In mammals a considerable 92% of genes contain introns, with hundreds and hundreds of these introns reaching the incredible size of over 50,000 nucleotides. These “large introns” must be spliced out of the pre-mRNA in a timely fashion, which involves bringing together distant 5′ and 3′ acceptor and donor splice sites. In invertebrates, especially Drosophila, it has been shown that larger introns can be spliced efficiently through a process known as recursive splicing—a consecutive splicing from the 5′-end at a series of combined donor-acceptor splice sites called RP-sites. Using a computational analysis of the genomic sequences, we show that vertebrates lack the proper enrichment of RP-sites in their large introns, and, therefore, require some other method to aid splicing. We analyzed over 15,000 non-redundant, large introns from six mammals, 1,600 from chicken and zebrafish, and 560 non-redundant large introns from five invertebrates. Our bioinformatic investigation demonstrates that, unlike the studied invertebrates, the studied vertebrate genomes contain consistently abundant amounts of direct and complementary strand interspersed repetitive elements (mainly SINEs and LINEs) that may form stems with each other in large introns. This examination showed that predicted stems are indeed abundant and stable in the large introns of mammals. We hypothesize that such stems with long loops within large introns allow intron splice sites to find each other more quickly by folding the intronic RNA upon itself at smaller intervals and, thus, reducing the distance between donor and acceptor sites. Introns are found ubiquitously in eukaryotic genomes and yet their role is still poorly understood and underappreciated. A range of recent studies have suggested that introns may have even existed in what some regard to be primordial eukaryotes About 92% of mammalian genes have exon/intron structures while only 8% of genes are intron-free. The average segmented gene of these species contain between 8 and 9 introns. The total length of introns represents 35–40% of the euchromatic portion of mammalian genomes. Many introns are extremely long. For example, there are over 3000 human introns larger than 50 kb, 1,234 longer than 100 kb, 299 longer than 200 kb, and 9 longer than 500 kb In particular, we concentrate on the problems that large introns (>50 kb) pose to their host genes. During the initial steps of splicing, the 5′-terminus of an intron is brought close to the downstream 3′-terminus by the spliceosome RNA-protein complex. This spatial formation allows the phosphodiester bond at the donor splice site to be attacked by the 2′-OH group of an adenosine residue from a so-called “branch point” located just in front of the acceptor splice site . The larger the intron, the more remote its ends are from each other. At first approximation, the difficulty of bringing an intron's termini together in our three-dimensional world is proportional to the cube of the intron's length. Therefore, for a large 100 kb intron, it is one million times harder to bring its ends together than for a medium-sized intron of 1 kb in length. In fact, a stretched 100 kb RNA molecule spreads out over a distance of 30 microns, which is larger than the size of mammalian nucleus (about 5 microns). Moreover, splicing of large introns already takes extra time because there are so many bases to transcribe in the first place. Indeed, it takes about 45 minutes for RNA polymerase II to transcribe a 100 kb gene region. Thus, there is a fundamental question: How do large introns manage to splice at all?et al.et al.recursive splicing; that is, several pieces of the intron are spliced consecutively starting from the 5′-end. According to Burnette and colleagues, recursive splicing is achieved using a combined donor-acceptor splice site called the “ratcheting point” (or RP-sites). These RP-sites have a consensus of nncag|gtaagt(y), where the splice junction is shown as vertical bar. The consensus sequences of the donor and acceptor splice sites are AG|gtaagt and nncag|GT(y) respectively . It is possible therefore that RP-sites may perform both functions–serving as either the 3′- or 5′- splicing junction—in order to facilitate recursive splicing. In 1998, Hatton et al.et al.Hatton et al.In 2006 Grellscheid and Smith Since mammals as well as many non-mammalian vertebrates have many more large introns than Drosophila, it follows then that there ought to be some aid to the removal of large introns if these species do not rely upon recursive splicing. Thus, we have performed a large-scale bioinformatics analysis to understand the possible splicing mechanisms for large introns in various vertebrate species. In particular, we predicted the number of stem structures within large introns, hypothesizing that periodic hairpins with stable stems and large loops may be a possible mechanism for pre-mRNA folding which could aid splicing efficiency.In a similar manner, we calculated the distribution of donor and acceptor splice site motifs within the same set of large introns and their complementary strands . Using RepeatMasker in this dot-plot analysis, we excluded all simple and low-complexity repeats  from the analysis since they have an ability to interact with the nearest neighbor repetitive units rather than with more remote ones. In human, as with other mammals, the dot-plot detected a good number of matched segments throughout the entire large intron while the fruit fly showed very few possible stem structures. Examination of the predicted stem sequences of large introns showed that these possible stems are primarily formed by interspersed repeats belonging to the SINE and LINE classes. In the case of humans, the vast majority of the predicted stems are formed by any two oppositely oriented Alu-repeats, and, to a much lesser extent, L1 or L2 LINE repeats  or (−) orientations and were randomly positioned along the intronic sequence. In short, we were unable to detect any pattern in the location and orientation of the repetitive elements compared to models where we randomly placed such elements along introns. It is also interesting to note that Alu elements were more common in human intergenic regions than human large introns and that, if transcribed, the number of predicted stems in intergenic regions would also be larger.The direct computational method for the prediction of secondary structures in long RNA sequences, such as large introns, is not feasible because of the enormous sequence length Our human intergenic region sample contained a total of 53.61% DNA repeats with 22.06% of the intergenic region being SINE compared to the sample of large introns which had 44.4% repeats with 12.36% SINE. For fruit fly, a 7.54% repeat composition in large introns jumped to 26.75% in intergenic regions (large retroviruses such as the ROO element appear in intergenic regions). Correspondingly, there were 19.56 unique, predicted stems per 50 kb in human intergenic regions versus 9.39 in human large introns (≤−60 MFE). Drosophila had 1.16 predicted stems per 50 kb in intergenic regions versus 0.03 in fruit fly large introns.We detected negligible numbers of predicted stem structures formed by the ancient mammalian-wide interspersed repeats (MIR repeats from SINE class) presumably because they have accumulated too many mutations within each repetitive element to be adequately paired. Drosophila's only source of predicted stems came from simple repeats. See It is interesting to observe the sheer difference in magnitude in the number of stems between human and fruit fly. The average number of unique, predicted stem structures per 50 kb of large introns in different species is presented in RepeatMasker program. The results are presented in Tribolium castaneum) has the highest number of unique stems predicted for any insect as well as the most stable predicted stem structures for all insects.  