From a screen for suppressors of the Caenorhabditis elegans unc-93(e1500) rubberband Unc phenotype, we identified mutations in genes that encode the C. elegans orthologs of two splicing factors, the U2AF large subunit (UAF-1) and SF1/BBP (SFA-1). The uaf-1(n4588) mutation resulted in temperature-sensitive lethality and caused the unc-93 RNA transcript to be spliced using a cryptic 3′ splice site generated by the unc-93(e1500) missense mutation. The sfa-1(n4562) mutation did not cause the utilization of this cryptic 3′ splice site. We isolated four uaf-1(n4588) intragenic suppressors that restored the viability of uaf-1 mutants at 25°C. These suppressors differentially affected the recognition of the cryptic 3′ splice site and implicated a small region of UAF-1 between the U2AF small subunit-interaction domain and the first RNA recognition motif in affecting the choice of 3′ splice site. We constructed a reporter for unc-93 splicing and using site-directed mutagenesis found that the position of the cryptic splice site affects its recognition. We also identified nucleotides of the endogenous 3′ splice site important for recognition by wild-type UAF-1. Our genetic and molecular analyses suggested that the phenotypic suppression of the unc-93(e1500) Unc phenotype by uaf-1(n4588) and sfa-1(n4562) was likely caused by altered splicing of an unknown gene. Our observations provide in vivo evidence that UAF-1 can act in regulating 3′ splice-site choice and establish a system that can be used to investigate the in vivo regulation of RNA splicing in C. elegans.The removal of introns from eukaryotic RNA transcripts requires the activities of five multi-component ribonucleoprotein complexes and numerous associated proteins. The lack of mutations affecting splicing factors essential for animal survival has limited the study of the  Caenorhabditis elegans, we found that mutations in two splicing factors, the U2AF large subunit and SF1/BBP suppress the rubberband Unc phenotype caused by a rare missense mutation in the gene unc-93. Mutations affecting the U2AF large subunit caused the recognition of a cryptic 3′ splice site generated by the unc-93 mutation, providing in vivo evidence that the U2AF large subunit can affect splice-site selection. By contrast, an SF1/BBP mutation that suppressed the rubberband Unc phenotype did not cause splicing using this cryptic 3′ splice site. Our genetic studies identified a region of the U2AF large subunit important for its effect on 3′ splice-site choice. Our mutagenesis analysis of in vivo transgene splicing identified a positional effect on weak 3′ splice site selection and nucleotides of the endogenous 3′ splice site important for recognition. The system we have defined should facilitate future in vivo analyses of pre–mRNA splicing.Eukaryotic genes contain intervening intronic sequences that must be removed from pre-mRNA transcripts by RNA splicing to generate functional messenger RNAs. While studying genes that encode and control a presumptive muscle potassium channel complex in the nematode  C. elegans, mRNA transcripts of at least 13% of predicted genes are alternatively spliced Drosophila gene Dscam (Down syndrome cell adhesion molecule), which through alternative splicing could potentially generate over 30,000 isoforms Eukaryotic genes contain intervening introns that are spliced from transcribed pre-mRNAs to generate functional coding mRNAs splicing factor one/branch-point binding protein) and the large and small subunits of U2AF (U2 auxiliary factor) Saccharomyces cerevisiae lacks a U2AF small subunit and a polypyrimidine sequence in its introns, and the recognition of a 3′ splice site is achieved by binding of SF1/BBP to a highly conserved consensus branch-point sequence Caenorhabditis elegans, there is no consensus branch-point sequence or long polypyrimidine sequence, and the recognition of a 3′ splice site is achieved by the binding of the U2AF large and small subunits to a consensus UUUUCAGR sequence in which “AG” is the 3′ splice site Pre-mRNA splicing involves five small nuclear ribonucleoprotein particles (snRNPs) and numerous associated factors rginine-Serine-rich RNA-binding SR proteins Splicing is also regulated by many AC. elegans is a genetically tractable organism and has been used to study a broad variety of biological problems. Our laboratory has analyzed a set of genes, unc-93, sup-9 and sup-10, that encode components of a presumptive C. elegans two-pore domain K+ channel complex and regulate muscle activity gain-of-function (gf) mutations in any of these three genes cause abnormal body-muscle contraction and are thought to activate the SUP-9 K+ channel. The gf mutant animals are defective in egg laying, sluggish and exhibit a rubberband phenotype: when prodded on the head, the animal contracts and relaxes along its entire body without moving backwards. Complete loss-of-function (lf) mutations of unc-93, sup-9 and sup-10 do not cause any obvious abnormalities Two-pore Acid Sensitive K+ channels TASK-1 and TASK-3 sup-10 encodes a novel single-transmembrane domain protein without identified mammalian homologs unc-93 encodes a multiple transmembrane-domain protein that defines a novel family of proteins conserved from C. elegans to mammals unc-93, sup-9 and sup-10 genes were not designed to identify genes essential for fertility or animal survival. To seek such essential genes, we performed a clonal genetic screen for suppressors of the locomotion defect caused by the unc-93 gf mutation e1500. In this paper, we describe our studies of two suppressors identified from this screen and the establishment of a reporter system for in vivo analysis of RNA splicing in C. elegans. We suggest that the U2AF large subunit affects 3′ splice site recognition and that some aspect of the function of the putative UNC-93/SUP-9/SUP-10 two-pore domain potassium channel complex depends on an unidentified gene the processing of which requires the functions of the U2AF large subunit and SF1/BBP.Previous genetic screens for genes that affect the activities of C. elegans genes unc-93, sup-9 and sup-10 cause a rubberband Unc phenotype, while lf mutations in these genes result in a phenotypically wild-type phenotype unc-93(e1500) mutation, with the goal of identifying mutations that also cause sterility or lethality  of P0 animals mutagenized with EMS (ethyl methanesulfonate) and isolated the suppressors n4588 and n4562. n4588 causes embryonic lethality at 25°C, and n4562 causes sterility at all temperatures. By mapping these mutations, we found that n4588 and n4562 are not alleles of any previously characterized suppressors of the rubberband Unc phenotype (see Rare gf mutations in the lity see . We scretype see .n4588 is a strong recessive suppressor of the locomotion defect and rubberband phenotype of unc-93(e1500) animals (n4588 is or is closely linked to a mutation that causes a recessive temperature-sensitive (ts) lethal phenotype and results in embryonic lethality at 25°C  C. elegans, together with the U2AF small subunit ortholog UAF-2, UAF-1 binds a consensus UUUUCAGR sequence, in which AG is the 3′ splice site RNA recognition motif) domains (U2AF homology motif) domain that binds the splicing factor SF1/BBP n4588 mutation lies between the U2AF small subunit-interacting domain and the first RRM domain of UAF-1a  unc-93(e1500) animals  in wild-type animals did not cause a rubberband Unc phenotype or any other visible abnormality (data not shown). Introducing stop codons or the n4588 T180I mutation into the full-length uaf-1a cDNA abrogated its rescuing activity (uaf-1a cDNA under control of a heat-shock promoter unc-93(e1500) by uaf-1(n4588) and the ts-lethality caused by uaf-1(n4588) (unc-93(e1500) animals with uaf-1 RNAi-expressing bacteria (uaf-1 is required for the rubberband Unc phenotype caused by unc-93(e1500).To test whether the point mutation found in the  animals . A predihenotype . Expressactivity . Heat-shbacteria  also parbacteria , suggestuaf-1 deletion mutation, n5222Δ, which removes the fourth exon (encoding part of the first RRM and part of the second RRM of UAF-1a) of the uaf-1a isoform (uaf-1a isoform are spliced together. uaf-1(n5222Δ)/+ animals grew and moved like the wild type, and uaf-1(n5222Δ)/+ did not suppress the rubberband Unc phenotype of unc-93(e1500) animals (data not shown). uaf-1(n5222Δ) homozygous mutants arrested and died at the late L1 to early L2 larval stages (based on body size), which precluded examination of the rubberband Unc behavior of n5222Δ homozygous animals (see uaf-1(n4588)/uaf-1(n5222Δ) suppressed the rubberband Unc phenotype of unc-93(e1500) animals as strongly as did homozygous uaf-1(n4588) (uaf-1(n4588) homozygotes, uaf-1(n4588)/uaf-1(n5222Δ) animals died embryonically at 25°C (data not shown). These results establish that n4588 is an allele of uaf-1 and that reducing the dosage of the uaf-1(n4588) allele by 50% does not affect the suppression of the rubberband Unc phenotype of unc-93(e1500) animals. These data suggest that uaf-1(n4588) causes either a reduction/loss of uaf-1 activity or an altered uaf-1 activity that is antagonized by the wild-type uaf-1 gene  . Similargene see .unc-93(e1500) are also caused by the unc-93(n200) mutation sup-9 and sup-10uaf-1(n4588) mutation could suppress the Unc phenotype caused by these other mutations (unc-93(n200) was suppressed by uaf-1(n4588) (sup-10(n983), which causes a rubberband Unc phenotype that is more severe than that of unc-93(n200) animals but less severe than that of unc-93(e1500) animals, was completely suppressed by uaf-1(n4588) (sup-9(n1550)uaf-1(n4588) (uaf-1(n4588) is an allele-specific suppressor of unc-93 but not a gene-specific suppressor of the rubberband Unc mutants and is distinct in its suppression pattern from other known suppressors of unc-93, sup-9 and sup-10 (see Locomotion defects similar to those caused by utations . Neither1(n4588) . sup-10(1(n4588) . The str1(n4588) . These dp-10 see .unc-93, sup-10 and sup-9 do not cause visible abnormalities in a wild-type background uaf-1, by generating double mutants containing uaf-1(n4588) and null mutations of unc-93, sup-10 or sup-9. We found that such double mutant animals grew and behaved indistinguishably from uaf-1(n4588) single mutant animals  can suppress gf mutations affecting other two-pore domain potassium channels, we generated double mutant animals containing the uaf-1(n4588) mutation and the unc-58(e665sd)egl-23(n601sd)twk-18(e1913sd)uaf-1(n4588)  .unc-93, sup-9, sup-10 or any of the other genes known to interact with these genes is reduced in uaf-1(n4588) animals, we examined unc-93, sup-10, sup-9, sup-18sup-11unc-93, sup-10 and sup-9, lf mutations in sup-18 and gf mutations in sup-11 can suppress the rubberband Unc phenotype caused by gf mutations in unc-93, sup-9 and sup-10. Using real-time qRT–PCR, we found no obvious reduction of the mRNA levels of these five genes (uaf-1(n4588) animals (data not shown), suggesting that the suppression of unc-93(e1500) by uaf-1(n4588) is not caused by a reduction of the level of the UAF-1 protein.To determine if the expression of ve genes . We alsounc-93 is altered by uaf-1(n4588). We examined the splicing of each exon of unc-93 in wild-type, uaf-1(n4588), unc-93(e1500), uaf-1(n4588) unc-93(e1500), unc-93(n200) and uaf-1(n4588) unc-93(n200) animals by RT–PCR  unc-93(e1500) animals (data not shown) using the PCR primer pairs at the 3′ end of exon 8 and the 5′ end of exon 9 (indicated in black in unc-93(e1500) animals the region between exon 8 and 9 corresponded to a weak but consistent RT–PCR product of a reduced length (unc-93(e1500) mutant animals (uaf-1(n4588) unc-93(e1500) animals, the RT–PCR product of reduced length was the most prominent product (unc-93(e1500) missense mutation, which has a G-to-A transition that