibcoBRL) was patched onto plates lacking Leu and Trp (LW plates), and then replica plated onto plates lacking Leu, Trp and His with 75 mM 3-amino triazol (LWH/3AT plates), onto plates lacking uracil (URA plates) and onto filters for β-galactosidase assays. Only clones that activated all three  reporter genes were kept for further analysis. Plasmid DNAs were purified from positive clones and retested for interaction with the bait plasmid. To study the interactions between UNC-69 and various ARL proteins, we used the yeast reporter strain L40, and followed Clontech's Yeast Protocols Handbook for o-nitrophenyl β-D-galactopyranoside (ONPG) and auxotrophic assays.Random- and dT-primed yeast two-hybrid phage libraries were generous gifts from R. Barstead . A further random-primed library was provided by M. Vidal . The In vitro transcription and translation of unc-76 was performed following protocols of the TNT-coupled reticulocyte lysate system (Promega) in the presence of 35S-labeled methionine . For pull-down experiments, purified recombinant GST and UNC-69-GST proteins were first immobilized on glutathione beads (Amersham). Immobilized proteins (2–10 μg) were incubated with 1–20 μl in vitro translated UNC-76 (depending on binding efficiency) in 1x interaction buffer ) at 4°C for 1.5–2 h. The beads were washed three times in washing buffer ), resuspended in 20 μl 2x SDS sample buffer, boiled and analyzed by SDS-PAGE and autoradiography.dpy-20(+) or pL15-EK lin-15(+) genomic fragments. To generate low-copy extrachromosomal arrays, plasmids were individually or co-injected each at 5 ng/μl together with 195 ng/μl pL15-EK. Transgenic progeny of injected animals were selected as non-Dpy or non-Muv animals at 25°C. Stably transmitting extrachromosomal arrays were integrated by γ-irradiation at 120 kV for 4.2 min.For high-copy overexpression, plasmids were injected into the germline of adult hermaphrodites  at 50 ngSCOCO, ChEST376p13, from the HGMP Research Center [in ovo RNAi was transcribed in vitro as described [We obtained a cDNA clone of chick h Center . Double-escribed . Embryosescribed  were staFor whole-mount immunostaining, staged embryos were dissected and fixed as described . EmbryosSCOCO expression as described previously [Embryos were sacrificed at different developmental stages (from stage 17 to 33) and fixed in 4% paraformaldehyde. The tissue was cryoprotected in 25% sucrose, and 20-μm-thick transverse sections of the lumbosacral spinal cord were used for the analysis of eviously .unc-69(e587) mutants  mutantsA figure showing that the overexpression of a full-length UNC-69(M1I)::GFP protein rescues locomotion defects of the Click here for additional data fileok339 deletionA figure showing the extent of the Click here for additional data fileA figure showing that RNAi knockdown of chicken UNC-69/SCOCO results in epaxial nerve pathfinding defectsClick here for additional data file


We used fluorescent in situ hybridization to investigate the dynamics of gene association with transcription factories during immediate early (IE) gene induction in mouse B lymphocytes. Here, we show that induction involves rapid gene relocation to transcription factories. Importantly, we find that the Myc proto-oncogene on Chromosome 15 is preferentially recruited to the same transcription factory as the highly transcribed Igh gene located on Chromosome 12. Myc and Igh are the most frequent translocation partners in plasmacytoma and Burkitt lymphoma. Our results show that transcriptional activation of IE genes involves rapid relocation to preassembled transcription factories. Furthermore, the data imply a direct link between the nonrandom interchromosomal organization of transcribed genes at transcription factories and the incidence of specific chromosomal translocations.Transcription in mammalian nuclei is highly compartmentalized in RNA polymerase II-enriched nuclear foci known as transcription factories. Genes in  Myc proto-oncogene and examined its location with respect to transcription factories and its common translocation partner, the immunoglobulin heavy chain (Igh) gene. We found that switching on the Myc gene leads to its rapid relocation to a transcription factory. Surprisingly, we found that the activated Myc frequently chooses the same transcription factory as the highly transcribing Igh gene. This close juxtaposition of translocation partner genes at a shared transcription factory may provide the opportunity for a chromosomal translocation, and thus may be the first step in the genesis of several types of cancers.Many different types of cancer result from gene translocations. Specifically, two different chromosomes can be joined that fuse growth control genes with powerful regulatory elements, leading to unrestricted control of cell growth. Translocation partner genes must physically encounter each other in the nucleus to undergo a translocation; how they find each other in the crowded nucleus is unknown. We showed previously that gene transcription occurs at a few hundred discrete nuclear sites called transcription factories. In the current study we investigated the effects of activation of the  Transcription in mammalian nuclei occurs in nuclear foci known as transcription factories. Genes from different chromosomes can share the same factory, suggesting that factories are preassembled and genes migrate to them. Nonraphocytes . Similarf nuclei . Any procis and trans can frequently share the same factory, which strongly suggests that genes migrate to preassembled transcription sites for transcription [Nascent transcription occurs at RNA polymerase II (RNAPII)-rich nuclear foci known as transcription factories –11. Thescription .Myc to the same transcription factory that is occupied by its frequent translocation partner, Igh. Our results suggest that this frequent and preferential juxtaposition may provide the opportunity for a chromosomal translocation, and may in part dictate the incidence with which specific chromosomal translocations occur.In this study, we investigated the positioning of immediate early (IE) genes relative to transcription factories and other B cell expressed genes during IE induction. We found that before activation the majority of IE alleles are not associated with transcription factories, whereas upon induction, IE genes rapidly relocate to preformed transcription factories. Remarkably, we observed preferential recruitment of the proto-oncogene Fos and Myc [Igh are present at approximately 90% of alleles both before and after induction  technique capable of quantitating the average absolute number of primary transcripts per cell . In thisIgh primary transcripts does not change during B cell induction . Since RNA FISH shows that 90% of Igh alleles are transcriptionally active, we estimate that there are approximately five transcripts being produced on each active allele.We found that the average number of nduction , consistFos gene is very different. In unstimulated cells we found that the extrapolated, average absolute number of Fos primary transcripts per cell is 1.73 copies. This is less than one primary Fos transcript per allele, indicating that not all Fos alleles are transcriptionally active in unstimulated cells. If we calculate the average number of Fos transcripts per active allele based on our RNA FISH data in which 20% of Fos alleles showed a transcription signal, we arrive at an average number of 4.3 Fos primary transcripts per active allele. In stimulated cells Fos primary transcript intron copies per cell increase approximately 2.7-fold, consistent with the 2.65-fold increase in the percentage of actively transcribed Fos alleles determined by RNA FISH. Comparison of the number of Fos primary transcripts per active allele indicates that the number of Fos transcripts per active allele does