The sequences of non-redundant, large introns (>50 kb) were obtained from the Exon-Intron Database et al. 2005 with the same 80% scoring threshold for counting the number of RP-sites. In this computation we assumed that all RP-sites must have an invariable core sequence of AG|GT representing the intron's dinucleotide termini. The consensus for intron splicing junctions was obtained from our purged sample of 11,315 non-redundant human gene sequences (with <50% sequence identities between each other) from the human Exon-Intron Database, release 35p1 Homo sapiens. Various scoring thresholds for human and Drosophila were used: 80%, 70%, and 60% with 80% being the highest quality RP-site recognition threshold. The intron size classes chosen for this analysis  had a total of between 203 and 212 million bases for human. The fruit fly intron size classes were held to the same intervals allowing direct comparison of intron class size between Drosophila and human. For human and fruit fly RP-site analysis in intergenic regions we used the respective human and fruit fly consensus matrices.For the recognition of RP-sites we used the same computational algorithms as published by Burnette The data used in the stem prediction and analysis see –6 was a blastn and matched only to the top or forward strand. From the blast2 alignments we actually predicted the stems using the RNAcofold program (Vienna package 1.6.1) with default parameters unique if the stem's strands did not overlap with any other stem's sequences in the predicted stem set. Moreover, if a new stem to be added overlapped only one stem in the set and if the new stem had an MFE within 10% of the old stem and a smaller loop size, the algorithm would replace the old stem with the new one. The results are presented in For the stem prediction, we initially gathered a rough pool of possible stems using blast2 alignments of large introns versus their complementary sequences. We used default parameters for RepeatMasker Open-3.1.8 (www.repeatmasker.org) using the sensitive/slow search mode and species/genus specific repeat libraries RepeatMasker was database release 20061006 with WUBlast 2.0MP (http://blast.wustl.edu) to perform the scanning Masking and characterization of repetitive elements inside introns (and intergenic regions) were performed with Cross-referencing between predicted stems sequences and RepeatMasker data for the large intron fragments of human and drosophila was performed using a custom perl program. The coordinates of the two sequences forming a predicted stem were each individually cross-referenced against the locations of all repeats in the respective 50 kb large intron fragment. For Dot-plot analysis was performed using a modified version of the Java applet “Nucleic Acid Dot Plot” All other computations were performed by programs written in perl and with queries performed in MySQL—all available upon request.Drosophila melanogaster there exists a special strategy named recursive splicing for the excision of large introns. Recursive splicing occurs via selective accumulation of combined donor-acceptor splicing sites called RP-sites et al.The timely removal of large introns from pre-mRNA poses a challenging problem to spliceosomal machinery. It has been experimentally and computationally proven that in Alu-repeats (81.7%). Other investigated mammals do not have Alu-elements, but other types of evolutionarily new SINEs specific for their taxa. These SINEs could also allow for the formation of multiple hairpin structures inside large introns. Only one of the studied vertebrates, chicken, does not have SINE elements in its genome. Instead, the chicken has very abundant and relatively short LINE elements that comprise over 60% of its repetitive elements. Thus in chicken large introns, possible stems may be formed solely by LINE repeats and not SINE repeats. One may observe, however, that the chicken has very few predicted stems, less than all studied vertebrate species and comparable to some insect species. It may be the fact that avian genomes deal with large intron splicing differently than other vertebrate species. Two facts though are clear: predicted stems for chicken are quite strong and stable  located in different orientations throughout large introns drive the potential formation of hairpins with large loops. For humans there were an average of about 9.4 possible hairpins per 50 kb of the analyzed large intron sequence fragments. A vast majority of these possible stems are formed by oppositely oriented primate-specific able see  and the able see .Interestingly, the beetle and especially the sea urchin contain the most predicted stems of all studied invertebrates. While the sea urchin may contain the most predicted stems, even comparable to zebrafish, the majority of these predicted stems (47.5%) overlap with simple and low complexity repeats that might form hairpin structures without loops instead of the stems with large loops that we predict in mammals. Curiously, beetle's predicted stems are not strongly associated with any particular kind of repeat. We suppose that the beetle predicted stems might be formed by as yet unidentified repeats, or that they are merely a part of more complicated RNA secondary structures.The average number of predicted long and stable stems in large introns of different mammals is 5.5 to 14 per 50 kb of large introns see . These sin vitro.Insertion of interspersed retrotransposon elements, such SINEs and LINEs, is a major force for the expansion of the genome size as a whole and intron sizes in particular Supplementary Figure S1RP-site enrichment with respect to intron size. Human and Drosophila RP-site enrichment ratio calculated for various scoring thresholds and intron size classes. The RP-site ratio is the count of RP-sites on the direct strand of introns divided by the count of RP-sites on the complementary strand of said introns. Thresholds for scoring or recognizing RP-sites to a consensus sequence are 80%, 70%, and 60% with 80% being the most stringent  score. Intron class sizes are the five sets with individual intron lengths: 1) 1–6 kb, 2) 6–17 kb; 3) 17–41 kb; 4) 41–100 kb; and 5) larger than 100 kb. Note: Drosophila large intron group 100+ kb with scoring threshold 80% ratio is estimated, since 8 to 0 cannot be divided, using a polynomial curve fit (Rˆ2  = 1) to the previous four points.(0.04 MB DOC)Click here for additional data file.Supplementary Figure S2RP-site ratio comparison. In various species, the ratios of the number of sites  on the sense strand of large introns (>50 kb) is compared to the number of sites on the anti-sense strand of large introns.(0.04 MB PDF)Click here for additional data file.Supplementary Figure S3Comparison of controls used to calculate RP-site enrichment ratios in large introns. The complementary strand of the large introns is used as the first control. The RP-site enrichment ratio is the frequency of RP-sites in the direct strand of large introns to the frequency of RP-sites in the direct strand of large introns. A set of 50 kb intergenic region fragments is used as a second control. The RP-site enrichment ratio here is the frequency of RP-sites in the direct strand of large introns to the frequency of RP-sites in intergenic regions. Both human and fruit fly species are considered with RP-sites being calculated at the 80% scoring threshold. The human consensus matrix was used for human and the fruit fly consensus matrix was used for fruit fly. All frequencies are per 100 kilobases.(0.03 MB DOC)Click here for additional data file.


