G-U-base pairings (secondary structure) and additional cross-serial base pairs. These interactions are called pseudoknots and are observed across the whole spectrum of RNA functionalities. In the context of studying natural RNA structures, searching for new ribozymes and designing artificial RNA, it is of interest to find RNA sequences folding into a specific structure and to analyze their induced neutral networks. Since the established inverse folding algorithms, RNAinverse, RNA-SSD as well as INFO-RNA are limited to RNA secondary structures, we present in this paper the inverse folding algorithm Inv which can deal with 3-noncrossing, canonical pseudoknot structures.RNA exhibits a variety of structural configurations. Here we consider a structure to be tantamount to the noncrossing Watson-Crick and http://www.combinatorics.cn/cbpc/inv.html.In this paper we present the inverse folding algorithm Inv. We give a detailed analysis of Inv, including pseudocodes. We show that Inv allows to design in particular 3-noncrossing nonplanar RNA pseudoknot 3-noncrossing RNA structures-a class which is difficult to construct via dynamic programming routines. Inv is freely available at The algorithm Inv extends inverse folding capabilities to RNA pseudoknot structures. In comparison with RNAinverse it uses new ideas, for instance by considering sets of competing structures. As a result, Inv is not only able to find novel sequences even for RNA secondary structures, it does so in the context of competing structures that potentially exhibit cross-serial interactions. The structure S motivates to consider a new adjacency relation within C[S]. Indeed, we may reorganize a sequence  into the pairLet uh denotes the unpaired nucleotides and the ph =  denotes base pairs, respectively, see Figure C[S].where the Accordingly, there are two types of compatible neighbors in the sequence space u- and p-neighbors: a u-neighbor has Hamming distance one and differs exactly by a point mutation at an unpaired position. Analogously a p-neighbor differs by a compensatory base pair-mutation, see Figure G-C ↦ G-U) or Hamming distance two (G-C ↦ C-G). We call a u- or a p-neighbor, y, a compatible neighbor. In light of the adjacency notion for the set of compatible sequences we call the set of all sequences folding into S the neutral network of S. By construction, the neutral network of S is contained in C[S]. If y is contained in the neutral network we refer to y as a neutral neighbor. This gives rise to consider the compatible and neutral distance of the two sequences, denoted by C and N. These are the minimum length of a C[S]-path and path in the neutral network between s and s', respectively. Note that since each neutral path is in particular a compatible path, the compatible distance is always smaller or equal than the neutral distance.Note, however, that a p-neighbor has either Hamming distance one  probabilities. The third routine constitutes a mechanism for minimizing the occurrence of undesired but favourable interactions between specific sequence segments. Following these subroutines, RNA-SSD derives a hierarchical decomposition of the target structure. It recursively splits the structure and thereby derives a binary decomposition tree rooted in T and whose leaves correspond to T-substructures. Each non-leaf node of this tree represents a substructure obtained by merging the two substructures of its respective children. Given this tree, RNA-SSD performs a stochastic local search, starting at the leaves, subsequently working its way up to the root.INFO-RNA constructs sequences folding into a given secondary structure by employing a dynamic programming method for finding a well suited initial sequence. This sequence has a lowest energy with respect to the T. Since the latter does not necessarily fold into T,  INFO-RNA then utilizes an improved  stochastic local search in order to find a sequence in the neutral network of T. In contrast to RNAinverse, INFO-RNA allows for increasing the distance to the target structure. At the same time, only positions that do not pair correctly and positions adjacent to these are examined.Cross is an ab initio folding algorithm that maps RNA sequences into 3-noncrossing RNA structures. It is guaranteed to search all 3-noncrossing, σ-canonical structures and derives some (not necessarily unique), loop-based mfe-configuration. In the following we always assume σ ≥ 3. The input of Cross is an arbitrary RNA sequence s and an integer N. Its output is a list of N 3-noncrossing, σ-canonical structures, the first of which being the mfe-structure for s. This list of N structures  is ordered by the free energy and the first list-element, the mfe-structure, is denoted by Cross(s). If no N is specified, Cross assumes N = 1 as default.Cross generates a mfe-structure based on specific loop-types of 3-noncrossing RNA structures. For a given structure S, let α be an arc contained in S (S-arc) and denote the set of S-arcs that cross α by α =  and α' = , we next specify the partial order "≺" over the set of arcs:α ∈ β-crossing if there exists no α' ∈ α' ≺ α. Note that α ∈ β-crossing, while β is not minimal α-crossing. 3-noncrossing diagrams exhibit the following four basic loop-types:All notions of minimal or maximal elements are understood to be with respect to ≺. An arc (1) A hairpin-loop is a pairi, j) is an arc and  is an interval, i.e. a sequence of consecutive, isolated vertices .where ((2) An interior-loop, is a sequencei2, j2) is nested in . That is we have i1 < i2 < j2 < j1.where ((3) A multi-loop, see Figure ωh, τh], subject to the condition that if all these substructures are simply stems, then there are at least two of them, see Figure where A pseudoknot, see Figure (P1) A set of arcsi1 = min{ih} and jt = max{jh}, such thatwhere (i) the diagram induced by the arc-set P is irreducible, i.e. the dependency-graph of P (i.e. the graph having P as vertex set and in which α and α' are adjacent if and only if they cross) is connected and(ii) for each  ∈ P there exists some arc β (not necessarily contained in P) such that  is minimal β-crossing.(P2) Any i1 <x <jt, not contained in hairpin-, interior- or multi-loops.Having discussed the basic loop-types, we are now in position to stateTheorem 1 Any 3-noncrossing RNA pseudoknot structure has a unique loop-decomposition  and bw =  ⊃ aw, being the maximal interval consisting of aw and its adjacent unpaired consecutive nucleotides, see Figure Tw <Tw + 1, we have two scenarios:projecting the loop bw and bw+1 are adjacent, see b5 and b6 in Figure • either bw ⊆ bw + 1, see b1 and b2 in Figure • or a1, b1, c1, ..., am', bm', cm'. If there are no unpaired nucleotides adjacent to aw, then aw = bw and we simply delete all such bw. Thereby we derive the sequence of intervals I1, I2, ..., Im. In Figure T1, T2 and we have I1 = , I2 = , I3 = , and I4 = .Let OCAL-SEARCH: Given the sequence of intervals I1, I2, ...,  Im. We proceed by performing a local stochastic search on the subsequences seq = seqmiddle and where s|x, y]    ← random nucleotide or pair, s.t. seq ∈ C[T] and seq ∉ C[Ch(λi-1)]27.          end if28.       end for29.    Tseq ← Cross(seq)30.    if d <dmin + 5 then31.    seqmiddle ← seq32.       else if Step III run less than 5 times then33.    goto Step III34.       end if35.    end for ▻ loop to line 336. 37.return seqmiddle38. 7.3 Algorithm 7.3 - LOCAL-SEARCHInput:seqmiddleInput: the target TseqOutput: Ensure: Cross(seq) = Tseq ← seqmiddle1. if Cross(seq) = T then2. seq3.    return end if4. T and derive the ordered intervals5. decompose I ← 6. w Iin I do7. for all 8.    ▻ Phase I: Identify positionsdmin9.    10.U1 via 11.    derive U2 via 12.    derive 13.14.    ▻ Phase II: Test and Updatefor all p in U1 do15.    T compatible mutate seqp16.       random end for17.    for all  in U2 do18.    T compatible mutate seqp19.       random end for20.    21.E ← ϕ22.    for all p ∈ U1, U2 do23.    d ← d)24.       if d <dmin then25.       dmin ← d, seq ← seqp26.          goto Phase I27.          else if dmin <d <dmin + 5 then28.       goto Phase I with the probability 0.129.          end if30.       if d = dmin then31.       E ← E ∪ {seq}32.          end if33.       end for34.    seq ← e0 ∈ E, where e0 has the lowest mfe in E35.    if Phase I run less than 10 n times then36.    goto Phase I37.       end if38.    end for39. return seq40. We are grateful to Fenix W.D. Huang for discussions. Special thanks belongs to the two anonymous referee's whose thoughtful comments have greatly helped in deriving an improved version of the paper. This work was supported by the 973 Project, the PCSIRT of the Ministry of Education, the Ministry of Science and Technology, and the National Science Foundation of China.