changes amino acid 388 from Gly to Arg unc-93 exon 9 in unc-93(e1500) animals and 68% in uaf-1(n4588) unc-93(e1500) animals (uaf-1(n4588), unc-93(n200) or uaf-1(n4588) unc-93(n200) animals, all of which lack the cryptic 3′ splice site caused by the unc-93(e1500) mutation.We tested whether the splicing of y RT–PCR . Every eexamined . However animals . Both no animals  and quan animals  analysesunc-93 transcript is predicted to encode a truncated protein lacking 12 amino acids in one of the predicted transmembrane domains unc-93 transcript in uaf-1(n4588) unc-93(e1500) animals encoded a functional UNC-93 protein, we expressed the cDNA in the body-wall muscles of sup-9(n1550); unc-93(lr12Δ) animals unc-93 cDNA in these animals restored the severe rubberband Unc phenotype. These results suggested that the alternatively spliced unc-93 transcript encoded a lf UNC-93 protein or possibly a dominant-negative UNC-93 protein. To test the latter possibility, we expressed either unc-93 wild-type cDNA or the alternatively spliced unc-93 cDNA in the body-wall muscles of unc-93(e1500) animals (unc-93(e1500)/+ animals have better locomotion than unc-93(e1500) animals unc-93 cDNA dramatically improved the locomotion of unc-93(e1500) animals (unc-93 transcript encoded an UNC-93 protein that could interfere with the endogenous UNC-93 function and cause the suppression of the rubberband Unc phenotype by uaf-1(n4588) , the transgene should also suppress the Unc phenotype of unc-93(e1500) animals. However, expression of the alternatively spliced unc-93 transcript in the body-wall muscles did not suppress the Unc phenotype of unc-93(e1500) animals  mutation, would alter the splicing of unc-93(e1500) exon 9, we fed animals with bacteria expressing dsRNA targeting uaf-1 and assessed unc-93 exon 9 splicing. As shown in unc-93(e1500) exon 9. The RNAi treatment did significantly reduce the level of UAF-1 protein  exon 9 similarly to that by the uaf-1(n4588) mutation is consistent with the hypothesis that uaf-1(n4588) does not reduce the function of UAF-1a but rather alters the function of UAF-1a, which leads to the recognition of the cryptic 3′ splice site of unc-93(e1500) exon 9 (see uaf-1(n4588) reduces uaf-1 function and that uaf-1(RNAi) does not reduce uaf-1 function as much.To examine whether reducing UAF-1 expression, like the  protein . That reon 9 see . Howeveruaf-1(n4588) suppressed the rubberband Unc phenotype of sup-10(n983) animals but did not suppress the rubberband Unc phenotype of unc-93(n200) and sup-9(n1550) animals (sup-10 mRNA in uaf-1(n4588) animals (sup-10(n983) transcript was alternatively spliced in uaf-1(n4588) mutants. uaf-1(n4588) did not cause the appearance of a sup-10 cDNA band different in size from the full-length sup-10 cDNA , uaf-1(n4588) and uaf-1(n4588); sup-10(n983) animals and failed to identify an alternatively spliced sup-10 transcript (data not shown). animals . Quantit animals . We exam-10 cDNA . We deteuaf-1(n4588) can affect the splicing of all genes known to be alternatively spliced, we tested for genetic interactions between uaf-1(n4588) and unc-52(e669). unc-52 encodes the C. elegans ortholog of human basement membrane-specific heparan sulfate proteoglycan core protein, and mutations affecting unc-52 cause adult paralysis unc-52(e669) can be suppressed by lf mutations of smu-1 and smu-2, genes that encode C. elegans homologs of mammalian splicing factors unc-52(e669) mutation causes a pre-mature stop in unc-52 exon 17 smu-1 and smu-2 lf mutations suppress unc-52(e669) by removing exon 17 and generating an alternatively spliced and functional transcript unc-52(e444) mutation causes a pre-mature stop in unc-52 exon 18, which is not removed in smu-1 and smu-2 mutant animals, leading to a transcript with a premature stop codon. Double mutants containing the unc-52(e444) mutation and the smu-1 or smu-2 mutations display an Unc phenotype unc-52(e669); uaf-1(n4588) and unc-52(e444); uaf-1(n4588) animals and found that uaf-1(n4588) did not suppress either unc-52(e669) or unc-52(e444) (uaf-1(n4588) mutation did not affect the alternative splicing of unc-52(e669) exon 17 and thus does not affect all cases of alternative splicing non-specifically.To test whether 52(e444) , implyinn4562 was also isolated from our clonal screen as a suppressor of the rubberband Unc phenotype of unc-93(e1500) animals. The suppressed phenotype was recessive, and n4562 caused a completely penetrant recessive sterility that was temperature independent and was tightly linked to its suppressor activity (see uaf-1(n4588), n4562 suppressed unc-93(e1500) and sup-10(n983) but did not suppress unc-93(n200) or sup-9(n1550)  . Therefon4562 to the right of LG IV (see unc-93(e1500) are located in this region. The genes uaf-2, encoding the C. elegans U2AF small subunit ortholog Y116A8C.32, encoding the SF1/BBP (splicing factor 1/branch-point binding protein) ortholog n4562. We determined the DNA sequences of coding regions of uaf-2 and Y116A8C.32 from n4562 animals and identified a nonsense mutation in Y116A8C.32, which we named sfa-1  (n4562 changed amino acid 458 from a Cys (TGT) to an opal stop (TGA) codon in a conserved C2HC-type zinc finger domain of the predicted SFA-1 protein (unc-93(e1500) by sfa-1(n4562) by expressing in body-wall muscles an SFA-1::GFP fusion protein driven by the myo-3 promoter unc-93(e1500) animals with bacteria expressing dsRNA targeting sfa-1 partially suppressed the rubberband Unc phenotype (We mapped G IV see . No know factor) . n4562 c protein . This muhenotype .sfa-1 deletion mutation, n5223Δ, which removes the third and fourth exons and a majority of the fifth exon  caused recessive embryonic lethality, and sfa-1(n5223Δ)/+ did not suppress the rubberband Unc phenotype of unc-93(e1500) animals (data not shown). sfa-1(n4562)/(n5223Δ) similarly caused embryonic lethalilty (data not shown), suggesting that the lethal phenotype of sfa-1(n5223Δ) homozygotes is caused by the sfa-1(n5223Δ) mutation. The embryonic lethality caused by sfa-1(n5223Δ) and sfa-1(n4562)/sfa-1(n5223Δ) precluded the use of n5223Δ for an analysis of the rubberband Unc phenotype, because our behavioral assay is performed with young adults (see We isolated an fth exon . Togetheults see .uaf-1(n4588), sfa-1(n4562) caused alternative splicing of unc-93(e1500) exon 9, we used RT–PCR to examine the splicing of unc-93(e1500) exon 9. As shown in sfa-1(n4562) did not cause increased alternative splicing of unc-93(e1500) exon 9. We tested the effect of sfa-1 on unc-93(e1500) exon 9 splicing by reducing sfa-1 expression using RNAi (sfa-1(RNAi) did not increase exon 9 alternative splicing  mutation causes a recessive sterile phenotype, which is less severe than the recessive embryonic lethality caused by sfa-1(n5223Δ)  or by sfa-1(n4562)/sfa-1(n5223Δ), (2) sfa-1(RNAi) phenocopies sfa-1(n4562) in the suppression of the rubberband Unc phenotype of unc-93(e1500) animals, and (3) sfa-1(RNAi) phenocopies sfa-1(n4562) in affecting the splicing of unc-93(e1500) exon 9, we propose that sfa-1(n4562) is a partial lf allele of sfa-1 and that the suppression of the rubberband Unc phenotype of unc-93(e1500) animals by sfa-1(n4562) is likely caused by reduced sfa-1 function.Because (1) the sfa-1(n4562) affects the alternative splicing of unc-93(e1500) exon 9 caused by the uaf-1(n4588) mutation, we generated uaf-1(n4588); sfa-1(n4562) animals with or without unc-93(e1500). Most uaf-1(n4588); sfa-1(n4562) animals (with or without unc-93(e1500)) died embryonically, and the few that hatched arrested at the L2 larval stage (based on body size) (To examine whether dy size) . We failsup-10 splicing in sfa-1(n4562) animals and failed to detect alternative splicing of the sup-10 transcript  offered a genetic approach to seek new regulators of RNA splicing by screening for suppressors of the ts-lethal phenotype. We performed a genetic screen for suppressors of uaf-1(n4588) ts-lethality at 25°C. From this screen, we isolated four intragenic suppressors, n5120, n5123, n5125 and n5127 (uaf-1(n5123) caused an I180F (ATT-to-TTT) change at the same residue mutated by n4588 (T180I) (ACT-to-ATT) (unc-93(e1500) (uaf-1(n4588 n5120) is predicted to cause a V179M (GTG-to-ATG) change in addition to the n4588 T180I mutation (uaf-1(n4588 n5120) weakly suppressed unc-93(e1500) (uaf-1(n4588 n5125) is predicted to cause a P177L (CCA-to-CTA) change in addition to the n4588 T180I mutation (uaf-1(n4588 n5125) also weakly suppressed unc-93(e1500) (uaf-1(n4588 n5127) is predicted to cause an M157I (ATG-to-ATA) change in addition to the n4588 T180I mutation (unc-93(e1500) (unc-93(e1500) suppressor activities of uaf-1(n4588 n5120), uaf-1(n4588 n5125) and uaf-1(n4588 n5127) were caused by uaf-1 mutations was confirmed by the observation that transgenes expressing uaf-1a (unc-93(e1500) suppressor phenotype of these mutants (unc-93(e1500) exon 9 in animals containing these uaf-1 mutations using Taqman RT–PCR. As shown in uaf-1 mutations exhibited differential effects on the splicing of unc-93(e1500) exon 9. uaf-1(n5123) had no apparent effect  alone). uaf-1(n4588 n5120) and uaf-1(n4588 n5125) weakly  and uaf-1(n4588 n5127) moderately (25%) increased the alternative splicing of unc-93(e1500) exon 9 (uaf-1(n4588) (68%)   and elim3(e1500) . uaf-1 . uaf-1 . uaf-1(nmutation  and was 3(e1500) . That thg uaf-1a  in the b mutants . We quan) exon 9 . None of8) (68%) .uaf-1(n4588) could suppress the Unc phenotype of unc-93(e1500) independently of unc-93 splicing, we generated transgenic animals that overexpressed in the body-wall muscles a full-length unc-93 cDNA containing the e1500 gf mutation. Since this cDNA generates a fully spliced form of unc-93 mRNA, if alternative splicing of unc-93(e1500) accounted for the suppression of the rubberband Unc phenotype, overexpressed unc-93(e1500) cDNA would not generate an alternatively spliced isoform and the animals would be as Unc in an uaf-1(n4588) background as in a wild-type background. As shown in unc-93(e1500) cDNA caused a strong rubberband Unc phenotype. The presence of the uaf-1(n4588) mutation reduced the severity of the rubberband Unc phenotype caused by the same transgenes. Similarly, overexpression of the unc-93(e1500) cDNA also caused a weaker rubberband Unc phenotype in sfa-1(n4562) animals than in wild-type animals (uaf-1(n4588) can suppress the unc-93(e1500) rubberband Unc phenotype through mechanism(s) other than by affecting the splicing of unc-93. This finding was consistent with our results showing that although sfa-1(n4562) suppressed the rubberband Unc phenotype of unc-93(e1500) animals, sfa-1(n4562) did not affect the alternative splicing of unc-93(e1500) exon 9, which also suggested that the suppression of unc-93(e1500) by sfa-1(n4562) was mediated by a mechanism other than by affecting the alternative splicing of unc-93  and the GFP reporter gene  and the exon 9 cryptic 3′ splice site (E9) in wild-type and ter gene  and placunc-93(e1500). In wild-type animals the splicing mimics that of the endogenous unc-93(e1500), with very little splicing at E9 (uaf-1(n4588) mutants occurred almost exclusively at E9 (>99%) (unc-93(e1500) was qualitatively but not quantitatively similar, occurring mostly at E9 (68%)  ; endogenE9 (68%) . These ruaf-1(n4588) animals, splicing occurred mostly at the new E9  (uaf-1(n4588) animals  (uaf-1(n4588) animals when two identical 3′ splice sites are present. We next switched the positions of I8 and E9 (uaf-1(n4588) animals, most splicing (80%) occurred at the new E9 (uaf-1(n4588) animals  . When I8ctively) , No. 3, ctively) . These r8 and E9 , No. 4. 