Drosophila, in contrast to most eukaryotic lineages, is still undergoing a dramatic rate of intron gain. These novel introns carry significantly weaker splice sites that may impede their identification by the spliceosome. Novel introns are more likely to encode a premature termination codon (PTC), indicating that nonsense-mediated decay (NMD) functions as a backup for weak splicing of new introns. Our data suggest that new introns originate when genomic insertions with weak splice sites are hidden from selection by NMD. This mechanism reduces the sequence requirement imposed on novel introns and implies that the capacity of the spliceosome to recognize weak splice sites was a prerequisite for intron gain during eukaryotic evolution.Intron number varies considerably among genomes, but despite their fundamental importance, the mutational mechanisms and evolutionary processes underlying the expansion of intron number remain unknown. Here we show that  The surprising observation 30 years ago that genes are interrupted by non-coding introns changed our view of gene architecture. Intron number varies dramatically among species; ranging from nine introns/gene in humans to less than one in some simple eukyarotes. Here we ask where new introns come from and how they are maintained in a population. We find that novel introns do not arise from pre-existing introns, although the mechanisms that generate novel introns remain unclear. We also show that novel introns carry only weak signals for their identification and removal, and therefore depend on nonsense-mediated decay (NMD). NMD maintains RNA quality control by degrading transcripts that have not been spliced properly. We propose that NMD shelters novel introns from natural selection. This increases the likelihood that a novel intron will rise in frequency and be maintained within a population, thus increasing the rate of intron gain. DrosophilaIntron number is highly variable among eukaryotes, ranging from about a dozen in some fungi to more than 100,000 in the human genome. Comparative genomics across broad phylogenetic distances have identified the importance of both intron gain and loss to the establishment of this variation While there is a general agreement that the very first spliceosomal introns arose from the degeneration of self-splicing group II introns Irrespective of the mutational mechanism, it is apparent that any new intron will require a number of key motifs including the 5′ and 3′ splice sites, and a set of auxiliary signals including the branch point and splicing enhancer and suppressor motifs In this study, we have investigated this alternative model in which novel introns are not required to be fully functional, relying instead on a back up mechanism of transcript quality control for incorrectly spliced introns Drosophila species, we show that novel introns have weaker splice sites and carry more stop codons than conserved introns. We propose that NMD may play an important role during the establishment of novel introns within a population, and in support of this we identified a significant deficiency of novel introns that would remain invisible to the NMD pathway upon intron retention.Using comparative genomics of nine Drosophila genomes  years in the melanogaster subgroup, being 6× greater than previously reported for DrosophilaDrosophila lineages have experienced far less intron gain. D. virilis underwent only 0.0022 intron gains/gene/Bya years and since the split between D. melanogaster and D. yakuba 10 million years ago not a single intron gain could be identified, demonstrating that the rate of intron gain may vary over orders of magnitude between closely related species.Here we have identified 307 novel introns amongst 284 genes across nine  genomes , present genomes . These eturnover . We obseD. pseudoobscura has undergone a gain of 218 bp, of which only 206 bp are spliced out, revealing an imperfect 8 amino acid repeat 5′ and 3′ of this novel intron  reported that several novel introns in Daphnia are flanked by short direct repeats et al., (2009).The previously proposed mechanisms of intron gain assume that new intronic sequence originates from elsewhere in the genome  of the cases. The remaining 13% (40/307) modified the adjacent coding sequence by only 1–3 amino acids . This observation is inconsistent with the intronisation model of intron gain Drosophila species (3.2% of variable nucleotides vs. 1.8% for SNPs Drosophila). This revealed 180 insertions  predicts that new introns are unlikely to arise with full strength splice sites. We observe that novel introns do in fact have weaker splice sites, with significantly reduced usage of the “strong” consensus motif at both the 5′ and 3′ splice site . Further2 = 331.5, P<0.0001) and not intron length . Phase 2 introns encode significantly more in-frame PTCs than either phase 0 and 1 due to the sequence requirements of the 5′ splice site. The canonical 5′ splice site GT(A/G)A restricts the first full potential codon of a phase 2 intron to either the TAA Ochre or TGA Opal stop codon. Only a minority of introns with non-canonical splice sites escape this constraint.Retention of 3n+1 and 3n+2 introns is expected to induce NMD due to the introduction of a frame-shift, but introns of length 3n require an in-frame PTC or they will remain invisible to the NMD pathway. Because of this, we reason that the failure to splice a new 3n insertion maybe deleterious, hence we predicted that novel 3n introns are more likely to encode a PTC as a backup mechanism for incomplete splicing. As the expectation for PTC occurrence is proportional to intron length, we fitted a logistic regression, modelling intron length, intron phase and a combined main effect of 3n class (3n vs. 3n+1 and 3n+2) and whether an intron is novel (n = 307) or conserved  . DespiteDrosophila that NMD carries a significant load caused by the weak splicing of introns Our analysis indicates that selection acts against introns that are invisible to the NMD pathway (if they undergo intron retention) leading to a deficiency of 3n PTC-free introns across the genome, as previously reported DaphniaDrosophila use weaker splice sites and are deficient for 3n PTC-free introns. Therefore, our evidence suggests that the establishment of these new sequences as introns is facilitated by NMD. Therefore, we propose a new model of intron gain   and therefore, should not show codon usage bias. We observed no correlation between the “codon” usage of novel introns and the expected codon usage for  0.8764) . Second,faux 3′ UTR” model suggests that PTC recognition depends on the distance to the downstream polyA tail 2 = 26.063, P<0.001) , consistent with previous reports do not show the 5′ bias among all intron seen in DrosophilaThe “P<0.001)  in suppoDrosophila phylogeny. In contrast, the mutational processes that generate repeat expansions, tandem duplications A significant question remains why does the rate of intron gain vary so much between closely related species? While differences in the action and potency of NMD are likely to exist between highly divergent taxa, we do not expect much variation on the fine scale of the Drosophila clade, predicting gene structure with GeneWise and using Dollo Parsimony to infer intron gain and loss events (Our approach to studying intron evolution is based on identifying gene orthologs across the s events .D. melanogaster (release 4.3) gene set as the basis of a best-bidirectional-blast-hit approach in the 11 other sequenced Drosophila species, namely; D. erecta, D. yakuba, D. ananassae, D. pseudoobscura, D. willistoni, D. virilis, D. mojavensis and D. grimshawi (obtained from http://rana.lbl.gov/drosophila/). We excluded D. sechellia, D. simulans and D. persimilis because of low sequence coverage D. melanogaster. We considered the HSPs to be orthologous when the reverse blast identified only the parental gene in D. melanogaster.We identified orthologous genes using the D. melanogaster gene together with 100kb of nucleotide sequence flanking the corresponding