ZNF9 transcript (ZNF9 encodes a zinc finger protein). In both pathologies the mutant RNA forms nuclear foci. The mechanisms that underlie the RNA pathogenicity seem to be rather complex and not yet completely understood. Here, we describe Drosophila models that might help unravelling the molecular mechanisms of DM1-associated CUG expansion toxicity. We generated transgenic flies that express inducible repeats of different type (CUG or CAG) and length  and then analyzed transgene localization, RNA expression and toxicity as assessed by induced lethality and eye neurodegeneration. The only line that expressed a toxic RNA has a (CTG)240 insertion. Moreover our analysis shows that its level of expression cannot account for its toxicity. In this line, (CTG)240.4, the expansion inserted in the first intron of CG9650, a zinc finger protein encoding gene. Interestingly, CG9650 and (CUG)240.4 expansion RNAs were found in the same nuclear foci. In conclusion, we suggest that the insertion context is the primary determinant for expansion toxicity in Drosophila models. This finding should contribute to the still open debate on the role of the expansions per se in Drosophila and in human pathogenesis of RNA-dominant diseases.Evidence for an RNA gain-of-function toxicity has now been provided for an increasing number of human pathologies. Myotonic dystrophies (DM) belong to a class of RNA-dominant diseases that result from RNA repeat expansion toxicity. Specifically, DM of type 1 (DM1), is caused by an expansion of CUG repeats in the 3′UTR of the DMPK protein kinase mRNA, while DM of type 2 (DM2) is linked to an expansion of CCUG repeats in an intron of the  DMPKDMPK encodes a serine-threonine protein kinase which phosphorylates various proteins including CUG-BP1, a CELF protein (CUG-BP/ETR3-like factor) whose dysfunction is involved in DM1 pathogenesis Myotonic dystrophy type 1 (DM1), also called Steinert myotonic dystrophy is a multi-systemic pathology originally described as a neuromuscular disorder DMPK adjacent loci; 3) toxicity of the CUG expansion containing RNA. Given the complexity of the pathology, it is possible that all these mechanisms contribute to the phenotype  haploinsufficiency of DMPK; 2) transcription repression at The finding that hnRNP H binds only to a specific sequence/structure , demonstrates that RNA expansions acquire new properties which are dependent on the sequences in which they reside. Indeed, the basic view that nuclear aggregates formed by CUG expansions are intrinsically toxic and/or that toxicity severity is determined only by the size of the repeats and their concentration have been already challenged by various studies 240.4 line that develops a deleterious phenotype upon ubiquitous or neuronal induction. Furthermore, all the (CUG)240 and (CUG)480 RNAs, but not the (CUG)16 RNA, form nuclear foci regardless of whether they have a toxic effect or not. We also demonstrate that in the (CTG)240.4 line, the expansion is inserted in an endogenous gene, CG9650, that encodes a zinc finger (ZNF) protein 240.4 expansion correlates with the fusion transcript that it forms with CG9650 RNA.To this aim, we generated fly transgenic lines to try to understand the role of repeat type, repeat length, RNA expression levels and RNA insertion context in the formation of nuclear foci and expansion toxicity. We show that CUG expansions do not have a toxic effect in most of the transgenic lines with the exception of the (CTG)Drosophila UASp vector. The (CTG)16 constructs were cloned in the SV40 3′UTR downstream the LacZ ORF. Five (CTG)240, three (CTG)480, one (CAG)240, two (CAG)480 and three (CTG)16 transgenic lines were generated.Our aim was to generate transgenic flies using the UAS/GAL4 system Drosophila n expression using RNA fluorescence hybridization (RNA-FISH) with a CAG-FITC probe (240 and (CUG)480 RNAs formed nuclear foci that were localized in a small area of the nucleus (16 transgenic RNAs did not form nuclear foci (240 and (CAG)480 lines (data not shown).Tissue-specific induction of the repeat expansions was achieved by crossing UAS-(CTG)TC probe . In sali nucleus . In cont nucleus  and ovar nucleus , we obseear foci . Using aTo unambiguously assign to RNA the localized signal observed in the salivary glands nuclei, we treated the specimens with ribonuclease before the RNA-FISH procedure. After ribonuclease treatment, we could not detect the nuclear foci any more, whereas the DAPI signal was not affected .240 and (CUG)480 expanded RNAs.Therefore, the nuclear foci were due specifically to the induced (CUG)Gal4 driver lines 480 lines produced nuclear foci, only the (CTG)240.4 had problems of viability. Indeed, upon ubiquitous and pan-neural induction the viability of this transgenic line was reduced to 0% and to 21% when the motoneuron-specific driver BG380-Gal4 was used 240.4 RNA toxicity, RNA rates were analyzed using quantitative RT-PCR (qRT-PCR) amplification. RNA extracted from adult heads of GMRnina-Gal4 induced flies was reverse-transcribed using either oligo-dT primer or random hexamers, and cDNA was amplified using transgene-specific primers, complementary to the 3′UTR of the transcript 16 lines which do not form nuclear foci, had an RNA rate variability within the same range as that of the lines with larger expansions, indicates that the RNA was quantitatively extracted from the foci. We consider that the RNA rate accounts for the level of expression.To further investigate the molecular basis of (CUG)anscript . A quanttrations . (CUG)n 240.4 RNA does not explain its toxicity.In conclusion, the level of expression of the (CUG)240.4 RNA could be accounted for by the transgene insertion context. We assessed this hypothesis by performing inverse PCR amplification on genomic DNA prepared from the different transgenic lines. All but three lines had the transgene inserted in a non transcribed region of the genome (240.4 line was one of the three exceptions. (CTG)240.4 insertion lies within the first intron of the CG9650 gene that encodes a zinc finger protein 240.4 line after induction of the transgene in the salivary glands using the MS1096-Gal4 driver 240.4 RNA (trans from an insertion that is located at a different locus (data not shown). Therefore, the (CUG)240.4/CG9650 foci could form only when CG9650 RNA and the CUG expansion lie in cis within the same transcript.We then investigated whether the toxic effect of the (CUG)e genome . The (CT40.4 RNA . We did 240.1 and (CUG)480.3 RNAs expressed by lines whose insertions lie also within a transcribed region d region , did not240.4 line and how the fusion transcript induced toxic effects is not clear. The important issue is that the formation of a fusion RNA in the (CUG)240.4 line results in a situation analogous to that observed in DM1 where the expansion lies in the DMPK 3′UTR.The formation of a fusion RNA must be an exceptional event as a classical SV40 3′UTR was used in these constructs that brings about a functional polyadenylation signal Drosophila genetics has proved to be particularly powerful in dissecting biological processes. As a result, Drosophila has been increasingly used to model human pathologies Drosophila models to try to understand the molecular basis of CUG expansions toxicity.In the present work we wanted to generate 16, (CUG)240 and 480, (CAG)240 and 480. These constructs were devoid of any of the human sequences used in various animal or cell models for myotonic dystrophy To address this complex question we generated transgenic flies with constructs that allow expression of RNA repeats of different type and length: (CUG)16 containing RNAs. (CAG)240 and (CAG)480 expansions did not form nuclear foci in spite of having a large number of repeats. The difference in the ability of the CAG and CUG expansions to form foci may reside in a difference in the stability of the secondary structures that are formed by these RNAs. Other Authors have described nuclear foci formation in cells expressing CAG expansions In this report, we confirm that CUG expansions are sufficient to generate ribonuclease sensitive nuclear foci provided they are long enough. Indeed we did not observe nuclear retention of (CUG)160 RNAs did not result in toxicity, in spite of co-localization with the Muscleblind double stranded RNA binding factor 480 expansion in transgenic flies recapitulated at least some of the disorders associated with DM1 whereas foci formation without toxicity was observed in (CUG)200 expressing flies Drosophila. However, using the same vector and repeat type 480 expansions in our transgenic lines. This was true also for all the (CUG)240 expressing lines but one, (CUG)240.4. Our results, therefore, rule out the possibility that a specific length of RNA repeats is the primary determinant in the gain-of-function toxicity in fly models.Nuclear foci have been considered as a hallmark of RNA gain-of-function toxicity. However, an increasing body of knowledge clearly suggests that the formation of nuclear foci is not sufficient n RNA expressing transgenes inserted randomly into the genome, therefore the insertion context could not be anticipated. Consequently its contribution to the RNA toxicity could be objectively assessed. Thus we were able to show that the only transgenic expansion that was toxic was expressed as a fusion transcript with an endogenous RNA, a situation that is similar to that of the RNA dominant diseases in humans. In human pathologies, expanded RNAs result from genomic amplification of endogenous repeats that lie within specific transcribed regions. In DM1, the expansion lies within DMPK and in DM2, it lies within ZNF9The (CUG)trans acting factors that bind the CUG repeats and that are reported to be implicated in DM1 Drosophila, like CUG-BP1 muscleblind gene was first identified in Drosophilaper se might not be sufficient. The expansion context or other elements might be major determinants of the RNA toxicity and the subsequent phenotype. Not only our findings but also previously published work strengthen this hypothesis. In a cell culture model large CUG expansions, in spite of their ability to form nuclear foci, do not affect myoblast differentiation. In contrast, myoblast differentiation is affected by the same expansions when they are present in the DMPK 3′UTR trans dominant neurological disease in which CUG repeat expansion was implicated One could argue that induction of toxicity in the fly transgenic lines and human pathogenesis might be supported by different mechanisms. However the 240.4 line cannot be explained by a mere loss or gain of function of CG9650, both (CUG)240.4 RNA induction (as shown in this work), and CG9650 over-expression affect the nervous system and the eye 240.4 expanded RNA analyzed in the present work. Similarly, in the SCA8 fly models mentioned above, no muscular disorder was induced by the CUG expansion The similarities that may be observed between the phenotypes that result from expression of CUG expansions and those that result from mis-expression of the RNA in which they reside deserve attention. Although the phenotype of the (CTG)trans dominant effects of the expanded RNA may be mediated by various factors among which some must be relevant to the pathway of the RNA in which the expansion resides. In favour of this hypothesis is the observation that DM1 and DM2, the two neuromuscular disorders linked to repeat expansions in DMPK and ZNF9 genes respectively, also show significant differences DMPK 3′UTR mRNA is over-expressed DMPK 3′UTR in the pathogenicity of CUG expanded RNA in DM1 patients.Our hypothesis based on this work and data reported in the literature trans dominant effect of the RNA expansion, that depends only on the repeat type, size or level of expression.All these reports and the present one definitely challenge the view of a ZNF9 RNA flanking sequences were found associated with the CCUG repeats nuclear foci Our hypothesis is, however, challenged by the finding that in DM2 no Drosophila models should provide an invaluable help in solving the central question: how the mutant RNA exerts its toxic effect. Since several laboratories are working on the production of fly models of RNA dominant diseases, a number of independent insertion lines should become available to the community for a systematic investigation to be undertaken.These contradictory findings and the complexity of the RNA dominant diseases, suggest that the molecular mechanisms of RNA expansion toxicity are still elusive and further work is needed to properly address this question. Gal4 drivers used in this work were MS1096-Gal424B-Gal4COG-Gal4Actin-Gal4 P{Act5C-GAL4}25FO1/BL#4414, elav-Gal4, daughterless-Gal4  and GMRnina-Gal4 drivers were used for ubiquitous, pan-neuronal and eye expression, respectively BG380-Gal4 driver was used to induce expression in motoneurons UASp transgenic lines were generated as described below. For germ line transformation, experimental conditions were as described earlier 16 sequence flanked by SpeI and BglII restriction sites was obtained by hybridizing the 5′ctagt(ctg)16a and 5′gatct(cag)16a oligonucleotides, called (CTG)16 and anti-(CTG)16. The recipient vector was prepared as following: Bluescript SK+ SpeI restriction site was destroyed, generating a SK/SpeI vector. To do so, the vector was first digested with SpeI, blunt ended with T4 DNA polymerase and religated. Eg5/EDEN/3′SV40 fragment from C06 plasmid SpeI vector using the EcoRI site. The EDEN element was substituted by the (CTG)16 sequence described above in the SK/SpeI/EDEN vector, thereby becoming the SK/SpeI/(CTG)16 vector. The Eg5/(CTG)16/3′SV40 fragment from