not change upon induction (4.3 versus 4.4 copies). These data show that our RNA FISH technique is very sensitive and is capable of measuring very small numbers of primary transcripts at a transcription site. In addition, these results show that when we do not see an RNA FISH signal over a particular allele, it is truly “off” and has no primary transcripts associated with it. Collectively, these results show that Fos IE gene induction occurs via transcriptional activation of additional, previously inactive alleles, rather than by simply increasing the “basal” rate of transcription of all alleles.The picture for the IE Myc gene has a well-characterized attenuation site that is thought to block passage of RNAP II, resulting in a stalled polymerase [Myc alleles may be pre-positioned in factories awaiting removal of a transcriptional block.Our previous studies in erythroid cells suggested that gene association with transcription factories is dynamic, with genes moving to preformed factories in order to transcribe . Induciblymerase . This obMyc RNA FISH signals are associated with strong RNAP II foci  Fos signal, before and after induction using double-label RNA FISH. In unstimulated cells approximately 7% of Igh signals had a colocalizing Fos signal   A. This sand Uros B and 6C.nduction D, most lMyc and Igh alleles in two other tissue types, adult kidney and fetal liver erythroid cells. We detected much greater separation distances between Myc and Igh in these tissues compared to unstimulated B cells  was used to detect ligation products in unstimulated and stimulated B cells. Over multiple experiments, ligation products were always detected in stimulated B cells, while in unstimulated cells the products were usually, but not always detected. In contrast, Igh/Myc ligation products were never detected in brain or kidney cells, indicating that juxtaposition of Myc and Igh is restricted to tissues in which both genes are expressed.To corroborate the FISH results, we used the capturing chromosome conformation (3C) assay ,26,27, w B cells . The priMyc and Igh are the two most common translocation partners in Burkitt lymphoma and mouse plasmacytoma. Of these cancers 80% harbor Myc-Igh translocations, while the remaining cases contain Myc-Igk (15%) or Myc-Igl (5%) translocations [Myc colocalized with Igk and Igl alleles in 10-min stimulated cells by RNA FISH. We found that 11% of transcribing Myc alleles colocalized with Igk and 7% colocalized with Igl, compared to 22% with Igh  genes t factory . Prefere factory ,37,39. GIgh translocations are presumed to occur through aberrant repair during programmed recombination, by recombinase activating gene protein (RAG) during V(D)J recombination, and activation-induced deaminase (AID) during somatic hypermutation and class switching [IGH translocations [Myc breakpoint region. Igh translocations within the Igh diversity and joining regions occur in the bone marrow pre-B cells, which undergo V(D)J recombination [Igh translocations to Myc are found in the Igh class switch region and are believed to occur in germinal center B cells, the site of class switching [MYC gene, at the 5′ end of the first intron [witching . Cleavagocations ,42. Howebination . Howeverwitching . There iwitching , which cwitching . Topoisowitching . Signifit intron . Furthert intron , and anot intron . Interest intron . In summMyc and Igh loci in transcription factories to persist, considering the potentially grave risks of such an organization. However, the apparent dangers of illegitimate recombinations may be outweighed by advantages of clustering transcribing genes, which may make efficient use of shared resources, or perhaps provide a degree of transcriptional coordination of subsets of genes.It is curious that evolution has allowed the interchromosomal juxtaposition of the − resting B cells were isolated from spleens of 6- to 8-wk-old BALB/c mice by magnetic cell sorting using CD43 microbeads  to deplete other cell types. Induction of B cells was done in PBS supplemented with 10 ng/ml recombinant mouse IL-4 , 20 μg/ml purified rat anti-mouse monoclonal antibodies to CD40  and 10μg/ml goat anti-mouse IgM μ chain, F(ab′)2 fragment  at room temperature for up to 15 min before fixation for FISH.CD43Igh transcription with a dinitrophenol-labeled single-stranded DNA probe to the intronic enhancer region [Fos, Myc, Eif3s6, Uros, Actb, Igk, and Igl, intron sequences as described [http://bacpac.chori.org) were used: 234 kb RP23-98D8 for Myc; 178 kb RP24-233K8 for Fos; 166 kb RPCI24-258E20 for Igh; 151 kb RP24-132K17 for Uros; and 213 kb RP23-97O1 for Actb. For double-label experiments, we labeled one of the DNA FISH probes directly with AlexaFluor 594 and labeled the other probe with digoxigenin, detected with fluorescein-conjugated antibodies. Immunofluorescence and immuno-FISH was carried out as described [http://www.upstate.com) that was raised against a Ser5-phosphorylated CTD. This antibody is specific to phosphorylated forms of RNAP II [RNA FISH was carried out as described previously ,51. We vr region , followeescribed . Primer escribed . The folescribed , using a RNAP II .Fos intron 1 sense, 5′-GCTTTGTGTAGCCGCCAGGT-3′; antisense 5′-AGAGGAAAGCGGAGGTGAGC-3′. Fos intron 2 sense, 5′-AAGTAGAGCTGGTGAGCAGCGATT-3′; antisense, 5′-AGAAAAGGACCAACATTCAGTTAAGG-3′. Myc intron 1 sense, 5′-AGCACAGATCTGGTGGTCTTTC-3′; antisense, 5′-CTCCTTCGAGCAGGGACTTAG-3′. Myc intron 2 sense, 5′-CTTCTCCACCACTCATTGGCATTA-3′; antisense, 5′-GGGAGGAAGTGGAAGATCACAGTT-3′. Eif3s6 intron 1 sense, 5′-GTGAGGAAGCTTTGAGAAGGAGGA-3′; antisense, 5′-ATTAATTTTGCTGTTCCCTGCTGA-3′. Uros intron 6 sense, 5′-TCAGCGCCACAGCAAGGGTT-3′; antisense, 5′-GCCTTCCCTCCTTTGTTCCCAGT-3′. Actb intron 1 sense, 5′-TCGCTCTCTCGTGGCTAGTA-3′; antisense, 5′-TGGCGAACTATCAAGACACA-3′. Igh Iμ intron sense, 5′-AGCTGTGGCTGCTGCTCTTA-3′; antisense, 5′-AGCCTCGCTTACTAGGGCTCTC-3′. Igl J-C intron probe 1 sense, 5′-TGAGTGACTCCTTCCTCCTTTG-3′; antisense, 5′-TGGAGGCAGTGTGTAAAGTGTC-3′. Igl J-C intron probe 2 sense, 5′-GTTGTCTTGCAAGGGTCTTTTT-3′; antisense, 5′-GTGCGAATAAAAGAAGGGATTG-3′. Igk J-C intron sense, 5′-AAGACACAGGTTTTCATGTTAGGA-3′; antisense, 5′-AATAGAATTATGAGCAGCCTTTCC-3′.Primers used to amplify RNA FISH probes were the following. Uros-Igh, for which 140 alleles were assessed. Transcription signals scored as colocalizing if the red and green signals overlapped to create a visible yellow signal. To assess the association of Myc DNA FISH signals and RNAP II foci, we captured image stacks of nuclei, using an Olympus BX41 epifluorescence microscope, equipped with a UPlanApo 100× oil objective to reduce chromatic aberration, and fitted with a motorized stage. Images were captured and analyzed using Analysis 3.2 image capture software, fitted with a RIDE module . The stacks were deconvoluted using a nearest-neighbor algorithm with 85% haze removal, and analyzed. We analyzed 81 and 88 alleles in unstimulated and stimulated B cells, respectively. Statistical analysis was carried out using a two-sided Fisher exact test. For Myc alleles that were not co-associated with an RNAPII focus, we measured the separation distance from the edge of the gene signal to the edge of the nearest RNAP II immunofluorescence signal, and analyzed 27 alleles.We examined RNA FISH signals on an Olympus BX41 epifluorescence microscope, and assessed 200 loci for each probe combination, except for Igh and genes in trans by DNA FISH, we collected image stacks using a Zeiss 510 Meta confocal microscope. Separation distances for each Igh allele and the nearest Myc, Fos, Uros, or Actb allele were measured on 3D-reconstructed image stacks using Volocity image analysis software (http://www.improvision.com/products/volocity). In all cases, we made measurements from center to center of the two gene signals. We analyzed at least 83 measurements