8 and E9 . Similare new E9 , No.4. H animals , No. 4 sunc-93 is expressed in body-wall muscles myo-3 promoter myo-3 promoter and the unc-93 promoter (unc-93(e1500) involves a mechanism that is not promoter-specific.The pattern of alternative splicing in cell culture can depend on the promoter used C. elegans consensus 3′ splice site TTTTcag uaf-1(n4588) animals (uaf-1(n4588) animals is similar to that of transgene No.1  animals, splicing occurred exclusively at the new E9 . We next changed each of the non-T bases to T from −7 to −4 of E9 (uaf-1(n4588) animals occurred exclusively at E9   animals, splicing at the new E9 was reduced  mutation by uaf-1(n4588) and sfa-1(n4562) remains to be determined. Four observations indicate that although uaf-1(n4588) causes alternative splicing of unc-93(e1500) exon 9, this alternative splicing is not the basis of the suppression. First, unc-93(e1500)/unc-93(lf) heterozygous animals are as Unc as unc-93(e1500) homozygous animals unc-93 expression by 50% does not reduce the rubberband Unc phenotype. By contrast, uaf-1(n4588 n5127) reduced unc-93(e1500) expression by 25% , and these animals were strongly suppressed. Also, in uaf-1(n4588) unc-93(e1500) animals, the unc-93(e1500) transcript was reduced by 68% , and these animals might have been expected to be slightly less Unc than unc-93(e1500)/unc-93(lf) animals but instead were strongly suppressed. Thus, the level of reduction of the unc-93(e1500) transcript does not correlate with the level of the suppression of the unc-93(e1500) Unc phenotype by uaf-1(n4588 n5127) and uaf-1(n4588). Second, the strong rubberband Unc phenotype caused by overexpression of the unc-93(e1500)-specific cDNA in body-wall muscles was partially suppressed by uaf-1(n4588), suggesting that unc-93(e1500) splicing is not needed for uaf-1(n4588)-mediated suppression. Third, sfa-1(n4562) suppressed unc-93(e1500) without affecting the splicing of unc-93(e1500) exon 9, and sfa-1(n4562) partially suppressed the rubberband Unc phenotype caused by overexpression of the unc-93(e1500) cDNA in the body-wall muscles. Again, suppression can occur without affecting unc-93(e1500) mRNA splicing. Similarly, we did not identify an alternatively spliced sup-10 transcript in either uaf-1(n4588); sup-10(n983) or sfa-1(n4562); sup-10(n983) animals, suggesting that sup-10(n983) was suppressed by uaf-1(n4588) and sfa-1(n4562) by a mechanism other than alternative splicing of the sup-10 transcript. Fourth, reducing the expression of uaf-1 and sfa-1 by RNAi suppressed the rubberband Unc phenotype of unc-93(e1500) but did not cause altered splicing of unc-93(e1500) exon 9. Based on these arguments, we propose that uaf-1 and sfa-1 mutations suppress unc-93(e1500) and sup-10(n983) by affecting the splicing of one or more unidentified genes required for the expression of the unc-93(e1500) and sup-10(n983) rubberband Unc phenotype. We cannot exclude the possibility that the alternative splicing of unc-93(e1500) contributed to the suppression of unc-93(e1500) by uaf-1 mutations.The mechanism(s) of the suppression of the Unc phenotype caused by the unc-93, sup-9 and sup-10 are of three classes. First, lf mutations in any of these three genes are recessive suppressors of the rubberband Unc phenotypes caused by gf mutations in any of these three genes, because the functions of all three genes are necessary for expression of the Unc phenotype sup-11 are strong dominant suppressors of unc-93(e1500) and unc-93(n200) and partial recessive suppressors of sup-9(n1550) and sup-10(n983)sup-11 suppression is unknown. Third, lf mutations of sup-18 are strong recessive suppressors of sup-10(n983) and weak recessive suppressors of unc-93(e1500), unc-93(n200) and sup-9(n1550)sup-18 suppression is also unknown. uaf-1(n4588) and sfa-1(n4562) define a new class of suppressors: they are recessive and allele-specific for unc-93 gf mutations (unc-93(e1500) was suppressed, but unc-93(n200) was not) but not gene-specific (sup-10(n983) was also suppressed). Previous genetic and molecular studies from our laboratory led to the hypothesis that UNC-93, SUP-9 and SUP-10 form a protein complex in the body-wall muscles in vivo functions regulated in different ways. As mentioned above, we propose that mutations in uaf-1 and sfa-1 affect the splicing of one or more unknown genes required for unc-93 and sup-10 activity. This unknown gene might be required specifically for the expression of the rubberband Unc phenotype caused by unc-93(e1500) or sup-10(n983) but have a negligible role in the expression of the rubberband Unc phenotypes caused by unc-93(n200) or sup-9(n1550). Similarly, sup-11 and sup-18 could affect functions of the UNC-93/SUP-9/SUP-10 complex distinct from that affected by uaf-1 and sfa-1.The known suppressors of gf mutations of Saccharomyces cerevisiae and Schizosaccharomyces pombe and the fruit fly Drosophila melanogaster have significantly facilitated the understanding of the in vivo function and regulation of these splicing factors S. cerevisiae identified genetic and biochemical interactions between the U2AF large subunit and SF1/BBP S. pombe provided in vivo evidence that the U2AF subunits are required for splicing Drosophila null mutations of the U2AF large or small subunits cause lethality C. elegans, reducing the expression of uaf-1 or sfa-1 by RNAi causes lethality uaf-1 and sfa-1 and allow the survival of animals in permissive conditions, such as at lower temperatures or when derived from heterozygous mothers. These mutations provide a valuable resource for analyzing the function and regulation of the U2AF large subunit and SF1/BBP genes in vivo in animals.The SF1/BBP and U2AF proteins are critical splicing factors that regulate splicing by binding the branch-point sequence and the 3′ splice sites in vivo. Distinguishing different 3′ splice sites is a critical aspect of alternative splicing.The recognition of 3′ splice sites is achieved by interactions between SF1/BBP and the U2AF large and small subunits, which together bind specific intronic sequences unc-93(e1500) missense mutation generates a new cryptic 3′ splice site (AG) within exon 9 (ACTGcag). This site differs from the consensus 3′ splice site for C. elegans (TTTTcag) C. elegans than is TTTTcag or the intron 8 endogenous 3′ splice site (AATTcag)  intron 8 and the cryptic non-consensus site in unc-93(e1500) exon 9 followed this prediction, as only 1.3% of the splicing events utilized this cryptic 3′ splice site. Strikingly, however, the n4588 missense mutation in uaf-1 shifted this specificity, causing splicing to occur mostly at the cryptic site, generating 68% of aberrantly spliced transcripts. This result suggests that UAF-1 might play an important role in determining the choice among alternative 3′ splice sites in vivo (see discussion below).The AATTcag) . Based on4588 mutation did not cause an apparent change of UAF-1 protein level, and reducing UAF-1 using RNAi did not increase the relative amount of alternatively spliced exon 9, suggesting that uaf-1(n4588) might alter UAF-1 function. However, RNAi-treatment did not abolish the expression of UAF-1 (unc-93(e1500) exon 9 because of residual UAF-1 protein in RNAi-treated animals. Thus, the altered splicing of unc-93(e1500) exon 9 in uaf-1(n4588) unc-93(e1500) mutants might reflect the consequence of the absence of UAF-1 activity. It is also possible that uaf-1(n4588) causes both a loss of function and an altered function of UAF-1, which cause the suppression of the rubberband Unc phenotype of the unc-93(e1500) animals and the altered splicing of unc-93(e1500) transcript, respectively.The of UAF-1 , and we unc-93 transcript appeared to be spliced similarly in wild-type and uaf-1(n4588) animals, suggesting that uaf-1(n4588) did not alter the recognition of most wild-type 3′ splice sites. We also found that uaf-1(n4588) did not suppress the Unc phenotype caused by the unc-52(e669) mutation, which can be suppressed by mutations in the splicing factor genes smu-1 and smu-2. We conclude that the uaf-1(n4588) mutation does not affect all cases in which alternative splicing is possible.The other 14 introns of the uaf-1(n4588). Three of the suppressors  carried both the original n4588 mutation and a second site mutation in uaf-1. These three new uaf-1 mutations reduced the alternative splicing of unc-93(e1500) exon 9 to levels intermediate between those of uaf-1(n4588) and wild-type animals. This finding supports the hypothesis that UAF-1 is important in 3′ splice-site choice. The fourth intragenic suppressor, n5123, affected the same site as the original n4588 mutation by generating a phenylalanine codon different from both the wild-type codon (threonine) and the codon generated by the n4588 mutation (isoleucine). The uaf-1(n5123) allele behaves like the uaf-1(+) allele, suggesting that this mutation restored the normal specificity of UAF-1. The amino acids affected by these uaf-1 mutations  are confined to a region between the U2AF small subunit-interacting domain We isolated four intragenic suppressors of the temperature-sensitive lethality caused by C. elegans, the first two nucleotides (−2 to −1) of 3′ splice sites are more highly conserved than nucleotides −7 to −3 in vivo.In C. elegans introns than there are copies of the strong 3′ splice site sequence TTTTcag (uaf-1(n4588) mutation does not appear to affect the splicing of most other introns of unc-93  was morf unc-93 , which hSecond, we conclude that the nucleotides at −7, −5 and −4 were more important than the nucleotide at −6 for wild-type UAF-1 to recognize the sequence of I8 , which iC. elegans  and E9 (ACTGcag) are both rare 3′ splice sites compared to TTTTcag, which is found in about 26% of the approximate 40000 introns analyzed, and is the most commonly used 3′ splice site in  elegans  [29],  was used to balance the uaf-1(n5222Δ) locus.unc-93(e1500) animals (P0) were mutagenized with EMS (ethyl methanesulfonate) as described 1 progeny from these animals were picked to single wells of 24-well culture plates with OP50 bacteria grown on NGM agar. F2 progeny were observed using a dissecting microscope to identify animals with improved locomotion. From ∼10,000 F1 clones screened, 100 independent suppressed strains were isolated. 97 of the isolates, including two weak recessive sterile suppressors and 95 recessive fertile suppressors, were kept as frozen stocks for possible later study. Three stronger suppressors that caused or were closely linked to mutations that caused sterility (n4562) or ts-lethality (n4588 and n4564) were chosen for further analysis. The analysis of n4564 is ongoing.Synchronized L4 n4588 to the left of dpy-1 on LGIII based on the suppression of unc-93(e1500) using standard methods. As the suppressor activity and ts-lethality were very closely linked, e.g., more than 500 n4588 unc-93(e1500)/+ unc-93(e1500) individuals failed to segregate Sup non-Let progeny, we then followed the phenotype of ts-lethality to further map n4588. We mapped n4588 to the right of nucleotide 186577 on BE0003N10 (cosmid BE0003N10 sequences refer to nucleotides of accession no. AC092690) using 10 Vab recombinants recovered after crossing vab-6(e697) n4588 hemaphrodites with males of the Hawaiian strain CB4856 n4588 dpy-1(e1) hemaphrodites with males from the Hawaiian strain CB4856. We determined the coding sequences of four genes in this interval, uaf-1, rab-18, kbp-4 and par-2, and identified a missense mutation in the third exon of the uaf-1a isoform.We mapped n4562 were very closely linked, e.g., over 500 unc-93(e1500); n4562/+ individuals failed to segregate Sup non-Ste progeny, we followed the sterility phenotype to map n4562 to the right of dpy-4 on LG IV using standard methods. We next mapped n4562 to the right of nucleotide 37163 on Y43D4A (accession no. AL132846) using 234 Dpy recombinants recovered after crossing dpy-4(e1166) n4562 with males of the Hawaiian strain CB4856. The sequences of coding exons of sfa-1 and uaf-2, both located in this region, were determined, and a Cys458Opal (TGT-to-TGA) mutation was identified in sfa-1.As the suppressor activity and sterility of uaf-1(n5222Δ) removes nucleotides 9786 to 11082 of YAC Y92C3B. sfa-1(n5223Δ) removes nucleotides 207818 to 208925 of YAC Y116A8C.Genomic DNA pools from EMS-mutagenized animals were screened for deletions using PCR as described uaf-1(n4588) L1 animals were prepared, and levels of control genes  and tested genes  were quantified from each biological replicate. For the Taqman probe-based assay , the probes , one sample for each assay was quantified. PCR primers and Taqman probes are listed in Total RNA was prepared using Trizol according to the manufacture's instructions (Invitrogen), treated with RNase-Free DNase I (New England Biolabs) and followed by incubation at 75°C for 10 minutes to inactivate DNase I. First-strand cDNA was synthesized with random hexamer primers (New England Biolabs) using the Superscript II or III First-Strand Synthesis Kit (Invitrogen). Quantitative RT–PCR was performed using either a DNA Engine Opticon System (MJ Research) or a Mastercycler realplex system (Eppendorf). For the SYBR green-based assay (DNA Engine Opticon System), each 30 µl PCR reaction contained 1 to 10 ng RT template, 0.5 mM PCR primers and 15 µl 2× SYBR Green PCR Master Mix (Applied Biosystems). Three independent samples of synchronized wild-type (N2) and e probes  were labuaf-1(n4588) animals (P0) at the L4 larval stage grown at 15°C were mutagenized with EMS as described 0 animals were allowed to grow to young adults at 15°C in a mixed population and bleached, and F1 progeny were synchronized at the early L1 stage by starvation in S medium 1 animals were placed on 50 Petri plates  with NGM agar seeded with OP50 and permitted to grow to young adults at 15°C and then moved to 25°C. After six days at 25°C, the animals were grown at 20°C for six days and examined each day for the presence of living F2 animals. About 50,000 F1 progeny from a mixture of more than 10,000 P0 animals were screened. From the screen, we recovered 13 surviving F2 animals from 13 different F1 plates. Six of the 13 suppressors were intragenic suppressors representing four different mutations: one was n5120, one was n5123, three were identical to n5125 and one was n5127. It is possible that the three isolates containing the n5125 mutation were derived from the same P0 animal, because all of the P0 animals were in a mixed population when bleached to release eggs. The other seven suppressors were extragenic mutations, i.e., n4588/+; sup/+ animals segregated n4588-like progeny. Some or all of the extragenic isolates could have been derived from the same P0 animal.Synchronized unc-93(e1500)) were fed HT115(DE3) bacteria containing plasmids directing the expression of dsRNAs targeting either uaf-1 or sfa-1 on NGM plates with 1 mM IPTG and 0.1 mg/ml Ampicillin 1 progeny of the unc-93(e1500) animals (escapers) were examined for suppression of locomotion defects. Animals were washed from plates, rinsed three times with H2O, and resuspended in Trizol (Invitrogen) for preparation of total RNA or in 2× protein loading buffer  for SDS-PAGE analysis. We generated the DNA construct expressing dsRNA targeting uaf-1 (see below). The bacterial strain expressing dsRNA targeting sfa-1 was obtained from a whole-genome RNAi library sfa-1 coding sequences.Young adult animals , 200 mM DTT, 4% SDS, 0.2% bromophenol blue, 20% glycerol), boiled for 5 minutes, and samples were then loaded onto 8% polyacrylamide gels containing SDS. Protein samples were transferred from polyacrylamide gels to Immobilon-P Transfer Membranes (Millipore). Primary and secondary antibody incubations were performed with 5% non-fat milk in TBST  at room temperature for one hour each. Signals were visualized using Chemiluminescence Reagent Plus (PerkinElmer Life Sciences) and X-ray film . Primary antibody was rabbit anti-UAF-1(1∶20000) Animals were washed from plates with Hunc-93(e1500) by uaf-1(n4588) or sfa-1(n4562), uaf-1 and sfa-1 cDNAs were subcloned to vector pPD93.97 using BamHI and AgeI restrictions sites. uaf-1b cDNA was amplified with PCR using uaf-1a cDNA as template and subcloned to pPD93.97 using BamHI and AgeI restrictions sites. uaf-1a cDNA was subcloned to pPD49.83 (for heat-shock induced expression of uaf-1a cDNA) using BamHI and SacI restriction sites. An XhoI/SpeI fragment of uaf-1a cDNA subcloned in a pGEM-TA easy vector (Promega) was subcloned to pPD129.36 (for the uaf-1 RNAi construct) using XhoI and NheI sites. To test whether the truncated unc-93(Δ) cDNA caused by altered splicing of the unc-93(e1500) transcript in uaf-1(n4588) mutants encodes a functional UNC-93 protein, unc-93 cDNA and unc-93(Δ) cDNA were subcloned to pPD93.97 using BamHI(blunt) and AgeI sites. To test whether uaf-1(n4588) or sfa-1(n4562) mutations could suppress the Unc phenotype caused by ectopic expression of the unc-93(e1500) cDNA, unc-93(e1500) cDNA was subcloned to pPD93.97 using BamHI(blunt) and AgeI sites. To examine the effect of nucleotide substitutions on the recognition of the intron 8 endogenous 3′ splice site and the exon 9 cryptic 3′ splice site, we fused the genomic sequence between exon 8 and exon 10 of unc-93(e1500) in-frame with the GFP gene of pPD93.97 using BamHI and AgeI sites. We replaced the myo-3 promoter of pPD93.97 with a 2 kb promoter of unc-93 using PmlI and BamHI sites. Point mutations in uaf-1(stop codons), the unc-93 (e1500 mutation) or mutated transgenes were introduced using QuickChange II or III Site-Directed Mutagenesis Kit (Stratagene) with primers containing corresponding mutations. PCR was performed using Eppendorf Cyclers, and DNA products were resolved using agarose gels. DNA sequence determination was performed with an ABI Prism 3100 Genetic Analyzer. PCR primers are listed in To rescue the suppression of the Unc phenotype of Arabidopsis genomic DNA and 10 µg/ml of the transgene of interest. When the transgene did not cause the expression of a GFP fusion protein, 10 µg/ml pPD95.86-GFP plasmid  or 5 µg/ml pmyo-2dsRED (expressing RFP in pharynx) was added to the injection mixture as a visible fluorescence marker to identify animals carrying the transgene.Germline transgene experiments were performed as described We downloaded approximate 40,000 unique intronic sequences from WormMart (WormBase Release 195) and processed the sequences using BBEdit and MS Excel softwares. Identical 3′ splice sites (positions −7 to −1) were grouped and counted.Figure S1uaf-1(n4588) does not reduce the mRNA levels of genes that genetically interact with unc-93. Real-time RT-PCR analyses of mRNA levels of constitutively expressed genes , a body wall muscle specific gene (myo-3) and genes involved in the rubberband Unc phenotype . The levels of endogenous rpl-26 mRNA of each sample were quantified in parallel real-time RT-PCR experiments and used as loading controls. Each data set represents the average of duplicate experiments of three biological replicates of synchronized L1 animals. Error bars, standard errors.(0.32 MB TIF)Click here for additional data file.Figure S2uaf-1 RNAi reduces UAF-1 protein level. (A) Animals were fed bacteria expressing control empty vector or dsRNA targeting uaf-1. A western blot was prepared using a UAF-1 polyclonal antibody uaf-1(RNAi) dramatically reduced the protein level of UAF-1. (B) Coomassie blue staining indicated that the loading of total protein was similar among the samples. (C) Quantification using NIH ImageJ software of UAF-1 levels in wild-type or unc-93(e1500) animals treated with either control empty vector or uaf-1(RNAi). uaf-1(RNAi) treatment reduced UAF-1 levels in both wild-type and unc-93(e1500) animals .(0.30 MB TIF)Click here for additional data file.Figure S3uaf-1(n4588) and sfa-1(n4562) do not cause obvious alternative splicing of the sup-10 transcript. (A) Total RNAs from animals of the indicated genotypes were prepared and RT-PCR experiments were performed to amplify the full-length sup-10 cDNA using PCR primers covering the 5′ start codon and the 3′ stop codon. No sup-10 transcript with an abnormal size was detected for wild-type, sup-10(n983), uaf-1(n4588) and uaf-1(n4588); sup-10(n983) animals. DNA sequences of the sup-10 RT-PCR bands from all the four genotypes were determined. No sup-10 transcript with altered splicing was observed. (B) No alternatively spliced sup-10 transcript was identified from wild-type, sup-10(n983), sfa-1(n4562) and sfa-1(n4562); sup-10(n983) animals using RT-PCR experiments and DNA sequence determination. Genotypes are indicated at the top.(0.37 MB TIF)Click here for additional data file.Figure S4uaf-1(n4588) and sfa-1(n4562) cause synthetic lethality. Anesthesized animals of the indicated genotypes were observed with Nomarski optics. For wild-type, uaf-1(n4588) and sfa-1(n4562), animals were photographed 24 hours after the mid-L4 stage . For uaf-1(n4588); sfa-1(n4562) double mutants, animals were observed each day for up to ten days after hatching. No animals grew beyond the size of a wild-type animal of the L2 larval stage. Two escapers of the uaf-1(n4588); sfa-1(n4562) genotype that survived embryonic lethality are shown. Scale bar: 200 µm.  sqv mutants of Caenorhabditis elegans are defective in vulval epithelial invagination. Proc Natl Acad Sci U S A 96: 968–973.)(0.79 MB TIF)Click here for additional data file.Table S1uaf-1(n4588) does not cause synthetic defects with loss-of-function mutations of unc-93, sup-9 and sup-10 and does not suppress other Unc mutants. To examine whether uaf-1 functions redundantly with unc-93, sup-9 or sup-10, we compared double mutants containing uaf-1(n4588) and lf mutations of unc-93, sup-9 or sup-10 to uaf-1(n4588) single mutants for locomotion, growth, and gross morphology. No differences were observed. Double mutants with uaf-1(n4588) and gf mutations of unc-58, egl-23 and twk-18 were compared to single mutants carrying these gf mutations, and no visible suppression of the Unc phenotypes was observed. Double mutants containing uaf-1(n4588) and unc-52(e444) or unc-52(e669) mutations were compared to single mutants of either unc-52(e444) or unc-52(e669) mutations, and no visible suppression of the Unc phenotypes was observed. Pvl: protruding vulva. Ste: sterile. Dpy: dumpy. Egl: egg-laying defective.(0.04 MB DOC)Click here for additional data file.Table S2unc-93 transcript does not encode a functional UNC-93 protein product. Transgenes driving the expression of the alternatively spliced unc-93 cDNA (unc-93 cDNA(Δ)) did not rescue the suppression of sup-9(n1550) by the unc-93(lr12Δ) mutation, while transgenes expressing a wild-type unc-93 cDNA rescued the suppression.The alternatively spliced (0.02 MB DOC)Click here for additional data file.Table S3unc-93 transcript likely does not encode a dominant-negative UNC-93 protein product. Tansgenes driving the expression of the unc-93 cDNA(Δ) did not suppress the rubberband Unc phenotype of unc-93(e1500) animals, while transgenes expressing a wild type unc-93 cDNA suppressed the Unc phenotype. As loss of function of unc-93 results in phenotypically wild-type animals, the lack of suppression of unc-93(e1500) by the unc-93(Δ) transgenes suggests that the function of unc-93 was not antagonized by unc-93(Δ), indicating that the unc-93(Δ) cDNA does not encode a dominant-negative UNC-93 protein.The alternatively spliced (0.02 MB DOC)Click here for additional data file.Table S4Sequences and distributions of the three 3′ splice sites we analyzed. Approximate 40,000 unique introns were analyzed, and the numbers and ratios of all types of 3′ splice sites were calculated. The list here includes the three sites we analyzed in our mutagenesis experiments shown in (0.03 MB DOC)Click here for additional data file.Table S5List of PCR primers and Taqman probes.(0.10 MB DOC)Click here for additional data file.