the SK/SpeI/(CTG)16 vector was cloned into the SK/LacZ plasmid EcoRI site NotI restriction site was inserted downstream the SV40 3′UTR sequence after ApaI/XhoI digestion of the vector and integration of a linker generated by hybridization of NADXAP1 (tcgaggcggccgcgggcc) and NADXAP2 (cgcggccgcc) oligonucleotides. This new LacZ/Eg5/(CTG)16/3′SV40 fragment was finally cloned into UASp NotI site. The resulting UASp/lacZ/(CTG)16 construct was used for germ line transformation.The double stranded (CTG)240 and Sp72(CTG)480 plasmids 240 and 480 fragments were cut out of the original plasmids by an XbaI/XhoI digestion, purified and cloned into pBluescript SK+ generating SK/(CTG)240 and SK/(CTG)480 plasmids. Flanking restriction sites were added at each side of the repeats using XbaI-SpeI-SalI and ApaI-BglII-XhoI linker sequences. These modifications allow the isolation of the CTG repeats as a SpeI-BglII cartridge that can be used as above to replace the EDEN sequence of the SK/SpeI/EDEN vector. SK/(CTG)240/3′Eg5 and SK/(CTG)480/3′Eg5 plasmids were generated. The repeat fragments were then cut out as BamHI fragments and inserted into a BamHI-digested UASp vector, thus generating UASp/(CTG)240/3′Eg5, UASp/(CTG)480/3′Eg5 and UASp/(CAG)480/3′Eg5 plasmids depending on the insert orientation. These plasmid constructs were used for germ line transformation.Sp72(CTG)XbaI-SpeI-SalI linker was obtained by hybridizing the XSpeIS1 (ctagactagtg) and XSpeIS2 (tcgacactagt) oligonucleotides. The ApaI-BglII-XhoI linker was obtained by hybridizing the XBglIIA1 (tcgagatctgggcc) and XBglIIA2 (cagatc) oligonucleotides.The 240/3′Eg5, a KpnI fragment was inserted into the KpnI site of UASp vector.To generate UASp/(CAG)The quality of the inserts was verified twice, first by sequencing the entire fragments before their cloning into the UASp vector and then, after the last cloning step, the sequence junctions were also verified by sequencing (Genome Express). The expansions proved to be highly unstable in bacteria and all these verifications were required. The cloning procedure required to grow the bacteria at 30°C.yw stock using standard methods 16, five UASp/(CTG)240/3′Eg5, three UASp/(CTG)480/3′Eg5, one UASp/(CAG)240/3′Eg5, two UASp/(CAG)480/3′Eg5 independent lines were generated and were analyzed for each construct.P element transformation was performed with the http://www.fruitfly.org). Genomic DNA was extracted from 30 flies, isopropanol precipitated and re-suspended in 150 µl TE as indicated in the BDGP procedure.Genomic DNA (gDNA) extraction and inverse PCR amplifications were performed following the protocol provided by the “Berkeley Drosophila Genome Project Resources” (Sau3A (Promega) or MspI (Biolabs), re-ligated with T4 DNA ligase (Promega) according to the manufacturer's instructions. After ethanol precipitation, PCR amplifications were performed according to the protocol instructions both with Pwht1 and Plac1 primers for the 5′ end and with Pry4 and Pry1 primers for the 3′ end of the inserted transgene. Sequencing reactions were performed either with Sp1 for the 5′ end or with Spep1 for the 3′ end using DYEnamic ET Terminator Cycle Sequencing kit and following the manufacturer's instructions (Amersham Biosciences). Sequencing reactions were run on ABI PRISM™ 377 DNA Sequencer (Applied Biosystems).Genomic DNA (10 µl) was then digested either with 16 transgene, 1 µl (0.1 µg) of total DNA samples was amplified in 50 µl reactions using GoTaq® DNA polymerase according to the manufacturer's instructions (Promega). For the (CTG)240 or 480 transgenes, 1 µl (0.1 µg) of total DNA samples was amplified in 20 µl reactions using JumpStart™ AccuTaq™ LA DNA polymerase according to the manufacturer's instructions (Sigma) with 5% DMSO and 50 mM betaine. PCR amplification reactions involved 3 cycles at 95°C (45 s), 67°C (45 s) and 72°C (1 min 30 s) followed by 30 cycles at 95°C (45 s), 62°C (45 s) and 72°C (1 min 30 s) in a MiniCycler™ (MJ Research). Amplified products (5 µl) were mixed with 1 µl of loading buffer and subjected to electrophoresis in a 1% agarose gel in 0.5× TBE with ethidium bromide and revealed by UV light. Size was determined by comparison with a 1 kb DNA ladder (Promega).For the LacZ (CTG)Salivary glands of third instar larvae were hand dissected in ice cold 1× PBT (PBS+0.1% Tween). They were fixed for 20 min in 4% paraformaldehyde in PBT, and then rinsed in PBT 5 times for 15 min. They were permeabilized for 5 min in a TE solution  containing 50 µg/ml proteinase K, and then rinsed twice in a PBT solution containing 2 mg/ml of glycine and twice for 10 min in PBT. A post fixation was performed for 20 min in 4% paraformaldehyde solution in PBT and then rinsed in PBT 5 times for 10 min. Salivary glands were then ready for RNA-FISH.Muscles from third instar larvae were hand dissected. Larvae were anesthetized by placing them for 5 min on ice. Each larva was immerged in a drop of 1× PBS containing 10 mM EGTA, then opened dorsally and pinned open on a siligar covered plate with minutiens . The cuticles with the attached muscles were then fixed, permeabilized, after post-fixation as indicated above they were ready for the RNA-FISH procedure. The ovaries were hand dissected from 3 day-old females and treated as above.6C oligonucleotide, FITC-labelled at both ends (PROLIGO). Samples were incubated in 40% formamide, 2× SSC for 30 min and hybridized overnight at 37°C with 1 ng/µl of the CAG-FITC probe in a 200 µl final volume of a solution containing 40% formamide, 2× SSC, 0.2% BSA, 2 mM vanadyl ribonucleoside complex and 1 mg/ml yeast tRNA. Samples were then washed in 40% formamide, 2× SSC for 30 min, rinsed twice in 1× SSC in PBS for 10 min and mounted on slides with Vectashield® DAPI-containing mounting medium . For double-labeling experiments with a CG9650 specific probe (C96), 1 ng/µl of two 5′ Rhodamin-labeled probes was added, rho-C96-1 (gtagcagtgggcaattggtt) and rho-C96-2 (tcgaacagagcctcattccg). When RNase-treated, samples were incubated for 30 min at 37°C in a solution containing 0.1 mg/ml of a DNase and Proteinase free ribonuclease A solution (Fermentas) prior to RNA-FISH.The CAG-FITC probe used was a (CAG)Slides were examined under a Leica DMRA2 microscope; images were captured with a photometrics coolSNAP™ HQ camera. Images from two or three wavelengths  were assembled and colored using Metaview Software.GMRnina-Gal4 driver. 30 heads from 3 day-old flies were cut and immediately put in RNA stabilizing buffer . Total RNA was extracted according to the manufacturer's instructions. RNA quantification was performed on 2 µl total RNA sample in a NanoDrop ND-1000 spectrophotometer .Flies were raised at 29°C for induction of transgene expression by 480 and different dilutions were used for (CUG)240  in a 20 µl total reaction with 50 mM betaine, 4 mM MgCl2, 0.5 µM of primer 3 (gctgctcgagatctaagcttc) and primer 4 (aatacacttttccataactaactcg) and 2 µl of master mix of LightCycler® FastStart DNA Master SYBER Green I kit (Roche Diagnostics). Transgene expression levels were normalized to the level of G-6-PDH expression using G-6-PDH-C2 (cgacattcgtgacgagaagg) and G-6-PDH-R1 (gttcgaatcgttgctaacgg) primers. G-6-PDH is an endogenous housekeeping gene 480 samples. For the (CUG)240 lines and controls, four independent experiments were carried out, with three different dilutions and two replicates for each. The mean value and standard deviation were determined using Microsoft Excel.2 µg of each of the total RNA samples were used for first strand cDNA synthesis using Superscript™ II Reverse Transcriptase (Invitrogen™) according to the manufacturer's instructions. For real-time PCR amplification, 5 ng of cDNA template was used for (CUG)Adult flies were frozen at −80°C, coated with Gold Palladium and examined with a Hitachi s-4000 scanning electron microscope.


Epithelioid sarcoma is an uncommon high-grade sarcoma, mostly involving the extremities.18F]-2-fluoro-2-deoxy-D-glucose-positron emission tomography (FDG-PET) detected multiple lesions with an increased uptake in the right neck, the bilateral upper arms and the right thigh, as well as in the left axillary lymph nodes, with maximum standardized uptake value (SUVmax) ranging from 2.0 to 5.5 g/ml. Magnetic resonance imaging confirmed that there was a lesion within the right thigh muscle which was suggestive of metastasis, even though the lesion was occult clinically.A 33-year-old man was referred to our institute with a diagnosis of Volkmann's contracture with the symptom of flexion contracture of the fingers associated with swelling in his left forearm. Magnetic resonance imaging (MRI) showed abnormal signal intensity, comprising iso-signal intensity on T1- and high-signal intensity on T2-weighted images surrounding the flexor tendons in the forearm. Diagnosis of epithelioid sarcoma was made by open biopsy, and amputation at the upper arm was then undertaken. [Increased uptake on FDG-PET might be representative of epithelioid sarcoma, and for this reason FDG-PET may be useful for detecting metastasis. Muscle metastasis is not well documented in epithelioid sarcoma. Accordingly, the frequency of muscle metastasis, including occult metastasis, needs to be further analyzed. Epithelioid sarcoma was first described in 1970 . The ove18F]-2-fluoro-2-deoxy-D-glucose-positron emission tomography (FDG-PET) has recently been used to assess various tumors. PET is evaluated using the standardized uptake value (SUV). An increased uptake of FDG-PET in cells reflects increased glucose metabolism as a result of various factors such as increased glucose transporters, high levels of hexokinase and a reduction in glucose-6-phosphatase [[sphatase ,6. FDG-Psphatase , head ansphatase . As for sphatase ,11. The sphatase .In this report, we present a case of epithelioid sarcoma with the symptom of Volkmann's contracture characterized by a claw-like deformity of the hand and fingers associated with contracture of the muscles in the forearm. Furthermore, FDG-PET detected occult metastasis to the muscle clinically, in addition to metastasis to the regional lymph nodes.A thirty-three-year-old man noticed extension disturbance of the left fingers, 7 months prior to the initial evaluation in our institute. He began to feel tension and pain in the forearm when he extended his fingers. He visited a local hospital 1 month after onset. A swelling in his left forearm appeared and this worsened gradually. The symptoms did not resolve, and the patient was then referred to our institute with a diagnosis of Volkmann's contracture Figure . Plain rAt our institute, surgery was undertaken not only to release the contracture, but also to obtain a biopsy specimen to diagnose the cause of the contracture. The surgery findings showed that the tendons had adhered to each other with cicatricial-like tissue without any obvious mass lesion. Then, release of the adhered flexor tendons was undertaken. The cicatricial-like tissue was sampled for analysis, and histologically, it was found to be composed of rounded or polygonal epithelioid cells, arranged in sheets or a solid trabeculae pattern. Degeneration and necrosis were also observed Figure . The neoCT showed no evidence of pulmonary metastasis, but it showed mild swelling of the axillary lymph nodes, which could have been possible metastasis, or simply non-specific swelling due to the biopsy procedure. Amputation above the elbow was undertaken. Three months after the amputation, bone scintigraphy showed no evidence of abnormal findings suggestive of metastasis to the bone (data not shown). However, CT showed increased size of the axillary lymph nodes, suggesting that these lymph nodes were actually metastasized Figure . For furA diagnosis of epithelioid sarcoma is challenging clinically, because epithelioid sarcoma is likely to be confused with a variety of benign and malignant conditions . Due to SUV in FDG-PET of malignant bone and soft-tissue lesions has been reported to be higher than that of benign bone and soft-tissue lesions, with the cut-off point ranging from 1.83 to 3.23 ,11. In aIn the current study, without FDG-PET, only the metastasis to the lymph nodes could be detected by CT, because metastasis to the muscle was occult clinically, even 3 months after the FDG-PET examination. The metastatic rate of epithelioid sarcoma has been reported to be 45%, and the most common sites of metastasis are the lung (51%), regional lymph nodes (34%), the scalp 22%) and bone 13%), while metastasis to the soft-tissue, including muscle, is thought to be less common [2% and bo%, while In conclusion, we have reported a case of epithelioid sarcoma with the symptom of Volkmann's contracture. The current case should act as a reminder that Volkmann's contracture can be a symptom of epithelioid sarcoma in the forearm, and this reminder should help avoid a delay in the commencement of treatment. Multiple metastases to the lymph nodes and muscle had an increased uptake in FDG-PET, and the increased uptake may be representative of epithelioid sarcoma. Metastasis to the muscle tissue has not been well described so far. Further reports are necessary for the precise frequency of muscle metastasis to be ascertained. In that case, FDG-PET might be useful for detecting such metastasis.The authors declare that they have no competing interests.AS drafted the manuscript. MH and MO are pathologists who helped with the discussion. AS, OJ and KA are surgeons who carried out the operation. YI is the Professor of the Department of Orthopaedic Surgery of Kyushu University who approves all relevant manuscripts. All authors read and approved the final manuscript.