for gene pairs in unstimulated and stimulated B cells, adult kidney cells, and E14.5 fetal liver cells. Changes in the distributions of measurements were assessed by two-sided Student t-test.To measure the distances between Igh and 236 bp Fos fragments that span 5′ exon-intron junctions. The plasmid containing the Igh fragment was digested with HindIII and AflII to release a 17 bp fragment, then religated. The Fos deletion was generated by BsmFI and NheI digestion to remove a 24 bp fragment. RNA was transcribed from linearized plasmids, then checked by gel electrophoresis and quantitated by UV spectrometry. Dilutions of controlled amounts of RNA was spiked into Trizol reagent that contained a known number of cells. RNA was extracted, reverse transcribed, and PCR amplified with nested primers.The assay was carried out essentially as described . RNA comIgh, the lanes with four copies of spiked competitor per cell was used for quantitation. For Fos, quantitation was obtained from the average from the lanes with 1 copy per cell and 0.5 copy per cell.To measure the numbers of primary transcripts over the length of the intron, the relative intensity of the endogenous RT-PCR product was compared to the spiked competitor RT-PCR product, measured using AIDA quantitation software. For Igh-f, 5′-CCTGGGAATGTATGGTTGTGGCTTC-3′; Igh-r, 5′-CCCCCTAAAGCAAT:GACTGAAGACTCA-3′; Igh nested-f, 5′-CCTCGGTGGCTTTGAAGGAACAAT-3′; Igh nested-r, 5′-CCCTAAAGCAATGACTGAAGACTCAGT-3′; Fos-f, 5′-AGCATCGGCAGAAGGGGCAAAGTA-3′; Fos-r, 5′-TGAAGTAGGAAGCTGTCAGGGAAACTG-3′; Fos nested-f, 5′-AGAAGGGGCAAAGTAGAGCAGGTGA-3′; Fos nested-r, 5′-TGTCAAAATCTGACAAGGGAGGGAAAG-3′.The primers used were as follows. 6 nuclei overnight with 600 units of BglII. We ligated digested chromatin (2 μg) with 2,000 units of T4 DNA ligase in a final volume of 800 μl. We cloned ligation product detected by primer pair d/g (see list below)and verified it by DNA sequencing. We tested the specificity of the 3C primers as previously described [Myc a forward, 5′-TCTACACCCCATACACCTCCA-3′; Myc a nested, 5′-CGAGAATATGCCATGAATTGG-3′. Myc b forward, 5′-GGGGAGGGAATTTTTGTCTATT-3′; Myc b nested, 5′-GGACAGTGTTCTCTGCCTCTG-3′. Myc c forward, 5′-TGCCCTCTCAGAGACTGGTAA-3′; Myc c nested, 5′-TTCCCCTTTCCTCTGTCATCT-3′. Myc d forward, 5′-ATTCTTCCAGGTGGTGATGTC-3′; Myc d nested, 5′-CTTCCCACAGCTCTCTTCCTT-3′. Igh e forward, 5′-AACCCATCTACCCATGTAGCC-3′; Igh e nested, 5′-CCTCTGACTGCCTCTTTTCCT-3′. Igh f forward, 5′-ACTGTGATCGGTTTTGGAGTG-3′; Igh f nested, 5′-CTGGGAGGGTTTGGTTCTTAC-3′. Igh g forward, 5′-CCCAGAACCTGAGAAGGAAGA-3′; Igh g nested, 5′-ACAGAACCGAACCATGACTTG-3′. Igh h forward, 5′-TTGGGCACTAAACACCACTTC-3′; Igh h nested, 5′-GGTGTGTGCAGGTTTTTGTCT-3′. Hbb-b1 forward, 5′-CTCAGAGCAGTATCTTTTGTTTGC-3′; Hbb-b1 nested, 5′-AGGATGAGCAATTCTTTTTGC-3′. Calreticulin Cal1, 5′-CTCCAGATAAACCAGTATGAT-3′; Cal2, 5′-AAACCAGATGAGGGCTGAAGG-3′. Actb Actb 1forward, 5′- CGGTGCTAAGAAGGCTGTTCC-3′; Actb 1nested, 5′- AGCAAGAGAGGTATCCTGACC-3′. Actb Actb 2forward, 5′- TGTGACAAAGCTAATGAGG-3′; Actb 2nested, 5′-TGAGTAGATGCACAGTAGG-3′.We carried out the 3C assay as described previously . We fixeescribed . The priFigure S1(807 KB PDF)Click here for additional data file.


KM is decreased by an order of magnitude and kcat increases 30 fold. Y14-Magoh do inhibit the MLN51-stimulated ATPase activity, but not back to background levels. The ATP-bound form of the eIF4AIII-MLN51 complex has a 100-fold higher affinity for RNA than the unbound form and ATP hydrolysis reduces this affinity. MLN51 stimulates the RNA-helicase activity of eIF4AIII, suggesting that this activity may be functionally important.The core of the exon-junction complex consists of Y14, Magoh, MLN51 and eIF4AIII, a DEAD-box RNA helicase. MLN51 stimulates the ATPase activity of eIF4AIII, whilst the Y14-Magoh complex inhibits it. We show that the MLN51-dependent stimulation increases both the affinity of eIF4AIII for ATP and the rate of enzyme turnover; the  The processes of transcription, pre-mRNA processing, mRNA export, translation and degradation are all interconnected. For example, during splicing, an exon-junction complex (EJC) is deposited at a defined location ∼20–24 nucleotides upstream of exon-exon junctions in a sequence-independent manner The EJC is a dynamic complex which changes its composition during mRNA export into the cytoplasm eIF4AIII is the third member of the eIF4A DEAD-box RNA-helicase family. It is functionally distinct from eIF4A isoforms I and II Previous biochemical studies have shown that MLN51 SELOR stimulates the RNA-dependent ATPase activity of eIF4AIII, whilst the Y14-Magoh complex inhibits this stimulation KM,ATP of eIF4AIII by an order of magnitude and increases the turnover (kcat) 30 fold. The RNA-binding affinity of a complex of MLN51 SELOR and eIF4AIII is increased about 2-fold in the presence of ATP, but about 100-fold if Y14 and Magoh are also present. Also we show that the helicase activity of eIF4AIII is stimulated by MLN51, demonstrating that the eIF4AIII-MLN51 complex has RNA-helicase activity that may be functionally important.In this study, we have further characterized RNA binding and the ATPase activity of the core components of the EJC. We show that MLN51 SELOR decreases the E. coli and used in subsequent ATP-hydrolysis assays. eIF4AIII has a low background rate of ATP hydrolysis, which is stimulated approximately 2 fold in the presence of whole yeast RNA  and the rate of enzyme turnover (kcat)  . In the Kd) of ∼1 µM. This result shows that in the absence of ATP, MLN51 SELOR alone or complexed with eIF4AIII interacts with RNA weakly. When ATP was also added, the amount of eIF4AIII-MLN51 SELOR bound to the RNA increased ∼50%  decreased by a similar amount, whilst the association rate remained unchanged, suggesting that ATP hydrolysis only affects dissociation from the RNA. Taken together, these data show that the ATP-bound eIF4AIII-MLN51 SELOR complex has a higher affinity for RNA than after the ATP is hydrolyzed. These results are similar to the related mammalian DEAD-box protein, eIF4AI We also examined the RNA interactions for eIF4AIII-MLN51 SELOR-AMPPNP and eIF4AIII-MLN51 SELOR-Y14-Magoh-ATP . Both ofKd of ∼2 µM. The RNA-binding affinity and kinetics for Y14-Magoh are similar to MLN51 SELOR. These data suggest that the Y14-Magoh complex can weakly interact with RNA so it may stabilize the RNA interaction within the EJC. This is supported by UV-crosslinking experiments that suggest Y14-Magoh crosslinks to RNA in the context of the core EJC (data not shown).It is still not clear whether the Y14-Magoh complex is involved in the interaction with RNA within the EJC. If it does bind to RNA it is probably not through the RBD since structures of the complex show that this domain forms an unusual protein-protein interaction with Magoh 32P. In contrast to the previous finding that eIF4AIII has RNA-helicase activity, we did not detect any helicase activity for eIF4AIII alone despite using a similar RNA substrate eIF4AIII has been previously shown to have RNA-helicase activity alone Complexes of eIF4AIII and MLN51 and of Y14 and Magoh are considerably more stable than the quaternary complex containing all four proteins Once the mRNA is transported to the cytoplasm, the constituents of the EJC change. Many of the components of the EJC are lost during transport Further research is required to determine whether the core EJC containing eIF4AIII, MLN51, Y14 and Magoh has RNA-helicase activity. It may form a stable complex with RNA relatively slowly in the absence of other components of the EJC in order to prevent the EJC from ‘locking’ onto RNA non-specifically. If it requires interactions with other components of the EJC to load it onto RNA in vivo this would ensure that the complex only binds tightly to a specific location on the RNA, 20–24 nucleotides