orthologous hit. We excluded any gene with a frameshift mutation . Intron gain and loss events were predicted using the Malin java application Drosophila genomes increases the power of Dollo Parsimony to accurately infer intron gain events, reducing the advantages of maximum likely methods D. willistoni and D. grimshawi) Dollo Parsimony remains sensitive to multiple losses being inferred as intron gain, but given the active debate about the best methods to infer intron turnover Exon-intron structure of orthologous genes was generated by submitting to GeneWise D. melanogaster lineage D. melanogaster, but required extensive validation to overcome the several limitations of GeneWise  and false negatives . Our experimental and informatic methods for their identification and exclusion are detailed in the http://i122server.vu-wien.ac.at/Drosophila_annotation/.This approach generated a high confidence set of 3,593 fully annotated orthologous genes  across nine D. melanogaster introns 10,000 times Click here for additional data file.Figure S2Drosophila species. Details as per Intron loss rates across (0.06 MB EPS)Click here for additional data file.Figure S3D. pseudoobscura associated with a novel intron. (A) Dotplot showing the subtle signal of direct repeats at either end of the novel intron. Window size = 8 bp, mismatch = 0. 50 bp of flanking exon are included. (B) Novel intron sequence (lower case) with the repeat underlined showing identity of 16/18 bp at the splice sites. The remaining intronic sequence finds no significant BLAST hit within NCBI. (C) Sequence alignment between three species showing that the gain of 218 bp resulted in an intron of only 206 bp, producing four novel amino acids in the 5′ exon. (D) A model showing that only four nucleotide substitutions within 48 bp are required to regenerate eight identical amino acids (LKLATTAT) at both ends of the intron.Duplication within the Bap170 gene  of (0.11 MB PDF)Click here for additional data file.Figure S4D. ananassae is associated with a novel intron of length 62 bp. (A) Dotplot with 50 bp of flanking exon. Window size = 8 bp, mismatch = 0. (B) Novel intron sequence (lower case) with the repeat (underlined) showing identity of 12/13 bp. The remaining intronic sequence finds no significant BLAST hit within NCBI. (C) Sequence alignment between three species.A direct repeat of length 12/13 bp in the Histone deacetylase 3 gene of (0.10 MB PDF)Click here for additional data file.Figure S5D. virilis. (A) Dotplot with 50 bp of flanking exon. Window size = 8 bp, mismatch = 0. (B) Novel intron sequence (lower case) with the repeat (underlined) and splice sites (bold). The remaining intronic sequence finds no significant BLAST hit within NCBI.A direct repeat of length 10/10 bp in the Autophagy-specific gene 9 (Atg9) gene of (0.04 MB PDF)Click here for additional data file.Figure S6D. grimshawi. (A) Dotplot with 50 bp of flanking exon. Window size = 8 bp, mismatch = 0. (B) Novel intron sequence (lower case) with the repeat (underlined) and splice sites (bold). The remaining intronic sequence finds no significant BLAST hit within NCBI.A direct repeat of length 8/8 bp in the CG2794 gene of (0.04 MB PDF)Click here for additional data file.Figure S7D. willistoni. (A) Dotplot with 50 bp of flanking exon. Window size = 8 bp, mismatch = 0. (B) Novel intron sequence (lower case) with the repeat (underlined) and splice sites (bold). The remaining intronic sequence finds no significant BLAST hit within NCBI.A direct repeat of length 11/12 bp in the CG3295 gene of (0.04 MB PDF)Click here for additional data file.Figure S8D. willistoni. (A) Dotplot with 50 bp of flanking exon. Window size = 8 bp, mismatch = 0. (B) Novel intron sequence (lower case) with the repeat (underlined) and splice sites (bold). The 5′ and 3′ splice sites are not within the direct repeat, but in close proximity. The remaining intronic sequence finds no significant BLAST hit within NCBI.A direct repeat of length 11/12 bp (or maybe 14/17) in the CG9536 gene of (0.04 MB PDF)Click here for additional data file.Figure S9D. yakuba (lower case) with the repeat (underlined) and splice sites (bold). The remaining intronic sequence finds no significant BLAST hit within NCBI.A direct repeat of length 8/8 bp in the CG5181 gene of the melanogaster subgroup. (A) Dotplot with 50 bp of flanking exon. Window size = 8 bp, mismatch = 0. (B) This novel introns was gained in the ancestor of mel, ere and yak, the sequence here is taken for the novel intron of (0.05 MB PDF)Click here for additional data file.Figure S10D. melanogaster. The flanking exonic sequence contains an imperfect CAG repeat, which is not present within the novel intron. This “new” intron pre-dates the split of D. melanogaster and D. ananassae and is therefore at least 14 million years old, sufficient time for any repeat structure to break down within non-coding sequence. (* indicate conserved amino acids.)A novel intron within the gene CG34382 has captured only part of the low complexity sequence. (A) The poly-Q region of the 5′ exon has continued to undergo length change in species with the novel intron. (B) The exon-2/intron-2 boundary from (0.02 MB PDF)Click here for additional data file.Figure S11Resampling analysis of splice site usage. We resampled (with replacement) 307 from the set of 307 novel introns (black) and 307 from the set of all 50,836 introns (gray) to obtain a distribution of the proportion of introns that carry the most common motif at the 5′ (A) and at the 3′ (B). The observed values for novel (black dot) and all (gray dot) are shown below each graph with 95% CI taken from the distributions above. The observed values for novel are outside the 95% CI for the distribution for all introns. Resampling from the set of novel is not actually required to establish significance, but does give an indication of the variation within novel introns. (C) The 307 novel introns use 83 different motifs at the 5′ splice site (black dot), outside the distribution of values obtained by resampling  from all , indicating that novel introns use a more diverse set of splice sites than expected (i.e. more rare/weak motifs).(0.04 MB PDF)Click here for additional data file.Figure S12D. melanogaster genes (http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=7227) (B) the 180 insertions excluded form our set of novel introns and (C) the 307 novel introns. Spearman Correlation Coefficients indicate significant codon usage bias in insertions  further justifying their exclusion from our data set, but no bias within novel introns .Novel introns show not codon usage bias. Distribution of codon usage values for all 64 codons for (A) all (0.08 MB PDF)Click here for additional data file.Figure S132 = 26.063, P<0.001) and conserved  both show a 5′ bias. This bias is stronger for novel introns . Lost introns  and insertions  do not differ from the uniform distribution.Novel introns are strongly biased towards the 5′ end of the gene. Empirical cumulative distribution of intron position across the gene for (A) novel and conserved introns, and (B) lost introns and insertions. Compared to a uniform distribution novel (X(0.04 MB PDF)Click here for additional data file.Text S1Supplemental methods.(0.06 MB DOC)Click here for additional data file.Dataset S1Includes Table S1, S2, S3, S4, S5, S6, S7, S8. Table S1. List of 3593 genes for which we identified a full length ortholog in all nine Drosophila species. Table S2. 307 novel introns, including length, length class, phase, PTC, and EST support. Table S3. 810 intron loss events. Table S4. 12 cases of concurrent intron gain and loss and cases of independent events. Table S5. 180 large protein coding insertions that do not undergo splicing . Table S6. 5′ and 3′ splice site usage for novel and control introns. Table S7. BLAST results against the EST data base. Table S8. 86 cases of failed intron prediction  in which the underlying intronic sequence and splice sites are still present, but GeneWise failed to predict an intron.(0.65 MB XLS)Click here for additional data file.