High throughput applications of the reverse transcriptase quantitative PCR (RT-qPCR) for quantification of gene expression demand straightforward procedures to isolate and analyze a considerable number of DNA-free RNA samples. Published protocols are labour intensive, use toxic organic chemicals and need a DNase digestion once pure RNAs have been isolated. In addition, for some tissues, the amount of starting material may be limiting. The convenience of commercial kits is often prohibitive when handling large number of samples.Arabidopsis thaliana tissues ready for RT-qPCR applications. Simple non-toxic buffers were used for RNA isolation from Arabidopsis tissues with the exception of seeds and siliques, which required the use of organic extractions. The protocols were designed to minimize the number of steps, labour time and the amount of starting tissue to as little as 10–20 mg without affecting RNA quality. In both protocols genomic DNA (gDNA) can be efficiently removed from RNA samples before the final alcohol precipitation step, saving extra purification steps before cDNA synthesis. The expression kinetics of previously characterized genes confirmed the robustness of the procedures.We have established protocols to isolate DNA-free RNA from Here, we present two protocols to isolate DNA-free RNA from Arabidopsis tissues ready for RT-qPCR applications that significantly improve existing ones by reducing labour time and the use of organic extractions. Accessibility to these protocols is ensured by its simplicity and the low cost of the materials used. Reverse transcriptase quantitative PCR (RT-qPCR) is a very useful and sensitive technique to quantitate changes in gene expression. Increasing number of labs routinely use the agarose-based or/and real-time versions of the technique. A significant number of replicates are required to ensure significance of the results, which adds a high-throughput component to the RT-qPCR. These requirements impose the need to have simplified protocols to reduce labour and costs without compromising RNA integrity or yield.Arabidopsis thaliana is a model organism for plant biologists, a number of RNA extraction protocols have been developed. Although there are several commercial kits available, the cost can be prohibitive when processing large numbers of samples. Published protocols use organic extractions with toxic chemicals, which involve the use of fume hoods to diminish health risks and lengthen the procedure [Since rocedure -5. Moreorocedure  and protrocedure -3,5 largArabidopsis thaliana that address the problems mentioned above. Both protocols start with small amounts of tissue and buffer volumes to fit the SMT format, and incorporate a DNase digestion before the final purification steps.Here we report improved RNA isolation protocols for the model plant 260/A230 and A260/A280) values  and the graphs obtained after plotting RNA species versus quantity  indicated the absence of undesirable contaminants (data not shown).The protocol presented here . AtDOF31 is a DOF transcription factor (TF) with very low relative transcript abundance, whose expression cannot be detected by in situ hybridizations or by promoter::GUS fusions . Gene-specific oligonucleotides were chosen to amplify regions near the 5' end to ensure RNA integrity and cDNA synthesis efficiency and a ubiquitin gene was amplified for sample normalization. cDNA was synthesized according to [AtDOF31 expression were detected in the wild type sample and several transgenic lines with different levels of AtDOF31 overexpression relative to the control (Col-0) were identified.To demonstrate the quality of the samples and sensitivity of the protocol, RT-qPCR was performed on RNA samples isolated from Arabidopsis leaves of the Columbia-0 ecotype (wild type) and leaves of transgenic plants overexpressing rding to , diluted260/A230 and A260/A280) values  and their electropherograms indicated the absence of undesirable contaminants  is expressed predominantly in the cortex and endodermis of the embryonic axis in germinating Arabidopsis seeds [AtBME3 (At3g54810) encodes a GATA-type zinc finger TF gene whose promoter is weakly expressed only at the micropylar end of the embryonic axis during germination [AtDOF31 is significantly induced during seed germination suggesting it may be an important player in the regulation of this seed phase since it is expressed at low levels outside this process. The kinetics observed for the AtGA3ox2 and AtBME3 genes . The only tissues not amenable for the previous protocol were seeds and siliques and we streamlined and improved existing protocols that use organic extractions for RNA purification from those recalcitrant tissues. This protocol has also been successfully used in the isolation of RNAs from developing barley seeds . Altogether, our results indicate that the use of these protocols can be extended to other plant species.We have described two simplified, inexpensive and robust protocols to isolate DNA-free RNA from Arabidopsis tissues. As far as we know, all non-commercial protocols for RNA isolation from plant tissues use organic solvents during the first steps. We present here a protocol that uses two simple buffers without organic solvents that includes a 30 min DNase digestion and requires only 90 min to obtain 24 DNA-free RNA samples. Although only data from leaves is presented here, RNA from most Arabidopsis tissues can be isolated since we have successfully used it with stems, roots and flowers (data not shown) and also from different vegetative tissues of the legume model plant AtDOF31 and AtBME3 are expressed at low levels and AtBME3 mRNA has a length (1700 bp) above the average for a TF. After cDNA synthesis using oligo dT and qPCR using specific oligonucleotides annealing as far as possible from the polyA, we were able to detect and quantify the expression levels of both genes in all samples analyzed, confirming sensitivity and specificity.We determined that the RNA obtained at an intermediate step of the isolation protocol was of sufficient quality to allow DNase digestion before the final purification steps. This modification shortens the protocol and maximizes yield bypassing the need to double precipitate the RNA. RNA integrity and purity are key factors influencing cDNA synthesis and ultimately detection and accurate quantification of RNAs, especially low abundance RNAs such as those derived from many TF coding genes. Accessibility to these protocols is ensured by its simplicity and low cost of the materials used. With the increase of genomic approaches involving the manipulation of large numbers of samples, we feel these protocols will be extremely useful for plant scientists.The authors declare that they have no competing interests.LOS was responsible for experiments and supervision of this study and JVC contributed to the supervision.


Escherichia coli involves the removal of the 5′-terminal pyrophosphate to generate a monophosphate group that stimulates endonucleolytic cleavage by RNase E. We show here however, using well-characterized oligonucleotide substrates and mRNA transcripts, that RNase E can cleave certain RNAs rapidly without requiring a 5′-monophosphorylated end. Moreover, the minimum substrate requirement for this mode of cleavage, which can be categorized as ‘direct’ or ‘internal’ entry, appears to be multiple single-stranded segments in a conformational context that allows their simultaneous interaction with RNase E. While previous work has alluded to the existence of a 5′ end-independent mechanism of mRNA degradation, the relative simplicity of the requirements identified here for direct entry suggests that it could represent a major means by which mRNA degradation is initiated in E. coli and other organisms that contain homologues of RNase E. Our results have implications for the interplay of translation and mRNA degradation and models of gene regulation by small non-coding RNAs.The best characterized pathway for the initiation of mRNA degradation in  Escherichia coli RNase E is required for the normal rapid degradation of many, if not most transcripts   that converts the 5′ group of primary transcripts from a triphosphate to a monophosphate  under the same reaction conditions , the BR13 G→A variant (see above) and then conjugated it to streptavidin  5′-hydroxylated BR13 appears sigmoidal, which is usually associated with positive cooperativity, a phenomenon that can be displayed by enzymes or receptors with multiple binding sites for a ligand. A sigmoidal curve and KM of >8 μM were also obtained for 5′-hydroxylated LU13, which has the G to A substitution that prevents quadruplex formation (data not shown).Modelling studies using the known dimensions of structures of quadruplexes, streptavidin, single-stranded RNAs and the NTH of RNase E indicated that the two RNA-binding channels in a principal dimer of RNase E can simultaneously contact single-stranded regions in the context of either the G quadruplexes or streptavidin conjugates . A schemectively . These pas 16 μM . Thus, tKM and kcat values that were obtained were 3.6 μM and 42 min−1 respectively , but that this could be offset under non-saturating conditions by the reduction in the maximum rate of turnover . This is consistent with the quadruplexed form of 5′-hydroxylated BR13 being cleaved at a similar rate to that of 5′-monophosphorylated BR13 under our initial assay conditions . Although footprints were not obtained using the acylation assay, the mapping of sites of secondary cleavage confirmed that NTH-RNase E can recognize multiple single-stranded segments within cspA mRNA. The contribution of the individual single-stranded sites to the rapid cleavage of cspA mRNA in its 3′ UTR is currently being investigated.We initially hoped that the activity of the D346N mutant would be sufficiently low to detect ‘footprints’ corresponding to sites of binding. However, at the micromolar concentrations of enzyme required to ensure that the majority of the RNA was bound, cleavage occurred to a detectable level: at several positions the primer extension reactions terminated independent of acylation . In addistograms . ConsistE. coli RNase E can cleave rapidly RNAs that lack a 5′ monophosphate group. Rapid cleavage of BR13 was facilitated by forming substrate multimers that modelling studies showed would allow simultaneous contacts between two single-stranded segments and two RNA-binding channels in the principal dimer of tetrameric RNase E  provides an important foothold for RNase E when there is a paucity of single-stranded segments that can facilitate