upstream of the exon-exon junction.E. coli strain BL21 (DE3) and purified from clarified crude cell extracts by immobilized metal-ion-affinity, ion-exchange and gel-filtration chromatography. MLN51 (137–283) was isolated by PCR amplification, cloned into the expression vector pGEX6P-1 and expressed as a GST fusion in the E. coli strain BL21 (DE3). It was cleaved from a glutathione-affinity column using PreScission protease (Amersham Biosciences), followed by gel-filtration chromatography. The nucleotide sequence of each expression clone was verified by automated DNA sequencing. Protein concentrations were determined from the absorbance at 280 nm using molar extinction coefficients derived by summing the contributions from tyrosine and tryptophan residues.The DNA sequence coding for Y14 (50–174) and full-length Magoh were isolated by PCR amplification from human cDNAs and cloned into pACYCDuet-1 (Novagen). Magoh was fused to an N-terminal hexa-histidine sequence using the EcoRI site and inserted 5′ to the native Y14 sequence (residues 50-174). eIF4AIII was isolated by the same method and cloned into pETDuet-1 (Novagen) and fused to the N-terminal hexa-histidine sequence using the BamHI site. The Y14-Magoh complex and eIF4AIII were expressed in the 4C)3UAGCACCGUAAAGCACGC and GCUUUACGGUGC.The RNA oligonucleotides were purchased from Curevac. The oligonucleotide used for surface plasmon resonance experiments was 5′ to 3′: Biotin-AUGUCAUUCGAGUACAGUCUGUUCAGCUAGUCUCC. The oligonucleotides used in the helicase assays were similar to those used previously Duplex RNA for RNA-helicase assays was formed by annealing 300 pmoles of labeled 12mer RNA with 400 pmoles of cold 40mer RNA in 10 mM Tris-HCl pH 8.0, 1 mM EDTA and 100 mM NaCl, by heating at 95°C and cooling slowly to room temperature.−1) using the extinction co-efficient for NADH of 6300 M−1.cm−1. Assays (200 µl) containing 2 mM ATP, 50 mM Tris-HCl pH 7.5, 50 mM NaCl, 5 mM DTT, 5 mM MgCl2, 1 mM phosphoenol pyruvate, 0.4 mM NADH, 1% (v/v) PK/LDH (Sigma) and 200 µg.ml−1 yeast RNA were measured at 25°C in a 96-well plate using a Benchmark Plus spectrophotometer (BioRad). eIF4AIII and MLN51 SELOR or Y14-Magoh were included at 400 and 800 nM respectively. For KM measurements the ATP concentration was varied from 0.01 to 2 mM and the data were fitted to the Michaelis-Menten equation. For eIF4AIII alone the protein concentration was increased to 1 µM.ATPase assays were performed using an enzyme-linked assay consisting of lactate dehydrogenase and pyruvate kinase to link ATP hydrolysis to the oxidation of NADH which causes a decrease in the absorbance at 340 nm. The absorbance was converted into rate  as described previously 32P-ATP and T4 polynucleotide kinase with standard protocols. The labeled RNA was purified from unincorporated ATP using G-25 Microspin columns (Amersham Biosciences) and then ethanol precipitated.1 nmole of the 12mer ribo-oligonucleotide was end labeled using γ-2, 3 mM DTT and 2 mM ATP and incubated at 37°C for 30 mins. 500 nM unlabelled 12mer RNA was also included in the reaction so that any free 40mer RNA that was generated was annealed to this excess of cold RNA. Reactions were terminated by adding 5 µl of 2% SDS, 50 mM EDTA, 50% glycerol and 0.01% bromophenol blue, then analyzed by electrophoresis at 12 mA for 45 minutes on native 12% acrylamide-TAE gels. The labeled components were visualized by autoradiography. Where indicated MLN51 SELOR and/or Y14-Magoh were included at 5 µM and reactions were terminated with 5 µl of 4% SDS, 50 mM EDTA, 50% glycerol and 0.01% bromophenol blue.Helicase assays (20 µl) containing 10 nM duplex RNA and 0.25 to 4 µM eIF4AIII were performed in 40 mM Tris-HCl pH 7.8, 50 mM NaCl, 2 mM MgCl


In eukaryotes, short RNAs guide a variety of enzymatic activities that range from RNA editing to translation repression. It is hypothesized that pre-existing proteins evolved to bind and use guide RNA during evolution. However, the capacity of modern proteins to adopt new RNA guides has never been demonstrated. Here we show that Rnt1p, the yeast orthologue of the bacterial dsRNA-specific RNase III, can bind short RNA transcripts and use them as guides for sequence-specific cleavage. Target cleavage occurred at a constant distance from the Rnt1p binding site, leaving the guide RNA intact for subsequent cleavage. Our results indicate that RNase III may trigger sequence-specific RNA degradation independent of the RNAi machinery, and they open the road for a new generation of precise RNA silencing tools that do not trigger a dsRNA-mediated immune response. Saccharomyces cerevisiae. In these organisms, most documented gene specific mRNA degradation events including those performed by RNase III are not sequence but structure dependentThe capacity of short RNA duplexes to direct sequence-specific RNA degradation provides an almost universal tool for design-based gene silencing. This technique termed RNA interference (RNAi), is initiated by either endogenous RNA duplexes generated by members of the RNase III family (e.g. Drosha and Dicer)Members of the RNase III family share a conserved dsRNA-binding domain (dsRBD) and a catalytic domainS. cerevisiae, we asked whether there is an independent strategy to target specific RNA sequences for degradation by RNase III. Our hypothesis was that Rnt1p could function as an RNP complex and use a small RNA guide to cleave a specific RNA sequence. To test this hypothesis we generated RNA transcripts containing a 5 bp hairpin that binds Rnt1p fused to sequences complementary to different RNA targets. As predicted, the different RNA guides successfully bound to Rnt1p and directed a specific cleavage at a fixed distance from the RNA hairpin in vitro and reduced the expression of abundant nuclear RNAs in vivo. Together, our data indicate that RNase III may function as a sequence specific RNP complex and reveal a new approach for the regulation of nuclear RNA.Since RNAi does not exist in Most RNase IIIs and other dsRBPs identify their substrate by recognizing the distance between the minor grooves generated by one turn (i.e. 11 bp) of an A-form RNA helixRNA footprinting, chemical interference, and binding assays indicate that a minimum of a 5 bp stem capped with a NGNN tetraloop is required for Rnt1p bindingin trans. Therefore, Rnt1p cleavage may also be guided by short RNA transcripts. To test this possibility, we generated RNA transcripts that contain an Rnt1p binding signal (5 bp stem) fused to RNA extensions complementary to the sequence of an independently transcribed single-stranded RNA fragment. One guide had long extensions (EL18-15) to allow cleavage in both guide and target RNA  and recombinant Rnt1p . As showin cis (d) of about 0.80 µM, while the guide RNA with only one single target complementary extension bound less efficiently with a K'd of 1.9 µM  was slightly reduced. We conclude that guide-mediated and classical Rnt1p substrates observe similar kinetic parameters.The cleavage efficiencies of the different substrates were tested by incubating each of them with Rnt1p and Mgnder low  and physnder low . Rnt1p canch RNA . SurprisGuide-induced RNA cleavage may be a unique feature of Rnt1p or it may be shared by other members of the RNase III family. We examined the guide's capacity to induce target cleavage by RNase IIIs from bacteria (RNase III), fission yeast (Pac 1) and human (Dicer) . As expein vivo, we tested its capacity to identify a given sequence in a natural mixture of yeast total RNA. The Rnt1p cleavage signal at the 3′-end of pre-U2 snRNA was replaced by a sequence complementary to the EL3′-11 guide and the new U2/target construct was expressed in vivo  or by primer extension  at the 3′-end. The addition of an inverted deoxythymidine reduces 3′ to 5′ exonuclease attackGuide RNA's potential to regulate