L mediates neural recognition byCa+2, Mg+2-independent homophilic binding. In the human and murine immune systems, L1 binds to the “classical” vitronectin receptor, αVβ3, and fibronectin receptor, α5β1, respectively, and abstains from homophilic binding. Homophilic L1 binding probably involves antiparallel alignment of several interactive domains. Integrin binding is mediated by a short segment of immunoglobulinlike domain 6, which includes two RGD repeats in rodent L1 and one RGD motif in human L1. L1 is modulated in activated leucocytes in vitro in parallel to L-selectin, and diverse cell types release intact L in vivo and in vitro. Released L1 can bind to laminin and adheres to the extracellular matrix of sciatic nerve, M21 melanoma, and possibly spleen and other tissues. It can support integrin-dependent cell migration and preliminary data implicate it in tumor development and transnodal lymphocyte migration.The cell-adhesion molecule L1 was originally described in the nervous system.It has recently been detected in CD4


Studies of mobile group II introns from a thermophilic cyanobacterium reveal how these introns proliferate within genomes and might explain the origin of introns and retroelements in higher organisms. Thermosynechococcus elongatus, which contains 28 closely related copies of a group II intron, constituting ∼1.3% of the genome. Here, by using a combination of bioinformatics and mobility assays at different temperatures, we identified mechanisms that contribute to the proliferation of T. elongatus group II introns. These mechanisms include divergence of DNA target specificity to avoid target site saturation; adaptation of some intron-encoded reverse transcriptases to splice and mobilize multiple degenerate introns that do not encode reverse transcriptases, leading to a common splicing apparatus; and preferential insertion within other mobile introns or insertion elements, which provide new unoccupied sites in expanding non-essential DNA regions. Additionally, unlike mesophilic group II introns, the thermophilic T. elongatus introns rely on elevated temperatures to help promote DNA strand separation, enabling access to a larger number of DNA target sites by base pairing of the intron RNA, with minimal constraint from the reverse transcriptase. Our results provide insight into group II intron proliferation mechanisms and show that higher temperatures, which are thought to have prevailed on Earth during the emergence of eukaryotes, favor intron proliferation by increasing the accessibility of DNA target sites. We also identify actively mobile thermophilic introns, which may be useful for structural studies, gene targeting in thermophiles, and as a source of thermostable reverse transcriptases.Mobile group II introns, which are found in bacterial and organellar genomes, are site-specific retroelments hypothesized to be evolutionary ancestors of spliceosomal introns and retrotransposons in higher organisms. Most bacteria, however, contain no more than one or a few group II introns, making it unclear how introns could have proliferated to higher copy numbers in eukaryotic genomes. An exception is the thermophilic cyanobacterium  Thermosynechococcus elongatus, a single intron has proliferated and constitutes 1.3% of the genome. Here, we investigated how the T. elongatus introns proliferated to such high copy numbers. We found divergence of DNA target specificity, evolution of reverse transcriptases that splice and mobilize multiple degenerate introns, and preferential insertion into other mobile introns or insertion elements, which provide new integration sites in non-essential regions of the genome. Further, unlike mesophilic group II introns, the thermophilic T. elongatus introns rely on higher temperatures to help promote DNA strand separation, facilitating access to DNA target sites. We speculate how these mechanisms, including elevated temperature, might have contributed to intron proliferation in early eukaryotes. We also identify actively mobile thermophilic introns, which may be useful for structural studies and biotechnological applications.Group II introns are bacterial mobile elements thought to be ancestors of introns and retroelements in higher organisms. They comprise a catalytically active intron RNA and an intron-encoded reverse transcriptase, which promotes splicing of the intron from precursor RNA and integration of the excised intron into new genomic sites. While most bacteria have small numbers of group II introns, in the thermophilic cyanobacterium  Mobile group II introns are bacterial and organellar retrotransposons that are hypothesized to be ancestors or closely related to ancestors of spliceosomal introns and retrotransposons in higher organisms  and chloroplast (cp) genomes of fungi and plants but are rare in archaea, with the few known examples of archaeal group II introns attributed to horizontal transfer from eubacteria trans-splicing Evolutionary scenarios for the evolution of group II introns into spliceosomal introns suggest that group II introns harbored by bacterial endosymbionts invaded the host's nuclear genome, where they proliferated and degenerated, with the group II intron RNA domains evolving into spliceosomal snRNAs that form the core of a common splicing apparatus for multiple dispersed introns Euglena cp DNA, which contains ∼150 group II introns Euglena introns are highly degenerate, lacking different domains, with some (referred to as group III introns) containing only DI-like and DVI-like structures and even lacking DV, which is catalytically essential. These degenerate introns presumably rely on protein factors and/or trans-acting RNAs to promote RNA splicing. Only two of the Euglena cp group II introns encode RTs, which may act in trans to promote splicing and mobility of the ORF-less introns Euglena group II introns can potentially provide insight into mechanisms involved in intron proliferation and generation of a common splicing apparatus, they have been largely intractable to detailed analysis.Although group II intron proliferation is rare in bacteria, it is evident in smaller organellar genomes, the most striking example being Thermosynechococcus elongatus strain BP-1, which contains 28 group II introns comprising ∼1.3% of the genome T. elongatus group II introns are closely related to each other and appear to have proliferated from a single group II intron. Here, we used bioinformatic analysis and intron mobility assays at different temperatures to identify four mechanisms that contribute to the proliferation of the T. elongatus introns. Our results provide insight into how group II introns proliferate within genomes; show how higher temperatures, which are thought to have prevailed on Earth during the emergence of eukaryotes, can contribute to this process; and identify actively mobile thermophilic group II introns, which may be useful for structural studies and biotechnological applications.Another instance of group II intron proliferation has been revealed by genomic sequencing of the thermophilic cyanobacterium T. elongatus introns classified according to intron family (F1–F6) by criteria described below, along with their insertion sites in the T. elongatus genome. All 28 introns are closely related to each other (85%–100% sequence identity in pairwise comparisons). Twenty-five of the T. elongatus introns are intact containing all six conserved RNA domains, but three are fragments that have undergone large deletions  with