Caenorhabditis elegans genome exists, however the application of these valuable biological tools has been limited due to the incomplete correlation between their genetic and physical characterization.A collection of genetic deficiencies covering over 70% of the We have applied oligonucleotide array Comparative Genomic Hybridization (oaCGH) to the high resolution, molecular characterization of several genetic deficiency and duplication strains in a 5 Mb region of Chromosome III. We incorporate this data into a physical deficiency map which is subsequently used to direct the positional cloning of essential genes within the region. From this analysis we are able to quickly determine the molecular identity of several previously unidentified mutations.Caenorhabditis elegans. Consequently we have generated a valuable physical mapping resource, which we have demonstrated can aid in the rapid molecular identification of mutations of interest.We have applied accurate, high resolution molecular analysis to the characterization of genetic mapping tools in  Caenorhabditis elegans genome has been generated by various research groups over the past three decades ), BC4638 (sDf127(s2428) [sDp3]) and BC4690 (sDf128(s2786) [sDp3])  known to cover the region under investigation in this study [To implement this strategy all of the is study . For thiis study .sDp3 from the background the ability to accurately resolve breakpoint positions in the ambiguously mapped deficiency strains improved significantly. A comparison of oaCGH data for sDf127 balanced by sDp3 and this same deficiency out-crossed to hT2 (qIs48) highlights this improvement  dpy-5(e61) I/hT2 [bli-4(e937) let-?(q782) qIs48] , BC4697 – sDf121(s2098) unc-32(e189) III; sDp3 , BC7162 – dpy-17(e164) sDf125(s2424) unc-32(e189) III; hT2 [bli-4(e937) let-?(q782) qIs48], BC4638 – dpy-17(e164) sDf127(s2428) unc-32(e189) III; sDp3 , BC7163 – dpy-17(e164) sDf127(s2428) unc-32(e189) III; hT2 [bli-4(e937) let-?(q782) qIs48], BC4690 – dpy-17(e164) sDf128(s2786) unc-32(e189) III; sDp3 , BC7164 – dpy-17(e164) sDf128(s2786) unc-32(e189) III; hT2 [bli-4(e937) let-?(q782) qIs48], BC7244 – sDf130(s2427) unc-32(e189) III; hT2 [bli-4(e937) let-?(q782) qIs48], BC7181 – dpy-17(e164) sDf135(s2767) unc-32(e189) III; hT2 [bli-4(e937) let-?(q782) qIs48], BC3737 – sDp8; eT1, BC4158 sDp3; dpy-17(e164) let-722(s2448) unc-32(e189) III, BC4160 – sDp3; dpy-17(e164) let-722(s2450) unc-32(e189) III, BC4228 – sDp3; dpy-17(e164) let-722(s2588) unc-32(e189), BC4262 – sDp3; dpy-17(e164) let-754(s2622) unc-32(e189) III, BC4173 – dpy-17(e164) let-766(s2463) unc-32(e189) III; sDp3, BC4167 – sDp3; dpy-17(e164) let-716(s2457) unc-32(e189) III, BC4266 – sDp3; dpy-17(e164) let-716(s2626) unc-32(e189) III, BC4192 – sDp3; dpy-17(e164) let-768(s2482) unc-32(e189) III, BC4232 – sDp3; dpy-17(e164) let-768(s2592) unc-32(e189) III, BC4268 – sDp3; dpy-17(e164) let-768(s2628) unc-32(e189) III, BC2842 – dpy-18(e364)/eT1 III; unc-46(e177) let-444(s1418)/eT1 [let-500(s2165)]V, MT3022 – nDf20/sma-2(e502) unc-32(e189)III.Nematodes were grown as previously described  on 150 msDp3 balanced strains into 5 ul of lysis buffer  and freeze cracked in Liquid N2 followed by digestion (1 hour at 60°C followed by 15 min at 95°C). 1 ul of this DNA preparation was subsequently used in a 25 ul PCR reaction.DNA preparation for oaCGH samples was done as previously described . For mutsDp3 balanced strains a C. elegans whole genome array was used, based on Wormbase release WS120, and available from NimbleGen Systems Inc. [hT2 (qIs48) balanced strains was performed with a whole genome C. elegans array designed with overlapping 50-mer probes targeting primarily annotated exons and micro-RNAs [For the analysis of cro-RNAs . oaCGH scro-RNAs . Copy nucro-RNAs  and are PCR amplification across the region of the breakpoint was performed with nested primers (Primer sequences and amplification conditions available upon request) and sequenced using standard molecular methods.MRJ carried out the experiments, data analysis and drafted the manuscript. JSM assisted in sample preparation. SF designed the oaCGH array. DGM helped to draft the manuscript. DLB conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript. Identified mutants of essential genes and their associated RNAi phenotype.Click here for file


These organisms possess an extended array of secondary metabolites that impair cell lysis, presenting particular challenges when it comes to nucleic acid isolation. Therefore, we used the NHM5 strain of Nostoc punctiforme ATCC 29133 to compare and improve existing phenol based chemistry and procedures for RNA extraction.The validity and reproducibility of gene expression studies depend on the quality of extracted RNA and the degree of genomic DNA contamination. Cyanobacteria are gram-negative prokaryotes that synthesize chlorophyll With this work we identify and explore strategies for improved and lower cost high quality RNA isolation from cyanobacteria. All the methods studied are suitable for RNA isolation and its use for downstream applications. We analyse different Trizol based protocols, introduce procedural changes and describe an alternative RNA extraction solution.It was possible to improve purity of isolated RNA by modifying protocol procedures. Further improvements, both in RNA purity and experimental cost, were achieved by using a new extraction solution, PGTX. Since txidation , their pxidation ,4 to varxidation .In order to develop this biotechnological potential, it is important to thoroughly understand different aspects of cyanobacterial physiology and metabolism. As a part of such a process, obtaining reliable gene expression data is vital. Several methods, from Northern blotting to microarrays, are routinely used to obtain such data. The validity and reproducibility of the data obtained depend on the quality of the extracted RNA and the degree of genomic DNA contamination. However, cyanobacteria present particular challenges when it comes to nucleic acid isolation – these organisms possess an extended array of secondary metabolites  that impIn order to assess the quality of RNA preparations two strategies are commonly followed: spectrophotometric analysis and ribosomal integrity verification by electrophoresis.From the spectrophotometric analysis three absorbance values usually are taken into consideration – 230, 260 and 280 nm. The ratio between the absorbance at 260 nm and 280 nm is used to evaluate the purity of the nucleic acid – for "pure" RNA a ratio around 2.0 is expected. A lower ratio may indicate the presence of proteins and peptides absorbing around 280 nm. Additionally, the ratio between the absorbance at 260 nm and 230 nm is expected to be 2.2 for "pure" nucleic acid samples. A lower ratio might be the consequence of contamination by peptides, phenols, aromatic compounds or carbohydrates.The integrity of the ribosomal RNA sub-units , the presence/absence of low weight RNA degradation products and the presence/absence of genomic DNA contamination are commonly visualized using agarose gel electrophoresis. Ideally, all expected ribosomal RNA sub-units should be observed, with no signs of RNA degradation products or presence of genomic DNA.The guanidinium thiocyanate-phenol-chloroform extraction ,9, commeThe most important factor when planning the composition of the extraction buffer PGTX (detailed below) was to give the extraction solution the ability to quickly inhibit ribonuclease activity. Both phenol and guanidine salts are very efficient protein denaturants, therefore ideal for fast ribonuclease denaturation, and their combined use has been previously described ,9. We alThe poor miscibility of phenol with water allows for easy phase separation at a later stage of the extraction procedure, but should be minimized at the beginning of the process. In order to avoid premature phase formation, glycerol was used to facilitate phenol solubility in the buffer. Later on, phase separation is achieved by adding BCP (bromochloropropane), as previously described .After phase separation, protection of the extracted RNA is reduced, since the phenol and guanidine salt concentrations will be lower. In order to avoid degradation from this point on in the process, we used both sodium acetate and EDTA as chelators to prevent divalent cation catalyzed RNA degradation.Triton X-100 is a non ionic detergent used for protein solubilisation, membrane permeabilisation and cell lysis. It has been previously demonstrated that its use, in combination with chloroform and heat, is a viable strategy for RNA extraction from both Gram-positive and Gram-negative bacteria . This meThe PGTX solution has the following composition : phenol (39.6 g), glycerol (6.9 mL), 8-hydroxyquinoline (0.1 g), EDTA (0.58 g), sodium acetate (0.8 g), guanidine thiocyanate (9.5 g), guanidine hydrochloride (4.6 g) and Triton X-100 (2 mL), and the final pH around 4.2. At room temperature, the PGTX extraction mixture forms a monophasic solution.The strategies followed for RNA extraction are summarized in Table The highest yields were achieved using the "PGTX beads" and "Trizol beads" methods – averaging 1.70 μg/μl and 1.63 μg/μl respectively. For "PGTX 95" the yield was 1.29 μg/μl while for "Trizol 95" the calculated concentration was 1.03 μg/μl. The "Trizol standard" procedure yielded 0.47 μg/μl while the previously published method "BPC" resulted in a solution containing 0.82 μg/μl of nucleic acids. The different yields are probably correlated with the ability of each method to promote cell lysis. For instance, when considering the use of Trizol, we observed that combining it with bead beating is more effective than using heat or Trizol alone . In Figure In order to verify integrity, four RNA samples for each of the extraction methods were analysed using an automated gel electrophoresis system , the diluted RNA samples were used as template for PCR using primers pairs for rbcL specific cDNA . For the PGTX based methods the cost is €0.29 . For Trizol based extractions the cost per sample is €2.35.Cyanobacteria have an important amount of polysaccharides that interfere in cell lysis and nucleic acid purification ,18, whilSynechocystis PCC 6803, Anabaena PCC 7120) and green algae .Potentially even more cost reducing is PGTX – this extraction mixture has equivalent potential to Trizol, while having only a fraction of the cost. We have also successfully used (data not shown) PGTX to extract RNA from several cyanobacteria . RNA integrity was verified using Biorad's automated electrophoresis system Experion , according to manufacturer's instructions.ftsZ (see Table Taq DNA Polymerase (Fermentas), according to manufacturer's instructions.For DNA contamination detection, 1 μg of RNA was diluted in water to a total volume of 20 μl. Then, 1 μl of RNase A/T1 Mix (Fermentas) plus 1 μl of RNase H (Fermentas) were added and the samples incubated at 37°C for 30 minutes. To inactivate the RNases, 28 μl of TE buffer (pH 8.0) were added and the solution incubated at 70°C for 10 minutes. Using 1 μl of the described reverse transcriptase simulated reaction as template, PCR was carried out using specific primers for the division related gene rbcL specific tagged primer [To perform RT, 1 μg of RNA was used as template for RevertAid H Minus M-MuLV Reverse Transcriptase (Fermentas), according to manufacturer's instructions. In order to avoid the interference of genomic DNA contamination during the subsequent PCR step, a d primer ,27 was ucDNA: complementary deoxyribonucleic acid; DNA: deoxyribonucleic acid; DNase: DNA ribonuclease; BCP: bromochloropropane; GSP: gene specific primer; mRNA: messenger ribonucleic acid; PCR: polymerase chain reaction; RNA: ribonucleic acid; RT-PCR: reverse transcription-polymerase chain reaction.FLP conceived the experimental setup and PGTX formulation, performed most experimental work and wrote most of the manuscript. AT performed spectrophotometric experiments, analyzed spectrophotometric data and actively contributed to the writing of the manuscript. WS was responsible for setting up culturing, harvesting and cell aliquoting, performed all BPC method extractions and participated in writing the manuscript. PL coordinated the project and contributed to the manuscript writing. All authors have read and approved the manuscript.