gene expression was evaluated by examining their capacity to restore cleavage to mutated Rnt1p cleavage site in both cell extract and in total RNA . The guiin vivo, we had to establish a method for RNA transfection. To do this, we adapted an electroporation based transformation strategy that is normally used for DNA transformationIn order to assay the guide activity Rapid RNA processing and difficulties detecting unprocessed RNA precursors suggest that RNA processing takes place co-transcriptionally. However, the importance of co-transcriptional RNA cleavage to RNA maturation remains unclear. We have taken advantage of the newly developed guide technology to assess whether the co-transcriptional Rnt1p cleavage of the pre-U2 3′-end is required for U2 maturation. We monitored the generation of mature U2 snRNA from a transcript that depends on the guide EL3′-11dT for cleavage . As expein cis independent of the target, which explains the reduced efficiency of target cleavage. These results indicate that RNA guides may act as helical scales marking the distance to the cleavage site. As expected, guide RNA with a mutation in the conserved second base of the Rnt1p tetraloop (EU2dT/ACUC) blocked cleavage (lane 5). Mutations altering the guide homing sequence complementary to the target site (EU2dT/2M and EU2dT/4M) also blocked cleavage (lanes 6 and 7) confirming the cleavage specificity.To evaluate the guide cleavage strategy as a tool for gene silencing, we designed and tested a series of guide RNAs targeting the branch site of U2 snRNAin vivo based on its performance in the in vitro cleavage assay. Incubation of EU2dT with total RNA and recombinant Rnt1p or cell extract introduces a single cleavage site at the predicted distance from the loop within the U2 branch site  was observed in 53±8% of the cells (data not shown). Therefore, the maximum expected inhibition level of the targeted RNA by any electroporated guide is about 50%. Induction of U2 snRNA cleavage by EU2dT in vivo was monitored by primer extension. As shown in in vivo.To examine the potential of Rnt1p guides as regulators of gene expression in trans makes it appear quite different from the largely non-specific ribonuclease-based RNA degradation. In this study, we have shown that a classical endoribonuclease may use small RNA guides for sequence specific cleavage in analogy to the RNAi degradation mechanism. Rnt1p, a member of the RNase III family, was shown to form an RNP complex with a specific RNA hairpin and uses it to guide the cleavage of targeted RNA in trans. This feature allowed us to transform Rnt1p from a structure-based enzyme into a sequence-specific RNP, supporting evolutionary models explaining the origin of RNA guided protein complexes. The capacity of short and largely single-stranded RNA to guide RNase III cleavage demonstrates the flexibility and tremendous potential of these enzymes as regulators of gene expression.RNAi and RNA turnover are often treated as two distinct mechanisms of gene regulation despite the fact that RNAi is in essence a process of targeted RNA degradation. The sequence specificity of RNAi-based gene silencing and its induction by small RNA in transThe debate about the evolutionary origin of RNA-protein complexes started soon after the discovery of the first catalytic RNAin vivo. Chemically modified RNA like the 2′-O-methylribonucleotide form that is very popular for gene silencing in mammalian cells may now be successfully introduced using electroporation and could be tested for the first time in yeast.Yeast is the most studied eukaryotic model mainly because of the powerful genetic and molecular biology tools available for both gene and genome analysisCurrently, RNAi is the most successful method for design-based gene silencing. In vertebrates and many eukaryotes the introduction of short RNA duplex with sequence specific to any target genes of choice has various success rates. However, the main problem with this approach is target specificity and secondary effects triggered by the introduction of dsRNA or by the induction of the RNAi mechanism. In contrast, targeting RNA for cleavage using guide RNA uses a largely single-stranded RNA and introduces a single cleavage site that leads to RNA degradation using the normal degradation machinery (e.g. exosome). Thus, this method should reduce secondary effects associated with RNAi. In yeast, this method is restricted to nuclear RNA since Rnt1p is localized in the nucleusMATa, trp1, ura3-52, ade2-101, his3, lys2, snr20::LYS2)Pvu II fragments from pRS315/U2 and pRS315/U2δStemNheI site of pRS314/U2δStem. The strain YHM111 was transformed with pRS314/U2 or pRS314/U2δStem/L2 to yield strains YHM111-U2 and YHM111-U2L2.Yeast was grown and manipulated according to standard proceduresE.coli RNase III were produced in bacteria and purified as described before32P] ATP as describedIn vitro cleavages were performed by incubating the different RNA substrates with 20–80 nM of enzyme for 20 minutes at 30°C (or 37°C for Dicer). The reactions were carried out in 20 µl of cleavage bufferE. coli RNase III. For Pac1, the substrates were incubated in the presence of 80 nM of enzyme for 20 minutes at 30°C in 20 µl reaction bufferRecombinant Rnt1p, Pac1, and 2, 0.1 mM dithiothreitol (DTT), and 0.1 mM EDTA pH 7.5) for 5 minutes at 30°C. After incubation, 20% glycerol was added and 4 µl of each reaction was fractionated on 12% non-denaturing polyacrylamide gels. Both bound and unbound RNA fractions were quantified using Instant Imager . Each experiment was repeated twice.RNA binding experiments were performed using guide concentrations that ranged between 0.08 and 0.64 µM and 0.32 µM of unlabelled target RNA spiked with a trace of labeled target in 20 µl reaction buffer . The growing cells were collected and the extracts prepared as previously describedPrimer extension was performed essentially as described beforeThe northern blots were performed essentially as described beforeDraI, 97 nt bases upstream the mature U2 3′-end. The probe covers 97 bases in the mature U2 snRNA and 480 bases downstream of the mature 3′-end. A probe complementary to actin was derived from T7 transcription of the plasmid pKS/Actin digested with HindIII. Total yeast RNA (5 µg) was incubated at 42°C for 12 h with ∼105 C.P.M. of probe in 80% formamide hybridization bufferA probe complementary to the 3′-end of U2L2 was derived from T7 transcription of the plasmid pRS314/U2δStem/L2 digested with 600 of 0.8. The culture was chilled on ice for 30 min and centrifuged at 5000 rpm for 5 min at 4°C. The supernatant was discarded and the pellet was washed twice in 50 ml ice-cold sterile water. After the second centrifugation, the pellet was resuspended in 20 ml ice-cold 1M sorbitol and centrifuged at 5000 rpm for 5 min at 4°C. The final pellet was resuspended in 500 µl of 1M sorbitol and used directly for electroporation. For electroporation, 40 µl of yeast suspension per transformation was used with RNA guide (0.2 to 4 nmols). The pulse was performed at 1.5 kV, 25 µF, and 200 Ω with the Bio-Rad MicroPulser . Immediately after the pulse, 1 ml of ice -cold 1M sorbitol was added and transferred into a tube containing 4 ml of YC-trp media for incubation at 30°C.Electrocompetent cells were prepared as described beforeFigure S1Mapping guide-induced RNA cleavage by RNase IIIs. The substrate 3′-Branch and the RNA target (TL) in the RNA/target complexes EL9-11:TL and EL3′-11:TL were 5′-end labeled and incubated with Rnt1p (A), bacterial RNase III (B), S. pombe Pac1 (C), and human Dicer (D) in presence of Mg2+ and the cleavage products were mapped. The black and gray arrowheads indicate cleavage sites when the reactions were performed at 10 and 150 mM monovalent salt concentration respectively.(0.23 MB TIF)Click here for additional data file.