a series of conserved motifs , and IBS (intron-binding site)). The latter are involved in a series of long-range interactions that help fold the intron RNA into the catalytically active tertiary structure. Notable regions are DI and DV, which together comprise the minimal catalytic core; DIV, which encodes the IEP in the loop of subdomain DIVb; and DVI, which contains the branch-point A-residue T. elongatus introns correspond to reciprocal changes in stem regions, but some introns have deviations in conserved structures or motifs that are expected to impair ribozyme activity . Inactive group II introns that have lost mobility functions are common in bacteria and organelles and presumably reflect selective pressure against intron mobility, which is deleterious to the host Except for the presence or absence of the intron ORF, the 25 intact TeI4a–h have long ORFs in DIV that encode predicted IEPs of 561 amino acid residues with RT, X/thumb, DNA binding (D), and DNA endonuclease (En) domains homologous to those of other group II IEPs  [1],[2],T. elongatus twintrons could potentially encode active IEPs initiated either from the first AUG after splicing of the inner intron or from the second AUG without splicing of the inner intron. In the remaining intron, TeI4g, the ORF is interrupted by an insertion element (TeI4g::ISEL2f) T. elongatus introns  could potentially encode active IEPs.Two introns (TeI4f and 4h) have continuous ORFs, while in five other introns  the ORF is interrupted by insertion of an ORF-less intron  between the first two ATGs, which are separated by only five codons . This coT. elongatus introns (TeI3a–t) have a ∼1.5 kb deletion in subdomain DIVb, which removes most of the ORF , while the 17 ORF-less introns are divided into four families F3–6; . The F1 T. elongatus introns are inserted within genes may reflect purifying selection against insertions that are deleterious to the host, but mechanisms for actively avoiding insertion within genes are also possible.F3 consists of seven identical ORF-less introns, five of which (TeI3a–e) are inserted at a conserved site between the two closely spaced start codons of the RT ORF of F1 introns, while TeI3i is inserted at the same site in the remnant ORF of TeI3h, and TeI3o is inserted in an intergenic region. F4 consists of three introns inserted at a conserved site in insertion element ISEL1, a member of the IS200 insertion element family Importantly, most of the ORF-less introns are inserted at sites with good EBS/IBS pairings, suggesting insertion via retrohoming, further evidence that the ORF-less introns are actively mobile . The larT. elongatus introns, we used an E. coli plasmid assay in which an intron with a phage T7 promoter inserted near its 3′ end is expressed from a donor plasmid and integrates into a target site cloned in a recipient plasmid upstream of a promoterless tetracycline-resistance (Rtet) gene, thereby activating that gene  intron-donor plasmid uses a T7lac promoter (PT7lac) to express a precursor RNA containing a ΔORF-derivative of the intron RNA (I-ΔORF) with short flanking exons and a phage T7 promoter (PT7) inserted in place of the intron ORF in DIVb. The IEP, which splices and mobilizes the intron, is expressed from a position downstream of the 3′ exon (E2). This configuration using an intron RNA with the ORF deleted and the IEP expressed from downstream of E2 gives high mobility frequencies for other group II introns R (ampicillin-resistant) recipient plasmid contains the intron target site  cloned upstream of a promoterless tetR gene. For mobility assays, the donor and recipient plasmids are co-transformed into E. coli HMS174(DE3), which contains an IPTG -inducible T7 RNA polymerase. After induction of donor plasmid expression with IPTG, cells are plated on Luria-Bertani (LB) medium containing tetracycline plus ampicillin or amplicillin alone, and mobility efficiencies are calculated as the ratio of (TetR+AmpR)/AmpR colonies.For the mobility assays with the IleCAU gene because it contains a continuous ORF and is inserted within an essential gene at a site with good EBS/IBS pairings. These characteristics imply insertion via retrohoming and active splicing to produce a functional tRNA. Nevertheless, TeI4h has features that deviate from the canonical group II intron structure, including a 5′ T residue and a mispairing in the δ-δ′ interaction in the intron RNA , but changing the IEP's YAGD sequence to YADD increased the mobility efficiency dramatically to 3.4%. This modified IEP, denoted IEP-4h*, was used in all subsequent constructs. Combining the TeI4h* IEP with a modified intron (denoted TeI4h*), which has the change C326T in δ′ to restore the δ-δ′ pairing, increased the mobility efficiency further to 39%. Surprisingly, however, combining the TeI4h* IEP with a modified intron in which the 5′-nucleotide residue was changed from T to the highly conserved 5′ G residue found in other group II introns decreased the mobility efficiency to 0.5%, and combining all three changes gave a mobility efficiency of only 8.4%. Thus surprisingly, the unusual 5′ T-residue is favored in TeI4h. Sequencing of retrohoming products from this experiment and from target-site definition experiments described below showed that the inserted TeI4h* intron begins with the unusual 5′ T-residue in all cases (>600 sequences), confirming use of a corresponding 5′ U-residue in the intron RNA for both RNA splicing and reverse splicing. The maximally efficient donor plasmid expressing the TeI4h*-ΔORF intron with the C236T mutation restoring δ-δ′ and the TeI4h* IEP, with the mutation changing YAGD to YADD at the RT active site, is designated pACD2-TeI4h*/4h* to denote the intron RNA/IEP combination.−2%), indicating that one or both of these suboptimal features inhibits mobility regardless of temperature , the remaining twintron IEPs were expressed only from the second ATG. The recipient plasmids contained the natural target site  for each intron . The other IEPs were less specific. Among these, the TeI4c IEP was the most active. Expressed from either the first or second ATG, it mobilized the TeI4c-ΔORF RNA with efficiencies of 2.6 and 2.3×10−2%, respectively, while it mobilized representatives of other intron families at comparable  or higher efficiencies . We note that low mobility efficiencies for some introns are a consequence of suboptimal natural target sites, as the TeI4c IEP could also promote mobility of either TeI4c-ΔORF or TeI3c at 60%–80% efficiency with other target sites (−2 to 2.1×10−4%) with all introns tested, and the TeI4f RNA showed little or no mobility with any IEP tested, likely reflecting mutations that inhibit ribozyme activity, which is required for RNA splicing and reverse splicing , while the outer F1 introns have diverged . Selection for mutations that decrease mobility is common for bacterial and organellar group II introns and presumably reflects that actively mobile introns are deleterious to the host because they can insert into essential genes or make harmful double-strand chromosome breaks T. elongatus introns enable them to partially overcome purifying selection, striking what is for them a more favorable balance toward intron accumulation.In order to proliferate to high copy number, the T. elongatus introns are not only actively mobile but also thermophilic  than does EcI5 (51.2%), possibly contributing to their greater stability at higher temperature. The actively mobile thermostable introns can potentially be used