This substance was also released from macrophage monolayers stimulated with IL-1β but not with TNF-α, IL-6 or IL-8. The substance secreted (3H-RNA) sediments in the 4–5S region of a 5–20% sucrose gradient. These results show that L-RNA induces cytokine secretion by macrophage monolayers and support the idea that, during inflammation, stimulated macrophages could release RNA which may further induce the release of cytokines by the resident cell population.The effect of exogenous RNA on many cellular functions has been studied in a variety of eukaryotic cells but there are few reports on macrophages. In the present study, it is demonstrated that cytoplasmatic RNA extracted from rat macrophages stimulated with Escherichia coli lipopolysaccharide (LPS), referred to as L-RNA, induced the release of TNF-α and IL-1 from monolayers of peritoneal resident macrophages. The activity of L-RNA was not altered by polymyxin B but was abolished by ribonuclease (RNase) pretreatment, indicating the absence of LPS contamination and that the integrity of the polynucleotide chain is essential for this activity. Both the poly A(−) and poly A(+) fractions obtained from L-RNA applied to oligo(dT)–cellulose chromatography induced TNF-α and IL-1 release. The L-RNA-induced cytokine release was inhibited by dexamethasone and seemed to be dependent on protein synthesis since this effect was abolished by cycloheximide or actinomycin-D. The LPS-stimulated macrophages, when pre-incubated with [5-


Evaluation of RNA quality is essential for gene expression analysis, as the presence of degraded samples may influence the interpretation of expression levels. Particularly, qRT-PCR data can be affected by RNA integrity and stability. To explore systematically how RNA quality affects qRT-PCR assay performance, a set of human placenta RNA samples was generated by two protocols handlings of fresh tissue over a progressive time course of 4 days. Protocol A consists of a direct transfer of tissue into RNA-stabilizing solution (RNAlater™) solution. Protocol B uses a dissection of placenta villosities before bio banking. We tested and compared RNA yields, total RNA integrity, mRNA integrity and stability in these two protocols according to the duration of storage.A long time tissue storage had little effect on the total RNA and mRNA integrity but induced changes in the transcript levels of stress-responsive genes as TNF-alpha or COX2 after 48 h. The loss of the RNA integrity was higher in the placental tissues that underwent a dissection before RNA processing by comparison with those transferred directly into RNA later™ solution. That loss is moderate, with average RIN (RNA Integration Numbers) range values of 4.5–6.05, in comparison with values of 6.44–7.22 in samples directly transferred to RNAlater™ (protocol A). Among the house keeping genes tested, the B2M is the most stable.This study shows that placental samples can be stored at + 4°C up to 48 h before RNA extraction without altering RNA quality. Rapid tissue handling without dissection and using RNA-stabilizing solution (RNAlater™) is a prerequisite to obtain suitable RNA integrity and stability. Molecular tools for tissue profiling, such as real-time quantitative RT-PCR, generally require collection of fresh frozen tissues as sources of high-quality RNA. The quality of qRT-PCR data analysis is strongly related to the integrity and stability of the mRNA extracted from the tissue which is in turn dependent on tissue sample processing.The fragile nature of RNA and the question of RNases enriched tissues such as placenta prompted us to examine the effects of storage time conditions with regard to RNA integrity and gene expression in non fixed human term placenta.Many parameters such as delay time, mode of tissue handling, processing protection from RNAses degradation, tissue hypoxia, might influence the quality of extracted RNA .Time of delivery cannot be predicted with accuracy and it is quite difficult to take in charge fresh placenta immediately after delivery. So it is difficult to standardize the delay time from delivery to sampling. The variability of gene expression also depends on the cellular homogeneity of the tissue. Placenta is an heterogeneous tissue with a large pattern of different cells with foetal and maternal areas immerged in blood. This requires a minimum of tissue washing and dissection before sampling .The mode of delivery might also be important for the quality of RNA. In fact, the duration of deliveries and the tissue hypoxia is not comparable between a placenta excised by cesarean and a placenta that follows the vaginal tray. Is has been found that the duration of labor might induce an hypoxia stress with a decrease of pH and the change of the expression of a large number of genes .The quality of total RNA is evaluated by the measure of its integrity and its stability. The integrity means that the pattern of total RNA ribosomal units 28S and 18S are abundant and that we have full length mRNA. The stability means an equal distribution of stable housekeeping genes despite different and heterogeneous sample conditions. It also means a stable amount of mRNA with a short half-life time.Our purpose was to test the influence of storage of placenta at different post partum intervals (up to 96 h every 24 h) for RNA integrity and stability, taking also in account the mode of delivery and tissues handling before banking.The comparisons of pH of placenta tissue at progressive delay time showed a significant decrease at T96 (mean pH = 6.71) compared to pH = 6.9 at T0  . The range varies from 0.14 μg/mg to 0.57 μg/mg. The overall yield was slightly but significantly lower in protocol B (dissection of tissue before transfer) compared to protocol A (direct transfer to RNA later™) at any time (p = 0.01)  and by 28S:18S values  compared to protocol B . The two protocols differ from each other with a mean delta of 1 unit of RIN. The difference was more marked at T72 and T96 (p < 0.008). No significant decrease of RIN values with delay time was observed for the protocol A. Samples extracted with protocol B showed a significant decrease of RIN values with delay time (p = 0.05). We did not report any difference of RIN values between vaginal and caesarean deliveries (data not shown).28S:18S ratios were similar in the two protocols with stable range (1.10–1.33) whatever handling state and delay time.mRNA integrity was evaluated by quantification of 5' and 3' fragments of selected large house keeping genes as fatty acid synthase (FASN) (8 kb) and glyceraldéhyde-3-phosphate deshydrogenase (GAPDH) (3 kb). 5'/3' ratios around 1 value account for the integrity of the transcript. A decrease of 5' fragment is predictive of mRNA degradation .FASN 5'/3' ratios varied with delay time from 1.54 to 0.62 (mean +/- SEM = 1.05 +/- 0.16) in protocol A and from 0.88 to 0.31 (mean +/- SEM = 0.63 +/- 0.18) in Protocol B  in protocol A and from 0.91 to 0.37 (mean +/- SEM = 0.53 +/- 0.11) in protocol B , cyclophilin, according to tissue handling and delay time. These genes are known for their stability in placenta and therefore are routinely used for RNA normalization [mRNA stability was first evaluated by the comparative expression of 3 house keeping genes: 5-aminolevulinate synthase (ALAS), βDelay time is associated with a progressive and significant decrease of ALAS relative values in the two protocols A and B from T0 to T96 (p = 0.007 and p = 0.03 respectively). There was no difference according to tissue handling. Up to 72 h, . Mean cyclophilin expression relative values were significantly different in the two protocols with lower values in protocol B: 0.66 +/- 0.08 (mean +/- SEM) in protocol A and 0.15 +/- 0.05 in protocol B (p < 0.007).The second test for RNA stability was performed only with samples extracted with protocol A as this protocol was found to give the best stability and integrity of mRNA. Tumor necrosis factor α (TNFα) and cyclooxygenase 2 (COX2) were chosen because of the short life time of mRNA. Moreover, these two genes are immediate early response genes induced by modification of tissue like hypoxia and apoptosis . Our resThe limiting factor for obtaining meaningful gene expression is the quality of the initial RNA preparation. RNA purity and integrity are of foremost importance to ensure reliability and reproductibility of qRT-PCR . AlthougReliable statistical analysis of these parameters leaded us to investigate a sufficient panel of tissues. Therefore our study was performed on 140 samples from 14 placentas, 2 protocols of preparation and 5 delay time points. Power calculations for qRT-PCR comparisons usually indicate that a sample size of at least 50 is required to detect difference. Hynd et al, reported that 13 cases by group yielded statistically differences on a range of widely disparate parameters .The first parameter studied was the pH of placental tissue. pH was of interest in the light of hypoxia related to tissue injury. Hypoxia is associated with an accumulation of lactates, a lower pH and a subsequent activation of acid lysosomial RNAses . There wThe absence of significant variation according to delay time shows that degradation of RNA seems to depend more on tissue handling than on delay time of storage. This agrees with several reports performed on various tissues -17.The assessement of total RNA integrity can be done by two main methods: the standard 28S:18S ratio and the recent RIN integration method. The standard method uses electrophoresis of RNA and the evaluation of 28S and 18S bands and ratios. It is commonly accepted that intact RNA has a rRNA band ratio > 1.8 . In soliOur observations suggest that despite the 28S:18S ratio is considered as the gold standard for the measure of integrity, it lacks precision and discrimination between preserve and partially degraded RNA.Following total RNA integrity, determination of mRNA integrity is important to assess. We analysed it by the quantification of 5' and 3' ends fragments of some gene transcripts. The fragments located towards the 5'end of the mRNA of housekeeping genes are used as indicator sequences for the degree of degradation ,22.FASN 5'/3' observed ratios are higher than those reported by Bauer in blood samples . This miEndogenous controls, usually housekeeping genes, are measured to better normalize between tissue samples -28. The The analysis of TNFα and COX2 mRNA PCR products show a stability of expression up to 48 h and thereafter an important up regulation . TNFα and COX2 are involved in cellular defense and stress response. Overexpression of these genes induced by storage of rat liver at 37°C has been already reported . The mecThis study, from the criteria of RNA yields, global RNA integrity, and RNA expression of some stable and some unstable mRNA, shows, for the first time, that it is routinely possible to obtain RNA of good quality from placentas stored intact at +4°C up to 48 h and transferred into a RNA stabilizing solution. However, one must be cautious for an extrapolation to placental expression of all the genes. Nevertheless, the delay time will be helpful for RNA preparation from this tissue, as an immediate processing is not always easy to plan. Dissection of placentas in order to obtain a tissue free of vessels and foetal membranes must be avoided or set up using a RNA-stabilizing solution.Our results are in agreement with those from other previous studies on various human and animal tissues showing that RNA degradation is a minor problem when intact tissues are stored either at +4°C or even at room temperature before biobanking.Placentas at full term (38–41 weeks of gestation), were obtained after caesarean sections or normal vaginal deliveries from 14 women with healthy babies and no complications of pregnancy. All the placentas from the patients were collected from one maternity hospital . This study is a preparative part of a PHRC approved by the Ethics Committee of LILLE (France), CPP . Data were recorded anonymously. Placentas were stored at +4°C until analysis.3) were removed and processed after storage of placenta at various delay times . For each placenta, tissue fragments were obtained from 4 various locations between the decidual and chorionic plates in order to limit the tissue heterogeneity [2 and PBS in order to remove blood, dissected in Petri dishes, made free of foetal membranes vessels and tissue from maternal origin, rinsed in sterile PBS solution, then adjusted to 150 mg, transferred to RNA later™ and stored at -80°C until processed (protocol B). (3) tissues were transferred to a tube containing 5 volumes of water, disrupted with a Qiagen tisssue disrupter, and the supernatant transferred to a new tube to measure the pH. All the experiments were performed on ice excepted tissue preparation for pH determination.Biopsy placental villi (about 1 cmEach frozen sample (stored at -80°C in RNAlater™) was placed in a 13 ml tube containing 4 ml of lysing buffer solution . Samples were homogenized with a Qiagen tissue disrupter using two 20–30 sec pulses, and processed for RNA isolation using the RNAeasy midi kit™ followed by an additional treatment with DNAse according to manufacturer's procedure (Qiagen). RNA concentration was evaluated by measuring the absorbance at 260 nm using the spectrophotometer Nanodrop. RNA samples extracted from placental tissues obtained from 4 various locations (see above) were pooled. Aliquots of 1 μg of pooled RNA samples were precipitated in ethanol for long storage. All tubes were RNAse free.Structural RNA integrity was evaluated using a microchip electrophoresis on an AGILENT 2100 Bioanalyser (Agilent technologies) . All chi20 anchored primer, 40 U Rnase out and 5 mM DTT in a final volume of 20 μl.Reverse transcription was performed using the instructions for the cDNA kit . A 20 μl reaction without reverse transcriptase was performed. A negative control without RNA was included in the reverse transcription reaction. 