Caenorhabditis elegans male exhibits a stereotypic behavioral pattern when attempting to mate. This behavior has been divided into the following steps: response, backing, turning, vulva location, spicule insertion, and sperm transfer. We and others have begun in-depth analyses of all these steps in order to understand how complex behaviors are generated. Here we extend our understanding of the sperm-transfer step of male mating behavior.The unc-18, a previously reported member of the Sec1/Munc-18 (SM) family of proteins that is necessary for regulated exocytosis in C. elegans motor neurons. Our allele, sy671, is defective in two distinct sub-steps of sperm transfer: initiation and continued transfer. By a series of transgenic site-of-action experiments, we found that motor neurons in the ventral nerve cord require UNC-18 for the initiation of sperm transfer, and that UNC-18 acts downstream or in parallel to the SPV sensory neurons in this process. In addition to this neuronal requirement, we found that non-neuronal expression of UNC-18, in the male gonad, is necessary for the continuation of sperm transfer.Based on observation of wild-type males and on genetic analysis, we have divided the sperm-transfer step of mating behavior into four sub-steps: initiation, release, continued transfer, and cessation. To begin to understand how these sub-steps of sperm transfer are regulated, we screened for ethylmethanesulfonate (EMS)-induced mutations that cause males to transfer sperm aberrantly. We isolated an allele of Our division of sperm-transfer behavior into sub-steps has provided a framework for the further detailed analysis of sperm transfer and its integration with other aspects of mating behavior. By determining the site of action of UNC-18 in sperm-transfer behavior, and its relation to the SPV sensory neurons, we have further defined the cells and tissues involved in the generation of this behavior. We have shown both a neuronal and non-neuronal requirement for UNC-18 in distinct sub-steps of sperm-transfer behavior. The definition of circuit components is a crucial first step toward understanding how genes specify the neural circuit and hence the behavior. C. elegans affords the opportunity to study a complex multi-step behavior in an organism with a relatively simple nervous system.To execute a complex behavior, an animal must perceive and integrate information from various sources, process this information and perform the appropriate task. In many animals, one such complex behavior is mating, as different aspects of this behavior must be coordinated to ensure sexual reproduction. Male mating behavior in C. elegans males exhibit a highly reproducible series of behavioral steps, culminating in the fertilization of hermaphrodite eggs  ; syEx302 . The following strains are from this work: PS4218 (him-5(e1490); unc-18(sy671)), PS4219 (plg-1(e2001); him-5(e1490); sy672), PS5107 (pha-1(e2123); him-5(e1490); syEx783[unc-18::YFP]), PS4894 (him-5(e1490); unc-18(sy671); syEx694[unc-18::UNC-18::YFP]), PS4902 (him-5(e1490); unc-18(sy671); syEx697[int2itr-1::UNC-18::CFP]), PS5081 (him-5(e1490); unc-18(sy671); syEx784[gpa-1::UNC-18::YFP]), PS4900 (him-5(e1490); unc-18(sy671); syEx695[unc-119::UNC-18::YFP]), PS4899 (him-5(e1490); unc-18(sy671); syEx691[myo-3::UNC-18::YFP]), PS5125 (him-5(e1490); unc-18(sy671); syEx785[aex-3::UNC-18::YFP]), PS5124 (him-5(e1490); unc-18(sy671); syEx786[unc-25::UNC-18::YFP]), PS5122 (him-5(e1490); unc-18(sy671); syEx800[unc-4::UNC-18::YFP]), PS5123 (him-5(e1490); unc-18(sy671); syEx787[unc-17::UNC-18::YFP]), PS5120 (him-5(e1490); unc-18(sy671); syEx788[cho-1::UNC-18::YFP]), PS4901 (him-5(e1490); unc-18(sy671); syEx696[acr-5::UNC-18::YFP]), PS5073 (him-5(e1490); unc-18(sy671); syEx789[acr-2::UNC-18::YFP]), PS5077 (pha-1(e2123); him-5(e1490); syEx790[acr-5::DsRed2]), PS5076 (pha-1(e2123); him-5(e1490); syEx791[acr-5::YFP]), PS5078 (pha-1(e2123); him-5(e1490); syEx792[acr-5::CFP]), PS5072 (pha-1(e2123); him-5(e1490); syEx793[acr-5::GFP]), PS5075 (pha-1(e2123); him-5(e1490); syEx794[acr-2::YFP]), PS5080 lin-39 (n1880); him-5(e1490); syEx795[acr-5::GFP]).rotocols . The wilon LGIII  and him-) on LGV  and was 1(e2123) , lin-39(9(n1880) , lin-39(lin-39n17 [60] LGI-31e169) LGIV and LGIV and15(n484) , unc-18(-2(e678)  LGX. Intunc-18 locus (unc-18(sy671) in trans to unc-18(b403)) we created the strain in a tra-2 background in which XX animals are males ..him-5 musy671 and sy672. The sy671 animals were defective in initiation, while sy672 animals were defective for continued transfer. sy671 animals were out-crossed at least three times with N2 strains before subsequent assays.Using this approach, approximately 95% of the initial F3 populations were eliminated as either plug-positive or sterile, leaving 5% enriched for mating-defective mutants. These 'plug-less' lines were subsequently assayed by observing mating behavior directly, and assigned a corresponding defect. From the 1400 haploid genomes screened, we isolated two sperm-transfer-defective mutants, For SNP mapping we used the Hawaiian CB4856 strain as the source of the polymorphisms . To anal• Chromosome I: ZC123:21628, Y71G12A:7551, W03D8:34384, F57C9:23691, T07D10:11633• Chromosome II: T01D1:3656, T13C2:22236, Y51B9A:7379, Y38F1A:41752• Chromosome III: H06104:15629, T28D6:5428• Chromosome IV: C45G7:33902, ZK792:2021, W02A2:25818, Y41E3:104573• Chromosome V: B0213:10033, T25E12:1056• Chromosome X: ZC449:15947, K10C2:8278, C01C10:13818, F26A10:1544, ZK154:6193, C54D2:9060, F45E1:9628, F11A1:9511, R04E5:10898, C05E7:28741.In addition, the region between ZK154 and F26A10 on chromosome X did not have any SNP data; therefore, we sequenced three intergenic 1.5-kb regions in this interval from the CB4856 strain to find polymorphisms. Two of the three sequenced fragments revealed the new SNP markers C47C12:6306 and F27D9:6408. Primers and information about these markers are detailed below.NdeII (GATC) gives two fragments in N2 (93 and 727 bp) and three fragments in CB4856  as there is an A (N2) to T (CB4856) change (underlined) in the following sequence: TCTTTCACTTTCCAATGTGAACTGATATATATTGATGGGGT.• C47C12:6306: amplification with the following primers, C47C12:6306A (5'-ATTTCTTGCTCTGCTTCAACATCC-3') and C47C12:6306B (5'-GAACATGACAGTAAGCAATCACG-3') amplifies an 820-bp fragment. Digestion with AGAAAAATCTGTATTATTGTA. As no restriction endonuclease exists that can resolve this change, sequencing is required in order to genotype.