the binding of RNase E. Such is most likely to be the case for ribosomal RNA precursors that are highly structured and associate with ribosomal proteins as well as for mRNAs that are translated efficiently and have short or highly base-paired UTRs. For some transcripts, it might be that RNase E can bind in the absence of a 5′ monophosphate, but that the presence of this group is required to position RNase E on the RNA at a site that can be cleaved. The latter might explain why 5′-triphosphorylated and circularized rpsT mRNA appears to be cleaved poorly by RNase E in vitro despite having multiple single-stranded regions  glycerol  (0.15 nmol) in 100 μl of RNase E reaction buffer  Coomassie Brilliant Blue R in 50% (v/v) methanol and 7% (v/v) glacial acetic acid and proteins visualized following destaining with 20% (v/v) methanol and 7% (v/v) glacial acetic acid. Products from the reaction with the lowest concentration of 5′-biotinylated RNA that shifted all of the streptavidin were used in subsequent experiments. This corresponded to an RNA concentration of 0.6 nmol, which was fourfold higher than the streptavidin concentration.Increasing amounts of 5′-biotinylated LU13  were incubated with streptavidin from n buffer  containibis-Tris-propane (pH 8.3), 100 mM NaCl. Measurements were made using a Jasco J-715 spectropolarimeter equipped with a Peltier temperature controller (Jasco PTC 351S). Samples of 240 µl were placed in a quartz cuvette with an optical path length of 1 mm. After transfer to the spectropolarimeter, the sample was allowed to equilibrate at 4°C for 10 min. Five CD scans were performed at 50 nm min−1 with a 2 s response time, 1 nm pitch and 1 nm bandwidth over the wavelength range of 220–320 nm. The sensitivity was set to 10 mHg and the slit width to 1000 μm. The average of the five scans was taken. Then the temperature was raised to 37°C and the sample was allowed to equilibrate for 15 min before scanning at this temperature. For each temperature, a CD spectrum of the buffer was recorded and subtracted from the spectrum obtained for the sample containing RNA. The high tension values were below 400 during all CD experiments, indicating that the obtained data have a high signal to noise ratio and are reliable. Data were zero-corrected at 320 nm. After all the measurements were made, the contents of the cuvette were analysed by denaturing gel electrophoresis to confirm the concentration and integrity of the RNA.RNAs were analysed by circular dichroism at a concentration of 7.5 μM in 25 mM in silico using RasMol (http://www.umass.edu/microbio/rasmol/). The PDB files for the NTH of RNase E and quadruplexes were 2c0b (plus 2c4r) and 1S9L (plus 2GRB) respectively.Potential molecular interactions were visualized cspA mRNA were 5′-ATCCTAATACGACTCACTATAGGGCCTTCGGGCCAAGGTTTGACGTACAGACC and 5′-GAACCGGACCGAAGCCCGATTTGGATCCGGCGAACCGGATCGAAAAATCCCCGCCAAATGG.Probing using selective acylation of 2′-hydroxyl groups was carried out essentially as described previously by others (bis-Tris-Propane (pH 8.3), 100 mM NaCl, 15 mM MgCl2, 0.1% (v/v) Triton X-100, 2.5% (v/v) glycerol and 1 mM DTT in the presence or absence of up to 12 μM of D346N NTH-RNase E. After incubating for 20 min, NMIA (N-methylisatoic anhydride) dissolved in DMSO was added to a final concentration of 13 mM and incubation continued at 37°C for a further 45 min. A negative control was included in which DMSO without NMIA was added. The RNA was then recovered by ethanol precipitation as described previously (RNA (1 pmol) was incubated in 25 mM 2, 0.5 mM of each deoxyribonucleotide triphosphate and 5 mM DTT. The reaction was warmed to 55°C before addition of 200 U of Superscript III reverse transcriptase (Invitrogen) and incubated for a further 30 min at 55°C. The final volume of the reaction was 20 μl. After reverse transcription, the RNA was degraded by adding NaOH to a final concentration of 200 mM and incubating at 95°C for 5 min. Next to be added was 29 μl of a 4:25 (v/v) mixture of 1 M unbuffered Tris-HCl: stop dye .Primer, radiolabelled at the 5′ end using T4 polynucleotide kinase, was added to the modified RNA that had been heated to 95°C for 3 min. Annealing was achieved by incubating at 65°C for 5 min, 35°C for 5 min and then placing on ice for 1 min. Sequencing was performed using unmodified RNA and dideoxyribonucleotide triphosphates that were added to a final concentration of 0.75 mM. The primer was extended in a buffer containing 75 mM KCl, 50 mM Tris-HCl (pH 8.3), 3 mM MgClbis-acrylamide, 1× TBE. Extended products were detected using a Phosphorimager FLA-5100 (FUJIFILM) with the laser set to 635 nm. Band intensity was quantified using the semi-automated footprinting analysis programme (cspA were obtained using the RNA structure programme (http://rna.ucsc.edu/rnacenter/xrna/). The sequences of the primers used in the analysis of cspA structure were 5′-GAACCGGACCGAAGCCCG, 5′-CCATCGTTCTGGATAGCAGAG and 5′-CATTTTACCGGACATAGTGTATTACC.The samples were incubated at 95°C for 5 min before being analysed in denaturing polyacrylamide gels: 7 M urea, 8–10% (w/v) 29:1 acrylamide : edp-pgk RNA was 5′-AAGTTTTGCAGACGCTGCTT. The sequence of the forward primer that produced an amplicon that identified the 3′ hydroxylated end generated by RNase E cleavage was 5′-ATCCTAATACGACTCACTATAGGGTTATTGATGCATACC.RNA samples were treated with poly(A) polymerase before ligation with an RNA adapter, which only ligates to a 5′-monophosphorylated end, as described previously (


Drosophila epithelia C. elegans Ena/VASP, UNC-34, display subtle defects in the leading edges of migrating epidermal cells but undergo normal epidermal morphogenesis. In contrast, embryos lacking both UNC-34 and the C. elegans N-WASP homolog have severe defects in epidermal morphogenesis, suggesting that they have parallel roles in coordinating cell behavior. GFP-tagged UNC-34 localizes to the leading edges of migrating epidermal cells, becoming redistributed to new junctions that form during epidermal-sheet sealing. Consistent with this, UNC-34 contributes to the formation of cadherin-based junctions. The junctional localization of UNC-34 is independent of proteins involved in Ena/VASP localization in other experimental systems; instead, junctional distribution depends upon the junctional protein AJM-1. We also show that Abelson tyrosine kinase, a major regulator of Enabled in Drosophila, is not required for UNC-34/Ena function in epithelia. Instead, our data suggest that Abelson kinase acts in parallel to UNC-34/Ena, antagonizing its function.Ena/VASP proteins are associated with cell-cell junctions in cultured mammalian cells  In wild-type embryos, the two pairs of ventral leading epidermal cells show extensive, relatively broad protrusions ; wsp-1(RNAi) embryos appear to be quite different from those in the wild-type  embryos.Protrusions in ild-type . Althougild-type , the LCPild-type . Pocket rphology E, pocketrphology . This phwsp-1 and unc-34 in several ways. First, we used an epidermal-specific RNA interference (RNAi) strain wsp-1 and unc-34 function, but we observed no lethality, perhaps because the promoter used in this strain is active too late in development to confer robust RNAi sensitivity. Attempts to drive expression of unc-34 in unc-34(gm104) mutants with a pan-epithelial promoter were likewise problematic because of selectively weak expression in the epidermis, although these results do suggest that at later stages, unc-34 acts specifically in epithelia (see below). Finally, we used a strain designed to reduce wsp-1 function in the epidermis, and this strain has been reported to result in some morphogenesis defects and lethality wsp-1 function alone in any other context, including the putative null deletion, wsp-1(gm324). However, we did observe sporadic lethality in F3 embryos derived from unc-34 homozygous mothers carrying the wsp-1 knockdown constructs, with phenotypes qualitatively similar to unc-34(gm104);wsp-1(RNAi) embryos (data not shown). This suggests that wsp-1 is specifically required in the epidermis. The synergistic genetic interaction between unc-34 and wsp-1 is consistent with several models at the molecular level ;wsp-1(RNAi) embryos display weaker migration phenotypes, in which all cells eventually reach the midline . This promoter is active in all major epithelia during embryonic and postembryonic development, as well as in an undefined set of neurons.A polyclonal antibody against UNC-34 shows broad cortical localization, as well as enrichment near apical junctions in epithelial cells A and 2A′GFP expression is first detected throughout the cytoplasm of most cells in early embryos. Once the epidermis is formed, UNC-34::GFP becomes enriched at cell-cell junctions. As leading cells approach the ventral midline, UNC-34 accumulates at the leading edge . UNC-34 hmr-1 germline mosaics, which lack both maternal and zygotic HMR-1/E-cadherin. These embryos display junctional localization of UNC-34::GFP that is essentially indistinguishable from wild-type embryos (hmp-1/α-catenin and hmp-2/β-catenin null mutants (data not shown). Thus, the recruitment of UNC-34::GFP to the apical junction is independent from the cadherin-catenin complex. Vinculin and zyxin have also been implicated as Ena/VASP recruitment factors C. elegans zyxin family member show normal junctional localization of UNC-34::GFP (data not shown), and vinculin is not expressed in C. elegans epithelial cells In primary keratinocytes, cadherin-catenin function is required for the recruitment of Mena and VASP to cell junctions  embryos K. We obtajm-1 null allele, ok160. Although UNC-34::GFP still localizes to junctions in ajm-1(ok160) homozygotes, the intensity of the junctional signal is greatly reduced relative to the wild-type  mutants rescued with a transgene that does not contain this region but is sufficient for rescue of essential AJM-1 functions ajm-1 null mutants, however, little or no UNC-34 is detectable at junctions via immunostaining, even though nonepithelial cell types express UNC-34 normally  and unc-34(gm104). hmp-1(fe4) mutants show variable defects in epidermal morphogenesis (unc-34 hmp-1(fe4) double-mutant combination exhibits completely penetrant maternal-effect lethality  hmp-1(fe4) embryos. Of 15 embryos that subsequently failed to undergo ventral enclosure, four showed complete retraction of the leading edge of the ventral epidermis dorsally and 11 showed partial ventral enclosure but extruded their internal organs once elongation began  double mutants  embryos, showing occasional punctate accumulations and discontinuities gm104 fe4 