for structural analysis, as well as for practical applications. The latter include use of thermostable group II intron RTs for RT-PCR and next-generation RNA sequencing and use of the thermophilic introns as gene targeting vectors for thermophiles, patterned after “targetrons” developed from mesophilic group II introns that can be reprogrammed to insert at desired sites by modifying the base pairing sequences in the intron RNA Importantly, we find that TeI4h and other mophilic , suggesttrans rather than cis as for other mobile group II introns de novo as a result of mutations or recombination events that introduce the minimal recognition sequences Finally, we speculate that the mechanisms elucidated here could have contributed to the initial proliferation of mobile group II introns in the nuclear genomes of ancestral eukaryotes. Estimates of paleotemperatures based on isotopic measurements and phylogenetic reconstruction of ancient enzymes suggest that eukaryotes evolved at a time of higher than present-day temperatures (50–65°C) T. elongatus introns and flanking exons were cloned via PCR of T. elongatus BP-1 DNA provided by Dr. T. Kaneko, Kazusa DNA Research Institute, Japan. PCRs were done using Taq DNA polymerase . For ORF containing introns, 5′ and 3′ segments of the intron were amplified separately by PCRs using an exon primer that appends a PstI, BamHI, or EcoRI site together with an intron primer that overlaps a unique site (HindIII or EcoRI). The PCR products were then cloned between compatible sites in the polylinker of pUC18 or 19.T. elongatus introns were swapped for the Ll.LtrB intron/IEP cassette in donor plasmid pACD2X T. elongatus introns, this was done via two PCRs that separately amplify 5′ and 3′ segments of the intron, while introducing a 1.5-kb deletion in the ORF coding sequence matching that in the ORF-less T. elongatus introns. These PCRs used end primers that append short flanking exons (∼15 nts) and unique cloning sites (SpeI (5′) and PstI and/or XhoI (3′)) and internal primers that replace the intron ORF in DIVb with a T7 promoter sequence and an MluI site. The two PCR products were then cloned between XbaI and XhoI sites of pADC2X. Twintrons were resolved by precisely deleting the internal intron using PCR primers spanning the insertion site and replacing the original fragment with the deleted one. The ORF-less T. elongatus introns were cloned directly into donor plasmid pACD2X by PCR of T. elongatus BP-1 DNA with primers that append SpeI and PstI sites. The T7 promoter and an MluI site were inserted into DIVb of the ORF-less introns by another round of PCR using the same external primer together with an internal primer that adds the T7 promoter and an MluI site and then swapping the PCR product back into pACD2X. Cloned introns were confirmed to have the sequence published for T. elongatus genomic DNA T. elongatus introns.Intron-donor plasmids for mobility assays were constructed in two steps to insert the coding sequences for the intron RNA followed by the IEP. In the first step, the Donor plasmids with point mutations in TeI4h  and the TeI4h IEP (YAGD to YADD) were derived by PCR amplifying the wild-type intron or IEP of previously constructed plasmids with primers that introduce the modification and then swapping the PCR product containing the mutation for the wild-type sequence to generate donor plasmids containing different combinations of mutations. The TeI4f intron's EBS1, EBS2, and EBS3 sequences were modified by quick-change site-directed mutagenesis  to GTTCTG, TTCAA, and A, respectively, in order to improve complementarity to IBS1, 2, and 3 in the 5′ and 3′ exons.T. elongatus introns contain ligated-exon sequences  cloned between the PstI site (or AatII site for the TeI4f target) and the EcoRI site of pBRR-tet Recipient plasmids for the 5′ GAATTCGACAACCCAACAG. The opposite strand was then synthesized with Klenow DNA polymerase (New England Biolabs) using a primer complementary to the tag, and the resulting double-stranded DNA target site was cloned between the PstI and EcoRI sites of pBRR-tet. The recipient plasmid library for TeI4h was constructed similarly by using Klenow DNA polymerase to fill in annealed, overlapping oligonucleotides that contain the randomized 5′- or 3′-exon sequences and append PstI and EcoRI sites.Recipient plasmid libraries for TeI3c and 4c were constructed by starting with synthetic DNA oligonucleotides that contain randomized target site positions −30 to −12 or −13 and +2 to +20 with a 5′ PstI site and a 3′ EcoRI site followed by the 3′ sequence tag All constructs were confirmed by sequencing the PCR amplified or modified region.E. coli HMS174(DE3)  grown in LB medium, with antibiotics added as required at the following concentrations: ampicillin, 100 µg/ml; chloramphenicol, 25 µg/ml; tetracycline, 25 µg/ml. Cells, which had been co-transformed with the CapR donor and AmpR recipient plasmids, were inoculated into 5 ml of LB medium containing chloramphenicol and ampicillin and grown with shaking (200 rpm) overnight at 37°C. A small portion (50 µl) of the overnight culture was inoculated into 5 ml of fresh LB medium containing the same antibiotics and grown for 1 h as above. The cells were then induced by adding 1 ml of fresh LB medium containing the same antibiotics and 3 mM IPTG  and incubating for 1 h at temperatures specified for individual experiments. In mobility assays with TeI4h and its mutant derivatives at 37°C, changing the IPTG concentration from 100 to 1,000 µM or induction time from 30 to 90 min gave at most a 2-fold increase in mobility efficiency. For determination of temperature dependence, the initial log-phase cultures (5 ml) grown at 37°C were mixed with an equal volume of fresh LB medium containing antibiotics and 1 mM IPTG  that had been pre-warmed to achieve the desired temperature. The cultures were then induced for 1 h at that temperature, placed on ice, diluted with ice-cold LB, and plated at different dilutions onto LB agar containing ampicillin or ampicillin plus tetracycline. After incubating the plates overnight at 37°C, the mobility efficiency was calculated as the ratio of (TetR+AmpR)/AmpR colonies.Mobility assays were done in R+AmpR colonies were picked into duplicate 96-well plates and used for colony PCR to separately amplify the 5′- and 3′-integration junctions using the primers Rsense (5′-ACAAATAGGGGTTCCGCGCAC) plus Te680rc (5′-GTTGGTGACCGCACCAGT) for the 5′ junction and Te420f (5′-AACGCGGTAAGCCCGTA) plus Rev2pBRR (5′-AATGGACGATATCCCGCA) for the 3′ junction. The PCR products were purified with Sera-Mag magnetic beads  and sequenced using the primers TargetSeq (5′-ATGCGAGAGTAGGGAACTGC) for the 5′ junction and Te500f (5′-AAACCGTAAGGAATGCTGATG) or Te420f (see above) for the 3′ junction.To verify correct insertion at the target site, TetE. coli HMS174(DE3) that had been co-transformed with the CapR donor plasmid and an AmpR recipient plasmid in which regions of the DNA target site had been randomized was induced with 500 µM IPTG for 1 h at the specified temperature and then plated on LB agar containing tetracycline and ampicillin, as described above for mobility assays. TetR+AmpR colonies were picked into 96-well plates for colony PCR and sequenced for target-site determination, as described above.Figure S1Multiple amino acid sequence alignments of T. elongatus and other group II intron-encoded proteins. The figure shows aligned predicted amino acid sequences of the IEPs of the group II introns