• F27D9:6408: amplification with the following primers, F27D9:6408E (5'-GAGACCTCCTTGCTCAATGACC-3') and F27D9:6408F (5'-ACGTGACAATATACGAGATTGAGC-3') amplifies a 786-bp fragment. The change is A (N2) to T (CB4856) at nucleotide 626 in the following sequence: AGTATAGAATTCCACGTGACsy671. These populations were used to map sy671 to the X chromosome, between the markers ZC449 and F11A1. The numbers adjacent to the marker shown below are the number of recombinant chromosomes/total analyzed for that marker:Our initial mapping populations included 18 mutant and six wild-type F3 populations  from a cross between the Hawaii strain (CB4856) and • Chromosome I: ZC123 (22/32), Y71G12A (18/48), W03D8 (15/48), F57C9 (12/36), T07D10 (13/34)• Chromosome II: T01D1 (14/30), T13C2 (25/36), Y51B9A (22/36), Y38F1A (20/34)• Chromosome III: H06104 (17/36), T28D6 (16/36)• Chromosome IV: C45G7 (8/36), ZK792 (14/38), W02A2 (10/48), Y41E3 (8/36)• Chromosome V: B0213 (14/48), T25E12 (13/48)• Chromosome X: ZC449 (4/48), F45E1 (0/48), R04E5 (0/22), C05E7 (11/36).lon-2(e678) unc-18(sy671) egl-15(n484) for further SNP mapping. To help avoid potential modifiers of mating behavior from the Hawaii strain, we intragressed the CB4856 sequence into the lon-2 egl-15 interval of N2 by crossing CB4856 with the lon-2 egl-15 double mutant and selecting a non-Lon non-Egl F3 population (three times). The intragressed CB4856 strain was checked with internal SNP markers and then crossed with lon-2 (e678) unc-18(sy671) egl-15(n484). In total, 99 Lon non-Egl recombinants were selected, made homozygous, and used for mapping sy671 to a five-cosmid interval.For generation of additional recombination events in our interval of interest, we generated the triple mutant unc-18(sy671) at 20 ng/μl with myo-2::GFP (5 ng/μl) as a co-injection marker and pBSKS(175 ng/μl) as a carrier. In addition, a rescuing 7.2-kb PCR fragment was amplified using the following primers F27D9P5F (5'-TCGTGACGATCTAGAAGTGGCATTCC-3') and F27D9P5R (5'-TTGGCTTCTCAACGTGGAATGACTGG-3') and purified using the QiaQuick PCR purification kit. The PCR fragment was injected at the same concentration as the cosmid. Transgenic animals were recognized by myo-2::GFP expression in the pharynx. Rescue was also achieved using a 5-kb fragment containing 2.6 kb of 5' upstream promoter sequence and the entire genomic region of unc-18.All cosmids, including the rescuing cosmid F27D9, were injected into unc-18 coding region were amplified from sy671 mutant DNA using PCR with the following primer pairs: F27d9.1A (5'-TTTCCGTCTCATGTTCTTCGCTCC-3') and F27d9.1B (5'-TTCTCTCAAAGTCAGCACGGTACC-3'), F27d9.1C (5'-TGCGCAACACTTGGAGAATATCC-3' and F27d9.1D (5'-TGATTGTTTCCTTATCAGCCATGG-3'), and F27d9.1E (5'-GATGGTGCCACTTTTGATTGACC-3') and F27d9.1F (5'-CTGGTGGGAGAATAAGAAAATTCC-3'). The PCR products from two independent reactions for each pair were separately purified using a QIAquick PCR purification kit and sequenced directly. The G→A nucleotide change causing the sperm-transfer phenotype in sy671 was found at nucleotide 346 using the E and F primer set. The mutation is in the following sequence: GCAAGTTTACCAATCTTCCCGCTGGGTTCCAGTTATCAAGG. As this mutation creates an additional site for restriction endonuclease digestion using TspRI, the mutation was rechecked by TspRI digestion of the PCR product. The Arg476His change is numbered according to UNC-18 isoform A. Currently there are three described isoforms A, B, and C  and Ex[Pint2itr-::unc-18::cfp] transgenes were scored as L4 males to determine if either YPF or CFP fluorescence was lacking in these animals. Animals completely lacking either Punc119::unc-18::yfp or Pint2itr-::unc-18::cfp were identified using a Zeiss M2Bio stereofluorescence microscope with the appropriate filter. These animals were then assayed for mating behavior, and then loss of fluorescence confirmed using conventional fluorescence microscopy (Zeiss Axioskop) after assay.Progeny of Individual males were placed in a 7-μl drop of sperm media  containiunc-18 reporter construction (unc-18::YFP), the unc-18 promoter was amplified by PCR using N2 genomic DNA as the template with the primers 5'-AAACTGCAGTGAAGGACAATGAACTAGAGGGAC-3' and 5'-CGCGGATCCGTGCCCAACGATTTGTTTGAGTGAC-3'. The PCR product was digested with BamHI and PstI, and ligated into BamHI/PstI-digested pSX95.77 (courtesy of S. Xu). This plasmid contains the yfp coding region followed by the unc-54 3' UTR from pPD136.64 (a gift of A. Fire). unc-18::YFP was injected at 75 ng/μl with pBX-1 [pha-1(e2123ts); him-5(e1490) animals [unc-18::YFP expression.For  animals . Males func-18 rescue construct (unc-18::UNC-18::YFP) was made using the following primers, 5'-AAAACTGCAGGTGACGATCTAGAAGTGGCATTCC-3' and 5'-CGCGGATCCTATGTCACGCGGTTTGTTCAGG-3'. We amplified by PCR a 5-kb fragment that contained a 2.4 kb sequence upstream of the unc-18 start codon through the entire coding region, except the stop codon, using cosmid F27D9 DNA as a template. This fragment was digested with PstI and BamHI, and was cloned in-frame into the corresponding sites of pSX95.77, creating a YFP-tagged form of UNC-18 (unc-18::UNC-18::YFP). This plasmid was injected into unc-18(sy671) at 100 ng/μl with myo-2::GFP (5 ng/μl) and pBSKS (95 ng/μl).The unc-18 through the end of unc-54 was amplified by PCR from the unc-18::UNC-18::YFP plasmid using the following primers: 5'-AAAACTGCAGAAAAATGTCACTCAAACAAATCGTTGG-3' and 5'-CCCAAGCTTTGATGCGGTATTTTCTCCTTACG-3'. The PCR product was digested with HindIII and PstI, and ligated into HindIII/PstI-digested pBSKS, creating pSOAunc-18.For site-of-action studies, a promoterless 4.2-kb fragment from the ATG of myo-3::UNC-18::YFP we amplified by PCR the myo-3 promoter region [NotI, ligated into NotI-digested pSOAunc-18, and the orientation checked.To create r region  using thmyo-3::UNC-18::YFP (above), except that the primers for amplification of the appropriate promoter regions were unique:The following were constructed as for • unc-17 : 5'-ataa• gpa-1 (Jiang LI and Sternberg PW unpublished observations): 5'-ATAAGAATGCGGCCGCTGCTTGAAGAACACAGTATCTACG-3' and 5'-ATAGTTTAGCGGCCGCCTGAAGTCTTCGAATAAATGACATTG-3'.unc-119::UNC-18::YFP plasmid, we amplified by PCR the unc-119 promoter region [NotI and PstI, and ligated into NotI/PstI-digested pSOAunc-18.To create the r region  using thunc-119::UNC-18::YFP (above), except the primers for amplification of the appropriate promoter regions were unique:The following were constructed as for • acr-2 : 5'-ATAA• acr-5 : 5'-ATAA• aex-3 : 5'-ATAA• int2itr-1: 5'-ATAAGAATGCGGCCGCTGAGGAACAAATTATGAGGAAATCCAGG-3' and 5'-CCAATGCATTGGTTCTGCAGGAAGCAATGGATGACTGTGCTTTTAGC-3'• unc-25 : 5'-ATAA• unc-4 : 5'-ATAAunc-119 reporter (unc-119::YFP) was created by digesting unc-119::UNC-18::YFP with PstI and SmaI to drop out the unc-18 coding region, followed by removal of the 3' overhang with T4 polymerase and re-ligation.The acr-5::YFP; acr-2::YFP; aex-3::YFP, and int2itr-1::YFP were all constructed by excising the unc-18 region from the appropriate construct above with PstI and SmaI, following which the resulting vector fragment was blunt-ended with T4 polymerase and the vector relegated. acr-5::GFP was constructed