double mutants compared to either single mutant  synergy occurs specifically in epithelial cells, we used the dlg-1 promoter to drive unc-34::gfp expression in epithelial cells (see unc-34(gm104) hmp-1(fe4) homozygote expressing unc-34::gfp. This result suggests that the loss of unc-34 function in epithelia is responsible for the observed synthetic lethality.To determine whether ells see . This coRecent work has led to the proposal that α-catenin acts to regulate actin dynamics at nascent adherens junctions, favoring the formation of unbranched, bundled actin filaments, and suppressing branched actin networks (reviewed in Drosophila Abelson tyrosine kinase (Abl) regulates epithelial morphogenesis, at least in part by regulating the localization of Enabled C. elegans Abl homolog, ABL-1. A putative null allele, abl-1(ok171), displays no obvious defects in epidermal morphogenesis hmp-1(fe4); abl-1(ok171) double mutants show no enhancement of the hmp-1(fe4) mutant phenotype. We examined the effect of the abl-1(ok171) mutation on unc-34(gm104) hmp-1(fe4) synthetic lethality by constructing unc-34(gm104) hmp-1(fe4); abl-1(ok171) triple-mutant homozygotes. The loss of abl-1 function partially suppressed the morphogenetic defects observed in unc-34 fe4 double mutants  embryos. Because unc-34(gm104) does not produce any detectable UNC-34 protein, abl-1(ok171) appears to act as a bypass suppressor, a genetic interaction that implies that unlike in Drosophila, Abelson kinase acts in parallel to UNC-34/Ena and that it antagonizes UNC-34 function. mutants D; a minoabl-1 loss of function could lead to a reduction of actin-capping activity and thereby partially relieve the requirement for UNC-34. In order to address this possibility, we attempted to suppress the synthetic lethality of unc-34(gm104) hmp1(fe4) by reducing the expression of the two C. elegans capping proteins with RNAi. However, even a mild reduction in the expression of these proteins resulted in arrest during early embryonic development, precluding assessment of genetic suppression (data not shown).In vitro experiments indicate that Ena/VASP proteins antagonize capping protein C. elegans epidermis. Future work aimed at identifying molecular components that recruit and modulate UNC-34/Ena function in these subcellular compartments should yield further insights into how this important actin regulator functions during morphogenesis.In summary, our studies have uncovered novel interactions between UNC-34/Ena and components of both the leading edge and cell-cell junctions in the


These findings provide direct evidence for a trans-initiation model, in which binding of proteins to internal sequences of a pre-existing positive-strand RNA affects the synthesis of subsequent copies of that RNA, most likely by organizing replication factors around the initiation site.RNA structures present throughout RNA virus genomes serve as scaffolds to organize multiple factors involved in the initiation of RNA synthesis. Several of these RNA elements play multiple roles in the RNA replication pathway. An RNA structure formed around the 5′- end of the poliovirus genomic RNA has been implicated in the initiation of both negative- and positive-strand RNA synthesis. Dissecting the roles of these multifunctional elements is usually hindered by the interdependent nature of the viral replication processes and often pleiotropic effects of mutations. Here, we describe a novel approach to examine RNA elements with multiple roles. Our approach relies on the duplication of the RNA structure so that one copy is dedicated to the initiation of negative-strand RNA synthesis, while the other mediates positive-strand synthesis. This allows us to study the function of the element in promoting positive-strand RNA synthesis, independently of its function in negative-strand initiation. Using this approach, we demonstrate that the entire 5′-end RNA structure that forms on the positive-strand is required for initiation of new positive-strand RNAs. Also required to initiate positive-strand RNA synthesis are the binding sites for the viral polymerase precursor, 3CD, and the host factor, PCBP. Furthermore, we identify specific nucleotide sequences within “ Enteroviruses are a subfamily of small, pathogenic, icosahedral viruses called picornaviruses. Poliovirus, the etiologic agent of paralytic poliomyelitis, is one of the most extensively studied members of this family. Poliovirus RNA replication utilizes a mechanism, common to all positive, single-stranded, lytic RNA viruses, which permits the amplification of a single initial molecule of RNA into thousands of RNA progeny in only a few hours. After entry, the viral genomic RNA is transcribed to generate a complementary RNA (negative-strand), which, in turn, is used as a template to synthesize new strands of genomic RNA (positive-strand). The specificity of the viral RNA template, and the relationship between translation and replication, are controlled by RNA elements present throughout the genome. Individual elements often carry out multiple, interdependent tasks, complicating the dissection of their precise roles in specific steps of replication. We employed a novel approach to overcome this roadblock. Our strategy demonstrated that an RNA element present at the 5′ end of the virus genome is the master regulator of the initiation of RNA synthesis. The genome of positive-strand RNA viruses has dual functions: as viral mRNA and as template for the synthesis of additional RNA genomes. Replication of the viral RNA occurs by a highly regulated, efficient mechanism, which produces tens of thousands of new RNA copies in only a few hours. Positive-strand RNA viruses follow a common strategy for replication: the viral genome is transcribed into a negative-strand intermediate, which, in turn, acts as a template for new positive-strand synthesis. The same RNA-dependent RNA polymerase synthesizes both RNA strands using viral and host factors. However, replication is a highly asymmetric process, resulting in the synthesis of many more positive- than negative-strands. Accordingly, a regulatory mechanism should exist to control levels of production of either strand, perhaps at the level of initiation.Several viral RNA structures present within negative- and positive-strand RNA are important for initiation of RNA synthesis. RNA elements that organize RNA replication initiation complexes are believed to form around the 3′-termini of both positive and negative strands, but, surprisingly, they have also been found at the 5′ end of the viral RNA or within the coding region of the virus RNA. Furthermore, recent evidences suggest that interaction of the 5′- and the 3′-ends of the viral genome is necessary to initiate negative-strand RNA synthesis Picornaviridae, as a model because both in vitro and in vivo systems are available to dissect the viral replication cycle cis-acting element for initiation of negative-strand RNA synthesis Here, we examine the structure and function of the initiation complex of positive-strand RNA synthesis. We chose poliovirus, a member of the family Initial evidence also implicated the cloverleaf structure as a critical RNA element for positive-strand RNA synthesis. Certain mutations in this element resulted in reduced accumulation of positive-strand RNA without a significant effect on negative-strand levels “stem a” that are essential for the initiation of positive-strand RNA synthesisWe thus developed a novel approach to analyze RNA elements that play multiple roles during virus replication. Our approach relies on the duplication of the cloverleaf structure so that one cloverleaf is dedicated to the initiation of negative-strand RNA synthesis while the other can mediate positive-strand synthesis. This allows the study of the function of the cloverleaf RNA on positive-strand RNA synthesis. Our studies demonstrate that the cloverleaf structure formed at the 5′-end of the positive-strand is required for initiation of positive-strand RNA synthesis. Also required to initiate positive-strand RNA synthesis are the binding sites for the viral polymerase precursor, 3CD, and the host factor, PCBP. Furthermore, we identified specific nucleotide sequences within “stem a” are critical for efficient positive-strand initiation “stem a” of the downstream cloverleaf were replaced with G-C pairs   and replicative intermediate (RI), in addition to RF , and a construct carrying “stem a” and “stem c” in front of the G-C cloverleaf (plus27). In the cell-free replication system these constructs were unable to produce positive-strand RNA , the sequence GGG was replaced by AAA. This mutation should disrupt the stem in the positive-strand but should maintain the structure in the negative-strand. The second construct, StemB-mut(−) (CCC to UUU) disrupted the duplex structure in the negative-strand without modifying the structure in the positive-strand. While StemB-mut(+) did not produce any detectable positive-strand RNA, StemB-mut(−) was able to produce small, but detectable amounts of ssRNA. Strikingly, in intact cells, StemB-mut(+) was not able to replicate, whereas, StemB-mut(−), after an initial delay, replicated with almost wildtype kinetics, reaching similar maximum luciferase expression ), or two G-U pairs (StemD-mut(−)), which should maintain the duplex structure on the positive-strand but should alter the structure on the negative-strand. In the cell-free replication system, StemD-mut(+) showed a reduced level of positive-strands as compared to dCL-PLuc (in vitro results. StemD-mut(−) replicated with wildtype kinetics and maximum luciferase expression, whereas, StemD-mut(+) showed a 10-fold decrease in replication  within the 2C-coding region of the poliovirus genome functions as a template for the covalent linkage of two UMP nucleotides to the viral peptide, VPg, resulting in VPg-pUpU 5 to C mutation) “stem b” duplex structure on the positive- or the negative-strand (StemB-mut(+) and StemB-mut(−)), the “stem a” structure (StemA-disr) or the binding-site for PCBP (StemB-ΔPCBP) (The uridylated peptide VPg-pUpU functions as a primer for both negative- and positive-strand synthesis B-ΔPCBP) , lane 8.B-ΔPCBP) . We conc“stem a” of the cloverleaf is needed for positive-strand synthesis “stem a” structure for positive-strand synthesis. We engineered a series of “stem a” mutations within the 5′- cloverleaf of the double cloverleaf construct. Since disrupting the cloverleaf structure itself leads to a decrease in the level of positive-strand synthesis, sequence alterations were introduced together with compensatory