1 μg of total RNA was reverse transcribed at 50°C for 45 min with a mixture containing 4 μl 5 × cDNA RT buffer, 15 U Thermoscript reverse transcriptase, 1 mM dNTPs, 2.5 μM oligo-dT-delta CtRelative expression levels of RNA per sample were quantified by SYBR Green I assay on Roche Light Cycler 2700 sequence detection assay . For each transcript, PCR was performed in duplicates with 10 μl reaction volumes of 1 μl of cDNA, 8 μl of mix, and 1 μl of each primer set. PCR was conducted using the following cycle parameters: 2 min at 50°C, 10 min at 95°C and 40 three steps cycles of 15 sec at 95°C, 20 sec at 50°C and 20 sec at 72°C. The assay was performed following the manufacturer's recommendations except that the reaction volume was reduced to 10 μl. A pool of cDNA from control placenta tissues prepared immediately after partum was used as a standard  for quantitative correction. All cDNA samples were applied in dilution of 1:5 to obtain results within the range of the standard. Each sample was evaluated in duplicate. Analysis of transcript level was carried out using first the determination of the threshold cycle Ct for each reaction corrected by the efficiency. Then the delta Ct was calculated by subtracting the mean Ct of the calibrator from each value of Ct for each gene. The amount of target relative to a calibrator was computed by 2 cDNA integrity was investigated by comparing the qRT-PCR 5'/3' ratio for 2 selected genes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fatty acid synthase (FASN). These two genes were chosen because of their ubiquitous expression as a so-called house keeping (HK) genes and for their various sizes, short for GAPDH (1 kb) and large for FASN (>8 kb). that generates fragments located at 3' and 5' ends of cDNA , and cyclophilin, and a second set of 2 genes with a short half life: TNFα (37 min) and COX2 (3 h). Primers were designed by Qiagen (GENGLOBE) for ALAS and B2M or by primer3 software for cyclophilin , βin Table . TNF andadeltaCt .To examine whether the variable qRT-PCR amounts, RNA concentrations, RIN, pH values 28S:18S ratios, were different between the groups defined by pre analytical conditions and the delay time of conservation, we used either the Kruskal-Wallis test (3 groups minimum) or the Mann-Whitney test (2 groups) which are non parametric alternative to one-way Anova.All statistical analyses were performed using ABI PRISM software. p values less than 0.05 were considered statistically significant.IF designed the study, carried out the analysis and interpretation of the results and drafts the manuscript, EM contributed to the development of methodology and performed the experimental procedure, AV and PD provide placenta tissues and are physicians involved in a common clinical research project on foetal growth and gene expression in placenta, MVM assisted in the preparation of RNA. JR participated in the conception and design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.Electrophoretic tracings of RNA according to different handling methods. the data provived presented AGILENT graphs of two RNA samples extracted from the same tissue and submitted to Protocol A: RIN 7.2 value (a) and protocol B: RIN 4.5 value (b).Click here for file


RNA transfection into dendritic cells (DCs) is widely used to achieve antigen expression as well as to modify DC properties. CD40L is expressed by activated T cells and interacts with CD40 receptors expressed on the surface of the DCs leading to Th1 polarization. Previous studies demonstrated that ectopic CD40L expression via DNA transfection into DCs can activate the CD40 receptor signal transduction cascade. In contrast to previous reports, this study demonstrates that the same effect can be achieved when RNA encoding CD40L is electroporated into DCs as evidenced by secretion of IL-12. To achieve higher levels of IL-12 secretion, a systematic approach involving modification of coding and noncoding regions was implemented to optimize protein expression in the DCs for the purpose of increasing IL-12 secretion.Site-directed mutagenesis of each of the first five in-frame methionine codons in the CD40L coding sequence demonstrated that DCs expressing a truncated CD40L protein initiated from the second methionine codon secreted the highest levels of IL-12. In addition, a post-transcriptional method of capping was utilized for final modification of the CD40L RNA. This method enzymatically creates a type I cap structure identical to that found in most eukaryotic mRNAs, in contrast to the type 0 cap incorporated using the conventional co-transcriptional capping reaction.The combination of knocking out the first initiation methionine and post-transcriptional capping of the CD40L RNA allowed for approximately a one log increase in IL-12 levels by the transfected DCs. We believe this is a first report describing improved protein expression of post-transcriptionally capped RNA in DCs. The post-transcriptional capping which allows generation of a type I cap may have broad utility for optimization of protein expression from RNA in DCs and other cell types. RNA transfection into Dendritic Cells (DCs) is widely employed to achieve antigen expression . RNA-traet. al. demonstrated that a poly(A) tail of 100 nucleotides and a 5'ARCA cap analogue act synergistically to produce high protein expression in dendritic cells [et al. reported that a longer poly(A) tail of 120 A residues as opposed to a more conventional poly(A) tail of 64 bases achieves higher protein expression levels. Furthermore, the 3' ends of the transfected RNA molecules were modified with a non-translated region from the β-globin gene of X. laevis. [Successful protein expression from transfected RNA depends on transfection efficiency, translation competence, and stability of the transfected RNA. A 5' cap structure and 3' poly(A) tail are essential components for RNA translation in eukaryotic cells. Mockey In addition to antigen-MHC complexes the immunopotency of DCs is dependent on high levels of co-stimulatory molecules and secreted cytokines. Together, these elements conspire to induce antigen-specific T helper cell 1 (Th1) and cytotoxic T lymphocyte (CTL) responses. Transfection of DCs with RNA encoding for a pro-inflammatory cytokine can modify the DC phenotype to enable these desirable properties. For example, DCs co-transfected with tumor antigen RNA and IL-12 mRNA were shown to induce higher numbers of tumor-specific CTLs with greater functional avidity compared to those transfected with tumor antigen mRNA alone .In the present study IL-12 secretion by DCs was achieved by a different approach involving transfection of the DCs with CD40L RNA. CD40L is normally expressed transiently on the surface of activated CD4+ T cells  and mediHere we demonstrate that expression of CD40L from transfected RNA likewise leads to increased maturation of DCs and induction of IL-12. Furthermore, additional optimization of the CD40L RNA was undertaken to achieve greater levels of the CD40L protein expression which in turn induce higher levels of IL-12 cytokine secretion. The first of these modifications was to the CD40L coding sequence where the initiation of translation from the second in-frame methionine codon led to greater levels of protein expression and consequently induced higher levels of IL-12 secretion. The other modifications we explored were not specific to the CD40L coding sequence and involved enzymatically adding a long poly(A) tail and post-transcriptional capping of the RNA.in vitro using most commercially available kits, 5' capping occurs by a very different mechanism. Capping does not occur after synthesis of the transcript, but rather occurs concurrently with the initiation of transcription and is referred to here as "co-transcriptional capping". During the in vitro transcription reaction, the dinucleotide cap analog m7G(5')ppp(5')G (referred to from here on as m7G) is substituted for a portion of the GTP nucleotide in the reaction and transcription templates from which the RNA is transcribed are engineered such that the first nucleotide transcribed is a guanosine. Consequently, RNA transcription can be initiated with the cap analogue instead of GTP in a fraction of the molecules. A molar excess of cap analogue relative to GTP drives the reaction toward preferential incorporation of the cap dinucleotide at the first position of the transcript. The products of this type of transcription reaction are always mixtures of uncapped and capped RNAs resulting from initiation of synthesis with either GTP or the cap analogue, respectively. Uncapped molecules present in a synthetic mRNA preparation are typically not able to be translated upon transfection into eukaryotic cells  GpppG. This anti-reverse cap analog (ARCA) is incapable of being incorporated in the reverse orientation and thus gives rise to co-transcriptionally capped RNA preparations in which all capped transcripts should be competent for translation [In eukaryotic cells, nascent mRNAs are modified with a cap structure early during their transcription, when the transcript reaches 20–30 nucleotides in length . First, iewed in ). In addiewed in . Therefoiewed in . Based uiewed in ,18. Consnslation . While mnslation  and are Since antigen loading of DCs is achieved via RNA transfection, the antigen payload can be conveniently supplemented with the CD40L-encoding RNA. Figure A dose response experiment between the mass of RNA electroporated into iDCs and the maturation phenotype was performed Figure . The levThese data support earlier observations that CD40L RNA expressed ectopically in DCs induce DC maturation and induction of IL-12 . The levCD40L mRNA stability is known to be regulated in T-cells through sequence elements within the 3' UTR . During In vitro transcribed RNAs were prepared from each of the three plasmid templates listed in Figure in vitro transcription from a poly(T) containing DNA template or were polyadenylated after the transcription reaction in which the poly(A) tails were greater than 200 nucleotides. Four hours post-electroporation into DCs, CD40L protein expression was measured and secreted IL-12 cytokine levels were assayed after overnight incubation  within the first 108 nucleotides transcribed . The protein expression level in cells transfected with ΔXE-MET1 RNA is approximately two-fold greater at both 4 and 10 hrs post-electroporation .A comparative analysis of the post-transcriptionally capped RNAs indicates that the post-transcriptionally capped RNA has greater potency than either co-transcriptionally capped counterpart. The molecular precursor for the cap in the post-transcriptional reaction was GTP and the resulting structure closely resembles the m7G cap. However, 100% of the products of the post-transcriptional reaction using Vaccinia virus Capping enzyme are in the correct orientation compared to 