by swapping the yfp from acr-5::YFP with a nuclear localized version of gfp from pPD95.70 (a gift of A. Fire).acr-5::DsRed2 reporter was constructed as follows. The acr-5 promoter was removed by linearization of acr-5::UNC-18::YFP by digestion with NotI, followed by Klenow treatment and subsequent digestion with PstI, creating a blunt/PstI promoter fragment. This fragment was ligated into a vector containing DsRed2 followed by the unc-54 3' UTR (PSX77-DsRed2 courtesy of S. Xu) that was prepared by digestion with HindIII, Klenow-treated, and subsequently cut with Sse8387 I.The cho-1 [BssHII fragment containing the ATG of unc-18 through the end of the unc-54 3' UTR from pSOAunc-18, treated it with T4 polymerase, and blunt-end cloned it into the EcoRV site in pBR322. We then PCR amplified the cho-1 promoter with the primers 5'-TCCCCGCGGATACTTCAATTATTCCCGTCTTACCAGG-3' and 5' '-TCCCCGCGGTAAGATAGAGGAACTCGCAAGATGTCG-3', digested with SacII, and cloned into the SacII site carried over to pBR322 and checked the orientation.Because the promoter region of cho-1  was not int2itr-1::UNC-18::CFP, we digested int2itr-1::UNC-18::YFP with NotI/SmaI to excise the int2itr-1 promoter followed by the unc-18 coding region (leaving the yfp and unc-54 3' UTR sequence) and ligated in frame into pBSKS containing the cfp coding region followed by the unc-54 3' UTR.To make Cell anatomy was observed with Nomarski optics, and GFP expression was analyzed by conventional fluorescence microscopy (Zeiss Axioskop) using a Chroma Technology High Q GFP long-pass filter set . CFP and YFP were visualized using a Chroma Technology CFP filter set  and a YFP set . Photographs were taken with a digital camera using Improvision Openlab software to obtain images, and images processed using Adobe Photoshop version 7.0.GS, AJW and PWS conceived the study. GS, AJW, JYT, and SG all contributed to the genetic screening and the initial characterization of isolated male mating-behavior mutants. GS performed laser ablations, genetic manipulations, and behavioral assays on transformants. JYT was involved in the behavioral assays on transformants. GS drafted the manuscript, with AJW, JYT, SG, and PWS revising it critically for important intellectual content. All authors read and approved the final manuscript.Initiation. The initiation step of sperm transferClick here for fileRelease and continued transfer. The release and continued transfer steps of sperm transferClick here for fileCessation. The cessation step of sperm transferClick here for fileResults from individual lines complied for Table 1Click here for fileResults from individual lines complied for Table 3Click here for fileNeuronal and non-neuronal expression of UNC-18 are necessary for sperm transfer.Click here for file


Ribonucleic acid (RNA) is a dogma breaker. The “central dogma” of cellular biochemistry mandates that deoxyribonucleic acid (DNA) stores information, and RNA copies this information and uses it to direct the assembly of amino acid building blocks into proteins, such as enzymes. Enzymes catalyze important chemical reactions in the cell, such as the breakdown of glucose or the synthesis of urea.When biochemists discovered catalytic RNA, they had to ditch the dogma. Because of its structure, it turns out, RNA can act as an enzyme and catalyze reactions. While two strands of DNA tend to zip up into the famous double helix, RNA usually goes solo. The single RNA strand folds back on itself to create myriad tangled arrangements. Some of these arrangements create an active center, the place on the RNA where the enzymatic magic happens. The many RNA enzymes and protein enzymes that use metal atoms to do their job are called metalloenzymes. One example of an important structural motif in RNA metalloenzymes is the group I intron, which can snip itself out of an RNA segment. Understanding exactly how the RNA and the metals interact will help to provide precise answers about how the enzyme really works.Ppro-S phosphoryl oxygen at nucleotide C262 in the intron from the unicellular Tetrahymena thermophila protozoan.Through X-ray crystallography, researchers have revealed many structural features of group I introns. But X-ray crystallography creates images of the enzyme frozen in time; it does not catch an enzyme in action. In a new study, Joseph Piccirilli, Daniel Herschlag, and colleagues discovered that a particular oxygen atom on a particular nucleotide in a group I RNA must bind to a particular magnesium ion in order for the reaction under study to proceed normally. The oxygen atom is known as the Since there's no way to watch the oxygen and metal hook up during the reaction, how do the researchers know they do? The researchers used the powerful techniques of metal ion rescue and atomic mutagenesis. Here's how it worked. They figured out how well the group I intron reaction works with a normal enzyme. Then, they replaced the oxygen in question with a sulfur atom. The reaction didn't work as well because, by the rules of chemistry, sulfur doesn't like to bind to magnesium. But sulfur does like manganese and cadmium ions. So they replaced the magnesium with one of these other metal ions and measured the reaction. The researchers saw that these other metal ions restored (or “rescued”) enzymatic activity. In short, the enzyme needs a bond where the oxygen and the magnesium are, but the bond doesn't have to be between oxygen and magnesium.As complicated as that is, plucking out one atom and trading it for another is itself a tricky business. Because most enzymes are made of stubborn amino acids and not nucleotides, atomic mutagenesis can be difficult. And usually when researchers have tried atomic mutagenesis, they've mutated the substrate (the molecule that the reaction acts upon) instead of the enzyme (the molecule that acts). Here, Piccirilli, Herschlag, and colleagues directed the applications of atomic mutagenesis to the molecule that does the work. To test that a specific oxygen in the intron binds to the magnesium ion, the researchers first had to compile a short list of potential atoms to which the magnesium might bind. By combining literature data from structural models and functional studies with a random sprinkling of sulfur atoms in the intron to find critical oxygen contacts, Piccirilli, Herschlag, and colleagues established a group of specific oxygen atoms to watch. They tried the metal rescue experiment with each of these oxygens, and the only enzyme rescued by the metal switch was the one in which they changed the C262 oxygen to a sulfur. Therefore, they concluded that this specific oxygen atom makes a critical contact with the magnesium ion. The strategy of atomic mutagenesis combined with metal ion rescue can be used to help understand the mechanism of other RNA and protein metalloenzymes.