mutations in the opposite side of the “stem a” to preserve the structure. The two 5′-terminal uridines were not mutated as they served as template for the 3′-terminal two A's on the negative-strand, which are believed to function as the binding site of the primer VPgpUpU. Instead we mutated a series of four A-U base-pairs (in vivo. We observed three types of replication phenotypes (in vitro and in vivo ((−) in “stem a” results in a defect in positive-strand synthesis. However, it seems that the bottom of the stem is more susceptible to sequence changes than the upper portion.It was previously shown that se-pairs . All mutenotypes . One groenotypes . These ienotypes . These m“stem a” in positive-strand RNA synthesis, we engineered the full-length poliovirus genome to carry two tandem cloverleaf structures (dCL-polio 1). A one-step growth curve demonstrated that dCL-polio 1 replicates with almost identical kinetics to wildtype poliovirus  “stem a” is also necessary for negative-strand synthesis “stem a” leads to a severe defect in positive-strand synthesis in the cell free system and completely abrogates replication in intact cells. Furthermore, the specific sequences of “stem a” are required for positive-strand synthesis (“stem a”. One single change (A), at the second base-pair of “stem a” suffices to greatly increase the fitness of a mutant with a severe replication defect . The titers of the virus were determined according to standard plaque assays HeLa S3 cells (ATCC CCL 2.2) were grown either (i) in tissue culture flasks in Dulbecco's modified Eagle medium-nutrient mixture/F-12 (Ham) (1∶1), supplemented with 2mM L-glutamine, 100 U of penicillin and streptomycin per ml, and 10% newborn calf serum or (ii) in suspension in suspension minimal essential medium (Joklik modified) supplemented with 2 mM L-glutamine, 100U of penicillin and streptomycin per ml, and 10% newborn calf serum. For virus production of rib(+)Xpa and double-Wt, stem a” of the cloverleaf (nucleotides A3-A6 of the poliovirus sequence were replaced by GGCC and nucleotides U91-U94 were replaced by GGCC) which resulted in pPLuc-GC. The complementary sequence in the hammerhead ribozyme was also altered to ensure efficient cleavage. A SacI-site was then introduced in front of the poliovirus sequence which resulted in pGC-SacI. The poliovirus Wt-sequence U1-C112 was then cloned into pGC-SacI, in front of the GC-pair cloverleaf, using the SacI-site, resulting in pdCL-PLuc. pGC-SacI was used as the parental construct for pPlus9, pPlus20, and pPlus27. The following sequences were inserted 5′ of the SacI-site: Poliovirus sequence U1-G9 in pPlus9; for pPlus20 poliovirus nucleotides U1-A8, followed by C38-U42, followed by U89-A95; and for pPlus27 poliovirus nucleotides U1-A8, followed by G35-C45, followed by U89-A95. prib(+)Luc-Wt was used as parental construct for pStemA-disr and all mutants depicted in 1-C112 of prib(+)Luc-N was cloned into pGC-SacI, in front of the SacI-site, resulting in pStemB-N, pStemD-N or pStemA-N, respectively. For each mutation that was engineered at the 5′ end of the poliovirus sequence, the complimentary sequence in the hammerhead ribozyme was also altered to ensure efficient cleavage. The sequences of the two cloverleaves in pdCLuc-PLuc were cloned into prib(+)Xpa resulting in pdCL-polio type 1. For stemA-mut8-virus, and stemA-mut10-virus, the sequence of the two cloverleaves and the hammerhead ribozyme in the respective replicons (p-stemA-mut8 and p-stemA-mut10), were cloned into prib(+)Xpa. CREmut-RNA has been transcribed from pCB3-CREmut, the luciferase-expressing, Coxsackie virus B3-derived replicon with a mutation within the CRE-region (A5 in the CRE-loop has been changed to C) as previously described PLuc-RNA is transcribed from prib(+)Luc-Wt, the luciferase-expressing, poliovirus-derived replicon; and WT-polio type 1-RNA is transcribed from prib(+)Xpa, containing the cDNA of the Mahoney strain of poliovirus, as previously described in vitro in reactions containing bacteriophage T7 RNA polymerase, 5×transcription buffer  and 7.5 mM NTP-mix. After incubation at 37°C for 3 h, DNaseI (Roche) was added and reactions incubated at 37°C for 15 min. RNA was precipitated by adding 50% (in volume) of LiCl2-solution  and incubation over night at −20°C. After centrifugation the pellet was washed once with 70% ethanol and then resuspended in RNA storage solution (Ambion) and stored at −80°C.Poliovirus-specific plasmid DNAs were linearized with ApaI. pCB3-CREmut was linearized with SalI. RNAs were transcribed 32P]-UTP (3000Ci/mmol). After incubation at 30°C for 2 h (if not indicated otherwise), the samples were mixed with 175 µl TENSK buffer  to stop the reaction. After incubation at 37°C for 2 h, RNA was extracted with phenol/chloroform, and precipitated with ethanol. The pellet was resuspended in RNA-storage solution (Ambion) and gel-loading buffer was added prior to loading on a 0.8% agarose gel. The gel was run at 20 V constant current over night. After drying the gel, products were visualized by using autoradiography. Bands were quantified using a phosphorImager Preparation of HeLa S10 cell extract and initiation factor has been described previously in detail 6 cells/ml. Then 800 µl aliquots were electroporated in 0.4 cm cuvettes with 20 µg of replicon RNA, using an Electro Cell Manipulator 600 (BTX Inc.) with the following settings: 300 V, 1000 µF, 24 Ω. Subsequently, 10 volumes of medium was added, the cells were divided in half, and guanidine hydrochloride (Sigma) was added to one half to a final concentration of 2 mM. 2×105 cells were plated per well in 12-well plates and incubated at 37°C in a 5% CO2 incubator.HeLa S3 cells were trypsinized, washed three times with phosphate-buffered saline, and adjusted to 5×10Replicon-transfected cells were scraped off, washed once with phosphate-buffered saline, and then lysed in 100 µl cell culture lysis reagent (Promega). Luciferase activity in 10 µl of lysate was determined in a luminometer using the luciferase assay system (Promega).4 buffer [pH8.0] and then resuspended in 500 µl of the same buffer. 125 µl of anti-VPg polyclonal antibodies were added to the Dynabeads-ProteinA and incubated rotating for 1 h at room temperature. The Dynabeads were washed twice again and resuspended in 500 µl 0.1 M NaPhosphate buffer [pH8.0]. After the 1 h incubation of the replication reaction as described above, 2.5 µl of 0.5 M NaPhosphate buffer and 25 µl of the Dynabeads-ProteinA coupled with anti-VPg antibodies were added. After incubation rotating at 4°C for 1 h, the Dynabeads were washed four times with phosphate-buffered saline. The Dynabeads were resuspended in 15 µl of tricine-sample buffer (Bio-Rad). The samples were heated at 94°C for 5 min and the supernatant was run on a 20% tris-tricine gel at 75 mA for 1 h and then for 72 h at 11 mA at 4°C. After drying the gel, products were visualized by using autoradiography using a phosphorImager .HeLa S10 cell extract was programmed with replicon RNA as described in the section “Replication in cell-free extract”. Pre-initiation complexes were resuspended as decribed above but incubated for 1 h rather than 2 at 30°C. The synthesis of VPg-pUpU was then analyzed by imunoprecipitation. Briefly, 500 µl Dynabeads-ProteinA (Invitrogen) were washed twice with 0.1 M Na PO6 HeLa S3 cells/well and incubated over night. Cells were washed with phosphate-buffered saline and infected with a multiplicity of infection of 10 of either WT-polio type 1 or dCL-polio type 1 viruses (P0-virus) in serum-free medium. After incubation at 37°C for 30 min, cells were washed twice with phosphate-buffered saline and fresh medium supplemented with 3% newborn calf serum was added to each well. The plates were incubated at 37°C and 5% CO2. At indicated time points viruses were harvested by three freeze/thaw cycles followed by centrifugation. The supernatant contained the virus and was stored at −80°C. The titers of the virus were determined according to standardplaque assays 6-well plates were seeded with 10In vitro RNA transcript of viruses were transfected into HeLa S3 cells as described above in section “Cells and viruses”. Cells were incubated for 72 hours or until total cytopathic effect was reached. Viruses were harvested as described under section “growth curve.” Standard plaque-assays were performed 6 cells/well the night before) before staining of the wells. The 6-well plates were incubated for 72 hours. Total RNA was purified by tryzol extraction (Invitrogen) and isopropanol precipitation. cDNA was synthesized using the Thermoscript RT-PCR system for First-Strand cDNA Synthesis (Invitrogen). Using standard PCR techniques and specific primers for the poliovirus genome, the viral genomes were amplified and the resulting PCR products were sequenced. For sequencing of the very 5′-end of the viral genome the 5′RACE system for rapid amplification of cDNA ends (Invitrogen) was used according to the manufacturer's instructions to amplify the 5′-ends and the resulting PCR products were sequenced.Figure S132P]ATP. The probe was digested with decreasing amounts of either RNase A, RNase T1 or RNase V1. The digested RNA was precipitated and separated on a sequencing gel. The gel was dried and autoradiographed for visualization of product using a phosphorimager. The autoradiograph of a representative mapping acrylamide gel is shown. The corresponding area of the two cloverleaves and the linker region is shown on the left side; the corresponding area within the cloverleaf structure is indicated on the right side of the autoradiograph. Major ribonuclease cleavages are indicated by the corresponding nucleotide of the cloverleaf RNA starting at the 5′-end. The very left lane (OH-Ladder) contains a hydroxyl radical 1bp produced from the same probe. The very right lane contains probe without any enzyme as a negative-control.Structural probing of the tandem cloverleaf structures of dCL-PLuc. RNA transcripts spanning the two cloverleaf region of dCL-PLuc were 5′-labeled with [γ(3.45 MB EPS)Click here for additional data file.Figure S232P]UTP for 1 hour. The radiolabeled RNA was immuno-precipitated using anti-VPg antibodies, separated on a Tris-Tricine SDS-Page gel and visualized by using autoradiography.VPg-uridylylation in a cell-free replication system. RNA transcripts corresponding to tandem cloverleaf replicons containing mutations in eitherStemB, StemD, or StemA were employed to program cell-free replications systems. VPg-pU(pU) formation was monitored by incubating the extracts with [α(0.44 MB EPS)Click here for additional data file.