67% of ARCA capped transcripts obtained in a co-transcriptional reaction. Since both types of caps are in a translation-competent form, the highest translation competency is observed in a population with greatest capping efficiency  cap [The majority of RNA made in e 0) cap .In addition to the synergistic effect on higher protein expression, both the cap structure and poly(A) tail also contribute to greater stability of the RNA . The pol111indium-labeled DC migration show accumulation in the lymph node after just 6 hours and continued accumulation at 24 hrs and even 48 hrs post-injection [Interestingly, the absolute level of protein expression measured at 4 hours post-transfection did not definitively predict the level of IL-12 secreted by the same cells at 24 hours post-transfection. It is possible that the induction of the IL-12 signalling pathway occurs at a time point later than 4 hrs post-electroporation and we are achieving greater steady-state levels as a result of increased RNA stability. Our routine Elisa assays studied IL-12 cytokine secretion at 24 hours post-electroporation. Human clinical studies examining intradermally administered njection ,34. It win vitro transcription reactions wherein the GTP nucleotide is not limiting. Transcription reactions that employ co-transcriptional RNA capping routinely include one-fourth the concentration of GTP that a standard reaction contains and consequently the final RNA yield from these reactions is approximately four times less than the theoretical maximum. In the process described here for CD40L MET1 RNA transcription, a four- to five- fold increase in yield from the same DNA template was obtained.Finally, another advantage of the post-transcriptional method is the ability to produce large amounts of uncapped RNA precursor using in vitro transcribed RNA and which may be applied to other RNAs for the purpose of enhancing protein expression.In summary we demonstrate various RNA modifications that greatly improve the expression of CD40L protein from Normal volunteer's T cells were stimulated with PMA for 1 hr. After stimulation and wash cells were used for RNA extraction. The RNA was taken into one tube RT-PCR reaction using Gene Amp Gold kit (Applied Bioscience) substituting Gene Amp Gold Taq polymerase with Advantage Polymerase (Clontech). One specific band of expected size of 0.8 kb was obtained in the PCR reaction using the following CD40L sequence specific primers: CD40L forward: 5'-GCATGATCGAAACATACAACC-3' and CD40L reverse : 5'-GTATTATGAAGACTCCCAGCG-3'.Purified PCR fragment was subcloned into pCR2.1 vector. Presence of specific sequence was verified by restriction digest and sequencing. Results of the BLAST analysis performed on the final sequence indicated identity to sequence with accession # NM_000074 which is encoding for CD40L. One exception was A to G transition at a codon 102 of open reading frame which is silent and does not result in a change in amino acid sequence and was left unchanged.E. coli DNA Polymerase I (IDT) in the presence of 30 μM dNTPs. Following gel purification of the 4738 base pair DNA fragment was re-ligated using the FastLink DNA ligation kit (Epicentre Biotechnologies).To construct the pCR2.1 CD40L ΔXE plasmid, the pCR2.1 CD40L plasmid was digested simultaneously with XbaI and EcoRV (Promega). The XbaI overhang was subsequently filled with Klenow fragment of To construct the pCR2.1 CD40L+5'UTR plasmid, the pCR2.1 CD40L plasmid was used as a template in a PCR reaction with the following oligonucleotide primers: CD40L 5'UTR Forward (5'ACGAATTCTTCTCTGCCAGAAGATACCATTTCAACTTTAACACAGCATGATCGAAACATACAACC) and CD40L 3'UTR Reverse (5'CCAGTGTGCTGGAATTCGGC) and Pfu Ultra Hot Start (Stratagene). The resulting 890 bp PCR was then digested with EcoRI (Promega), gel purified and was ligated to EcoRI-digested pCR2.1 CD40L ΔXE plasmid. These manipulations resulted in the addition of 39 bp of human CD40L 5' UTR sequence 12 base pairs downstream of the transcription initiation site.Construction of the pCR2.1 CD40L+Kozak plasmid involved PCR amplification of the pCR2.1 CD40L ΔXE plasmid template with the primers CD40L Kozak F (5'TCTGCAGAATTCGGCCACCATGATCGAAACATA) and CD40L 3'UTR Rev using Pfu Ultra Hot Start. The 880 base pair PCR product was digested with EcoRI and ligated to EcoRI site of pCR2.1 CD40L ΔXE.The accurate translation initiation site in pCR2.1 CD40L ΔXE was mutated to create pCR2.1 CD40L ΔXE-MET1 using the QuickChange method described by Stratagene. The oligonucleotides used for mutagenesis were CD40L First Methionine F (5'CTGCAGAATTCGGCTTGCGCGATCGAAACATACAACC) and CD40L First methionine R (5'GGTTGTATGTTTCGATCGCGCAAGCCGAATTCTGCAG). This manipulation resulted in substitution of GC for AT in the CD40L first ATG codon. The changes were confirmed by DNA sequencing.nd & 3rd methionine F (5'CACTGGACTGCCCATCAGCGCGAAAATTTTTGCGTATTTACTTACTG) and CD40L 2nd & 3rd methionine R (5'CAGTAAGTAAATACGCAAAAATTTTCGCGCTGATGGGCAGTCCAGTG). The template for pCR2.1 CD40L ΔXE-MET1–4 was pCR2.1 CD40L ΔXE-MET1,2,3 and the oligonucleotides used were CD40L Fourth methionine F (5'CTGTTTTTCTTATCACCCAGGCGATTGGGTCAGCACTTTTTGC) and CD40L Fourth methionine R (5'GCAAAAAGTGCTGACCCAATCGCCTGGGTGATAAGAAAAACAG).All subsequent methionine mutagenesis experiments followed the same procedure and resulted in a similar GC/AT substitution. The template for the pCR2.1 CD40L ΔXE-MET1,2 plasmid was pCR2.1 CD40L ΔXE-MET1 and the oligonucleotides used were CD40L Second methionine F (5'CACTGGACTGCCCATCAGCGCGAAAATTTTTATGTATTTACTTACTG) and CD40L Second methionine R (5'CAGTAAGTAAATACATAAAAATTTTCGCGCTGATGGGCAGTCCAGTG). The template for pCR2.1 CD40L ΔXE-MET1,2,3 was pCR2.1 CD40L ΔXE-MET1 and the oligonucleotides used were CD40L 2in vitro transcription were prepared by digesting each plasmid with SpeI (Roche). The linear template was purified by phenol:chloroform extraction followed by precipitation in ethanol and then resuspended in water.Linear templates for in vitro transcribed RNAs were purified using Qiagen RNeasy Purification kits as directed by the manufacturer.Transcription reactions of co-transcriptionally capped RNAs were performed using mMessage mMachine T7 Ultra (Ambion). Transcription reactions to produce uncapped RNA were performed using T7 Flash Kit (Epicentre). Reactions were assembled as specified by each kit's manufacturer. All Post-transcriptional capping modification of uncapped with type I cap CD40L MET1 RNA was performed using ScriptCap Kit (Epicentre Biotechnologies) components using capping buffer, GTP, SAM, Vaccinia Capping and Vaccinia 2'O-Methyltransferase Enzymes.The polyadenylation was performed on purified RNA using A-plus Poly(A) Tailing Kits (Epicentre Biotechnologies). Final RNAs were purified as described above and the length of the poly(A) tail was determined by comparing sizes of pre- polyadenylated and polyadenylated RNAs on denaturing gel electropheresis. A typical preparation of post-transcriptionally polyadenylated RNA contained polyA tail of greater than 150 nucleotides.35S-labeled methionine (Amersham) was assembled using Wheat Germ Extract Kit (Promega) according to manufacturer's instruction. 1 μL of each reaction was resolved on SDS Denaturing gel electrophoresis and transferred onto PVDF membrane. The membrane was exposed to phosphoimager screen and image obtained by scanning using Storm imager (Amersham).1 μg of RNA was heat denatured at 65°C for 3 minutes and immediately cooled on ice. 25 uL reaction containing the RNA and 1. 25 μL of Redivue E. coli RNaseH (Ambion) for 1 hour at 25°C. The products of the digestion were extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated with sodium acetate and ethanol in the presence of 20 μg glycogen (Roche). The precipitate was washed with 70% ethanol, allowed to air dry and then resuspended in water. 3'end labeling was performed with 10 mCi/mL α32P-pCp (NEN Perkin Elmer), T4 RNA ligase buffer and 1.25U T4 RNA ligase (Ambion) for 12 -16 hours at 4°C. Labelled RNA was purified using NucAway spin column (Ambion). RNA samples were diluted with urea loading dye and resolved on an 8M urea/15% polyacrylamide gel. Reaction products were visualized in the wet gel by phosphorimager analysis and quantified using ImageQuant software.2 μg of CD40L RNA and 32 pmol of the DNA oligonucleotide 5'CGCTCGAGCATGCAT/3ddC/3' (IDT) were first denatured at 85°C for 4 minutes and then allowed to hybridize for 4 hours at 42°C in 10 μL of RPA Hybridization III Buffer (Ambion). Immediately after the hybridization the RNA:DNA duplex was digested with 10 U 3 plastic flasks for 2 hours at 37°C. Non-adherent cells were removed and remaining cells cultured in X-vivo 15 medium, supplemented with 1000 U/ml GM-CSF (Leukine) and 1000 U/ml IL-4 (R&D systems), for 6 days at 37°C, 5% CO2.Human PBMCs were isolated from Leukapheresis collections from healthy volunteers by Ficoll-histopaque density centrifugation. PBMCs were re-suspended in AIM-V medium (Invitrogen) and allowed to adhere to 150 cm6/ml. Maturation was achieved by addition of 10 ng/ml TNF-α, 10 ng/ml Il-1β, 100 ng/ml IL-6 and 1 μg/ml PGE2 followed by culture for 24 hours at 37°C in low adherence six well plates (BD Biosciences).Prior to electroporation, DCs were harvested and washed in PBS and then re-suspended in chilled Viaspan. DCs were mixed with 2 μg of mRNA encoding GFP per million cells.and placed in a 4 mm gap electroporation cuvette and electroporated using BioRad apparatus. Immediately after electroporation, DCs were washed in X-vivo-15 medium and finally re-suspended in X-vivo-15 supplemented with 800 U/mL GM-CSF and 500 U/mL IL-4 at 1 × 102 for a further 24 hrs.As described above, washed immature DC were re-suspended in chilled Viaspan and mixed with varying amounts of mRNAs encoding CD40L as described in Results, and electroporated using BioRad apparatus. Post-electroporation, DC were cultured in X-vivo-15 in the presence of GM-CSF (800 U/ml) and IL-4 (500 U/ml) plus 1000 U/mL IFN-γ and 1 μg/mL PGE5 DCs were harvested post-transfection with CD40L mRNA and re-suspended in 250μL of Cytofix/Cytoperm solution (BD Biosciences) at 4°C. Cells were washed twice with 2 mL staining buffer , re-suspended in 0.5 mL staining buffer and stored over night at 4°C. Cells were re-suspended in 2.0 mL Perm/Wash solution (BD Biosciences) for 15 minutes, centrifuged and re-suspended in 100μL Perm/Wash solution. 20μL of mouse anti-human CD40L PE and anti-human CD40 APC (BD Biosciences) or mouse IgG1 PE and IgG1 APC (BD Biosciences) was added to each DCs preparation collected and permeabilised at each time point, and incubated at 4°C for 30 minutes in the dark. Cells were washed twice with 1 mL Perm/Wash solution and re-suspended in staining buffer prior to flow cytometric analysis.2 × 106 cells/ml and supernatants collected after 24 hrs for the measurement of Il-12 or Il-10 secretion using BD Pharmingen IL-12 or IL-10 ELISA kits. Briefly, ELISA plates (BD Biosciences) were coated with anti-IL-12p70 or anti-IL-10 ELISA capture antibody in coating buffer for 24 hours at 4°C using BD Opt EIA reagent set B pH 9.5. Plates underwent blocking with 200μL per well 10%FCS/PBS for one hour prior to the addition of standards (BD Pharmingen) and supernatant samples, in duplicate, at 100μL per well and incubated at room temperature for 2 hrs. Plates were washed and anti-cytokine detection antibody added, incubated for one hour, the plates washed and solutions replaced with 100 μL of streptavidin-HRP and further incubated for one hour at room temperature. Again plates were washed and colour development substrates applied for 10–20 minutes, followed by cessation of colour development with stop solution. Plate analysis undertaken using Bio-Tek instruments ELx800 plate reader with KC junior software.Mature DCs generated as described above, were immediately cultured post-electroporation at a concentration of 1 × 10IT conceived of the study, carried out its coordination, conducted experiments, drafted the manuscript and revised for important intellectual content. MA carried out molecular biology side of most experiments and analyzed the data, participated in the design of the study. XF created site-directed constructs as well as prepared the RNAs.AH, JH and DC carried out the immunoassays. DH participated in the design and coordination of the study. CN contributed to the drafting of the manuscript and approved the final version to be published.