Massively parallel pyrosequencing is a high-throughput technology that can sequence hundreds of thousands of DNA/RNA fragments in a single experiment. Combining it with immunoprecipitation-based biochemical assays, such as cross-linking immunoprecipitation (CLIP), provides a genome-wide method to detect the sites at which proteins bind DNA or RNA. In a CLIP-pyrosequencing experiment, the resolutions of the detected protein binding regions are partially determined by the length of the detected RNA fragments (CLIP amplicons) after trimming by RNase digestion. The lengths of these fragments usually range from 50-70 nucleotides. Many genomic regions are marked by multiple RNA fragments. In this paper, we report an empirical approach to refine the localization of protein binding regions by using the distribution pattern of the detected RNA fragments and the sequence specificity of RNase digestion. We present two regions to which multiple amplicons map as examples to demonstrate this approach. Mutations disrupting function of either cis-acting RNA elements or trans-acting RNA binding proteins (RBP) form the basis for many heritable human diseases [RNA-protein interactions influence virtually every step of post-transcriptional gene expression including pre-mRNA capping, splicing, polyadenylation and mRNA export, stability, localization and translation . These rdiseases -5. Despiin situ RNA binding specificity are required to elucidate the functions of RNA binding proteins in an efficient and comprehensive manner.The analysis of RNA binding proteins is further complicated by the observation that many are multifunctional . Elucidain vivo may be the immunoprecipitation (IP)-microarray assay [The most widely used technique for studying the interactions of RNA binding proteins with their RNA targets ay assay ,5. RNA bay assay . The croay assay ,8. CLIP ay assay ,8.in vivo, we have coupled the CLIP purification strategy from with high-throughput pyrosequencing of CLIP amplicons; we call this pyro-CLIP technology (pCLIP). The 454 sequencing platform  is an integrated system of emulsion-based PCR amplification of hundreds of thousands of DNA/RNA fragments linked to high throughput parallel pyrosequencing in picoliter-sized wells [In order to conduct global profiling of the RNA sites bound by RNA binding proteins ed wells -11. PyroPyro-CLIP experiments generate hundreds of thousands of RNA fragments (CLIP amplicons) to which an RBP binds. Substantial bioinformatics analysis is required to accurately identify the protein binding regions genome-wide. When the experimentally-detected RNA fragments (CLIP amplicons) are aligned to the human genome, many regions are observed to be encompassed by multiple fragments. In this study, we developed an empirical approach to refine the detection of protein binding regions by using the distribution pattern of the detected RNA fragments and the sequence specificity of RNase digestion along the amplicon-enriched genomic region.cis-acting element recognized by the studied RNA binding protein. The number of detected amplicons in each genomic region depends on various factors including the abundances of RNA transcript. Fig. Pyro-CLIP experiments result in hundreds of thousands of amplicons (RNA sequences with 50 to 70-bp in length). Preliminary data suggested that in a pyro-CLIP experiment, dozens of amplicons were usually detected within one genomic region. Hypothetically, all the detected amplicons should encompass at least one In the preliminary data, we found that the distribution of detected amplicons in each genomic region is similar to the pattern shown in Fig. The overall procedure includes three key steps: (1) pre-processing of CLIP amplicons, (2) estimation of sequence specificity of RNase digestion, and (3) prediction of RNA binding regions.blastn program [In the pyro-CLIP experiment, two short RNA linker sequences were ligated on both 5′- and 3′-end of each amplicon. These linker sequences were used as nested primer binding sites for reverse transcription and amplification. After removing both 5′- and 3′-end linker sequences, the remaining amplicons were mapped back into human genome using  program . In the blastn program [In practice, the number of genomic regions that match with the query amplicon varies depending on the selection of parameters while using the  program . We remoIn the pyro-CLIP assay, RNase digestion was used to trim the cross-linked RNA fragments to 50-70 nucleotides. Instead of cutting RNA fragments at random locations, RNase digestion has sequence specificity. The RNases used in the experiment were RNase I and RNase T1 .After mapping the detected amplicons back to the genome, we observed a large number of RNA fragments that shared identical 5′- and 3′-end genomic locations as in Fig 6=4096 potential 6-bp motifs based on all the detected amplicons and their mapped genomic locations.Each detected amplicon was generated by two RNase digestion sites Fig. . After mThe distribution of 6 nt sequences in the human transcriptome is not random, so in order to estimate sequence specificity of RNase digestion, the number of cutting events on each 6-bp motif should be corrected based on the frequency of the site. For instance, all the motifs that contain a CpG di-nucleotide appear in a much lower frequency than the ones without. Therefore, a lower number of detected cutting events on these motifs do not necessarily imply a low sequence specificity of RNase digestion. Based on this principle, we estimated the likelihood of RNase digestion on each 6-bp motif by normalizing the number of detected cutting events to the frequency of occurrence of the motif in the human transcriptome:kL depicts the likelihood of RNase digestion on the k-th 6-bp motif; if denotes the number of cutting events detected on the i-th 6-bp motif; and 0if represents its frequency of occurrences in the human transcriptome .where The size of detected amplicons ranges from 50 to 70 nucleotides. If there were no sequence specificity to the RNase digestion, the overlapped pattern of multiple amplicons would provide very good resolution of the detection of protein binding Fig. . HoweverOur strategy is based on several assumptions:cis-acting element to which the protein binds;1) All the detected amplicons should encompass the 2) The probability of each being selected as an RNase digestion site is proportional to the estimated likelihood of RNase digestion on its 6-bp motif, calculated in Eq. 1;3) Each genomic region where multiple amplicons are detected is located within one RNA transcript;For each genomic region in which multiple amplicons are detected, we simulate the distribution of amplicons that would occur based upon RNase sequence specificity, and then simulate the subset that would be selected if a protein bound to a specific site. This simulation is repeated as the binding site is moved along the RNA. We then compare the distribution of experimentally-detected amplicons Fig.  and of s1) Randomly generate a large number of RNA fragments based on the estimated likelihood of RNase digestion (Eq.1) along the genomic region in which multiple amplicons were detected plus a 70-bp extension towards both 5′- and 3′-side Fig. .i), randomly select M simulated amplicons from among the RNA fragments generated in the previous step that encompass the current site (i). M is the number of experimentally-detected amplicons  For each possible protein binding site , in both experimentally-determined group and simulated group; for each group, F(i) is a vector whose number of elements is equal to the length of genomic region being searched.3) For each genomic locus Pearson correlation coefficient of the resultant F(i) vector between experimentally-detected amplicons and simulated amplicons assuming that the protein binds on the i-th genomic locus, C(i).4) Calculate the 5) Continue steps 2 to 4 for the entire region. The region that results in highest correlation coefficient will be regarded as the region with the highest binding probability.In order to evaluate the robustness of the proposed approach to various noises resulted from multiple biological and computational steps, we created a simulated sets of virtual amplicons based on the sequences of a randomly-selected RNA region, and a pre-defined protein binding site. While generating these testing data, we deliberately included noises from various sources, including:1) Antibody non-specificity;2) Inaccurate estimation of the likelihood of RNase digestion on different 6-bp motifs;3) Noises associating with BLAST search.We tested the performance of the proposed approach under different levels of measurement noise based on the capability of the model to predict the pre-defined protein binding site.ip*logNpi), where ip is the probability that 6-bp motif i is selected as cutting site ip=if/ Σif. By definition, the resultant entropy should be a number from 0 to 1. A larger entropy implies less sequence specificity of the RNase digestion. The entropy for the frequencies of enzyme digestion on 6-bp motif is 0.42. This suggests that RNase digestion has significant preference for certain motifs. This finding is different from the reported cleavage specificity by the manufacturer, where RNase A cleaves at 3′ of single strand C's and U's, and RNase T1 cleaves at 3′ of single strand G's .In the preliminary dataset (unpublished data), a total of 29,071 RNA fragments ranging from 50-70 bp in length were used to estimate the sequence specificity of RNase digestion. We calculated the number of occurrences of all the potential 6–bp motifs at both ends of the RNA fragments, totaling 58,142 6-bp elements. We observed significant preference of RNase digestion on some 6-bp motifs. Only 967 of the 4,096 potential 6-bp motifs (24%) were observed as digestion sites. Strikingly, 26% of RNase digestion occurs at AATAAA sites. Fig. In vertebrate genomes, some sequences appear less frequently than others. For example, the number of occurrences of motifs that contain a CpG dinucleotide is significantly reduced. Therefore, we calculated the frequencies of all the 6-bp motifs in human transcriptome, and further estimated the likelihood of RNase digestion on each potential 6-bp motif based on Eq. 1. With this adjustment, the entropy level of cutting likelihood becomes 0.56. Top 1% 6-bp motifs with highest cutting likelihood are listed in Table The 29,071 RNA fragments cover 52 non-overlapping genomic regions that contain at least 10 fragments. Most enriched regions contain one or two protein binding sites. In this report, we use two examples to demonstrate the potential application of the proposed approach.1) Genomic regions that contain one protein binding sitePPEF2 .One enriched region on chr4: 77,026,287-77,026,348 contains 20 identical RNA fragments Fig. . This isWe generated a distribution of RNA fragments based on the likelihood of RNase digestion along the local chromosomal region  Genomic regions that contain two protein binding sitesThe proposed approach can also be used to refine the detection of protein binding regions that contain two or more binding sites. Instead of searching for the position of one binding site, as shown in Fig. i, j) represents the similarity of the distribution pattern between the experimentally-detected fragments , is shown in Fig. nts Fig.  and simutes Fig. . The numtes Fig. , where iblastn mapping errors.To evaluate the robustness of the proposed approach to various sources of experimental noises from both biological and computational steps, we created a simulated set of virtual amplicons by assuming known protein binding sites and deliberately introducing three different types of noise: antibody non-specificity, inaccurate estimation of sequence specificity of RNase digestion, and a) Antibody non-specificityBiological noise caused by antibody non-specificity may potentially undermine the ability of the proposed approach to accurately identify protein binding regions. Within the genomic region that is enriched by the detected RNA fragments, antibody non-specificity may cause false detection of RNA fragments that are not really bound by the proteins. These fragments may or may not encompass the real protein binding loci.N virtual fragments (50bp to 70bp) that were generated from two sources:We tested the ability of the proposed approach to detect the pre-defined protein binding site while introducing different levels of antibody non-specificity. Within a certain genomic region, we composed a set of n fragments that appeared randomly within the region, with no association with the pre-defined protein binding site and no correlation with 6-bp motif composition of local sequence. Hypothetically, these n fragments result from antibody non-specificity.1. N-n fragments that were simulated based on the 6-bp motif composition of local sequence and pre-defined protein binding site. These N-n fragments simulate the desired RNA fragments that associate with the studied protein.2. n/N). It is clear, however, that the proposed approach retains reasonably good robustness against antibody non-specificity while the proportion of detected RNA fragments contains less than 30% noise, with the correlation coefficient > 80%.Fig. b) Inaccurate estimation of sequence specificity of RNase digestion.The inaccurate estimation of the sequence specificity of RNase digestion may potentially decrease the accuracy of the model prediction. In this step, we deliberately added measurement noise to the estimated likelihood of RNase digestion on each 6-bp motif and tested the ability of the proposed approach to recover the pre-defined protein binding site. The correlation coefficient of the sequence frequency (number of RNA fragments that encompass each genomic locus) between RNA fragments simulated with and without added noise are shown in Fig. c) Noise from BLASTblastn is not always accurate. This is in part due to the sequencing error in the experimental stage, or due to the blastn parameters used. The consequences of BLAST noise on the model prediction are similar to the prediction inaccuracy caused by the antibody non-specificity, i.e. RNA fragments are inaccurately assigned to the studied genomic region. These RNA fragments usually have no association with the real protein binding sites, and have no correlation with the 6-bp motif composition of the studied genomic region. The effect of such noise on the model prediction is evaluated in the Fig. Mapping the detected fragment back to human transcriptome using de novo binding regions of DNA/RNA binding proteins in a genome-wide scale.In this study, we propose a computational approach to refine the detection of protein binding regions from pyro-sequencing-derived RNA fragments. As an ultra-high-throughput technology, pyro-sequencing allows sequencing of more than 100,000 amplicons in a single experiment [Using pyro-CLIP experiment as an example, we present a computational framework to increase the detection resolution within an RNA-fragment-enriched region by using (1) the distribution patterns of the detected amplicons Fig.  and 6B, Multiple RNA fragments can be detected in each enriched region. Intuitively, protein binding region can be localized by finding the common genomic regions that are encompassed by all the local fragments. This is based on the assumption that only those RNA fragments that contain the protein binding site can be immunoprecipated by the antibody, and therefore can be detected by the system. Localization is compromised by the sequence selectivity of RNase digestion. Therefore, the distribution pattern of the RNA fragments contains less information than the one that would result from random sequence cutting. Incorporating the sequence preference of the RNase digestion into the model can partially recover the information. In conclusion, the proposed approach demonstrated great potential in refining the detection of protein binding region using pyrosequencing-derived RNA fragments.The authors declare that they have no competing interests.YL and JRS contributed to the design of the study. JRS and XW performed the CLIP and pyrosequencing assays. XW and YL designed and performed the bioinformatics analysis and drafted the manuscript. CS, LL, SDM, HJE, and JRS participated in coordination, discussions related to result interpretation and revision of the manuscript. All the authors read and approved the final manuscript.