TeI4a, b, c, e, f, g, and h, EcI5, and Ll.LtrB (LtrA protein). The boundaries of conserved RT sequence blocks and the X/thumb, DNA-binding, and DNA endonuclease domains are delineated above the aligned sequences. Identical amino acid residues in the T. elongatus group II IEPs are shown as white letters on a black background, and similar amino acid residues, based on the matrix of Henikoff and Henikoff (1.37 MB TIF)Click here for additional data file.Figure S2Alignment of DIV sequences of T. elongatus group II introns showing deletion breakpoints in the ORF-less introns. The sequences are grouped by intron family and extend from the first to last nucleotide residue of the DIV stem. The positions of the AUG start and UGA stop codons of the intron ORF are indicated. The arrow at the top and asterisks in the alignment indicate the insertion site of TeI3c and other F3 introns in the TeI4a, b, c, d, e, and TeI3h introns. Bases identical in all sequences are in red. Regions with deletions or insertions in the ORF-less introns are highlighted in gray. All the ORF-less introns contain an additional U residue just downstream of the second inframe AUG of the RT ORF (highlighted in red). The alignments were done with ClustalX (2.38 MB TIF)Click here for additional data file.Figure S3Phylogeny of T. elongatus introns. (A) Phylogram of full-length ORF-containing introns. (B) Phylogram of intron-encoded proteins. RNA and protein sequences were aligned with ClustalX (0.19 MB TIF)Click here for additional data file.Figure S4Effect of mutation at DNA target site position C–16 on the mobility efficiency of TeI4h* at 37°C and 48°C. Mobility assays with donor plasmid TeI4h*/4h* and recipient plasmids containing either the wild-type or C–16G mutant DNA target site were done in E. coli at 37°C and 48°C, as described in R+AmpR)/AmpR colonies. The bar graphs show the mobility efficiency with the C–16G mutant DNA target site normalized to that with the wild-type DNA target at 37°C and 48°C. Mobility efficiencies with the wild-type target site in these experiments were 11.5±1.7 and 98±3% at 37°C and 48°C, respectively. The data are the mean for three experiments, with the error bars indicating the standard deviation.(0.02 MB TIF)Click here for additional data file.Figure S5Sequence alignment of F1 and F3 introns. (A) Alignment of F1 ORF-containing introns. Mutations relative to the TeI4c sequence are indicated in red. (B) Alignment of F3 ORF-less introns. The alignments were done with ClustalX (0.06 MB PDF)Click here for additional data file.Table S1Mobility efficiencies for donor plasmid TeI4c/4c with recipient plasmids containing different target sites. Mobility assays were done in E. coli, as described in T. elongatus). Donor plasmid expression was induced with 500 µM IPTG for 1 h at 48°C. Mobility efficiencies were calculated as the ratio of (TetR+AmpR)/AmpR colonies and are the mean of two independent experiments. The two repeats for the TeI4b target site gave mobility efficiencies of 83% and 72%, and the variation between repeats for the other target sites was less than 3-fold. Similar results were obtained with the Te4Ic IEP expressed from the first ATG (not shown). Insertion of the intron at the expected target site was confirmed by PCR (12 colonies) and sequencing (3 colonies) in all cases, except for the TeI4d and TeI4e target sites, where 100% and 60%, respectively, of the colonies did not give the expected PCR product and thus may not correspond to be bona fide mobility events. In a related experiment, donor plasmid TeI3c/4c, which expresses the TeI3c intron and TeI4c IEP, likewise gave high mobility efficiencies (60%–80%) with alternative target sites (unpublished data).(0.07 MB PDF)Click here for additional data file.


The 20, 40, 60, 80 and 100% concentrations of neem seed oil were found to kill all  the larvae after 27, 27, 27, 27 and 24 h respectively.The  Azadirachta indica A. Juss, family: Meliaceae) is a very popular tree in most tropical and sub-tropical regions of the world. It is known as neem all over the world and Dogon yaro in Hausa language, widely spoken in Nigeria. Neem is a hardy tree that grows vigorously in desert areas and in a harsh climate. It possesses versatile medicinal properties along with antibacterial and antifungal activities. The people of India have been using neem leaves for centuries for various aspects.The neem , Epidermophyton (a ringworm that invades both skin and nails of the feet), Microsporum , Trichosporon , Geotrichum  and Candida [Amblyomma variegatum (a three-host tick)[Rhipicephalus sanguineus (a three-host tick)[Boophilus decoloratus (a one-host tick)[Boophilus decoloratus, the blue tick is a one-host tick occurs throughout the Ethiopian region and Southern Asia especially in humid areas. It is parasitic chiefly on cattle and equines, but also found on sheep and goats, wild ungulates and dogs. The engorged females have a slaty blue colour and pale yellow legs. The females lay around 2500 eggs[Babesiosis [Boophilus decoloratus collected from cows.Neem has proved effective against certain fungi that infect the human body and are difficult to control by synthetic fungicides. Neem preparations showed toxicity to 14 common fungi, including members of the following genera. ost tick), Rhipiceost tick) and Boopn, death). There i2SO4.The kernels were collected from the ground under the neem tree in the Ahmadu Bello University campus, Zaria, Nigeria, depulped, washed with water and dried under sun. The kernels were crushed using a crucible and blender, then boiled with water for 1 h when the oil separated and floated to the top. The two phases were separated using a separating funnel and the oil, a viscous liquid was collected from the upper phase and dried over anhydrous NaBoophilus decoloratus were used in this study. The engorged female ticks were collected from Zangon Shunu area in Zaria, Nigeria by hand picking from the bodies of the cows. Each engorged female tick was placed in a clean plastic tube plugged with cotton wool and incubated at 29° with relative humidity of about 80%. The eggs laid in each tube were transferred into a clean tube with cotton plug and dead female ticks discarded. The eggs were kept under same incubating conditions until they hatched into larvae and then starved in the incubator at 29° for two weeks before use.The larvae of a one-host tick,Petri dishes (9 cm diameter) with one sheet of filter paper inside of the same size were used for the experiment. Five different concentrations of neem oil, 100, 80, 60, 40 and 20% were used for the study. One milliliter (100%), 0.8 ml (80%), 0.6 ml (60%), 0.4 ml (40%) and 0.2 ml (20%) of neem oil were evenly soaked (spread with a spatula) on different filter papers of each Petri dish. Distilled water soaked in filter paper was used as a control (0% concentration). Then different numbers of larvae were put inside each plate with the help of a soft brush and then monitored at certain interval of time. The number of larvae found dead in each plate was recorded and the results were summarized in From the results obtained, it is clear that the mortality of larvae was concentration and time dependent. With 0% concentration of neem oil (control), there was no mortality at any time. 100% mortality was observed with 20, 40, 60, 80 and 100% concentrations after 27, 27, 27, 27 and 24 h, respectively. Thus the neem oil can be safely used for the control of ticks in animals due to its non- adverse effect in animals. Moreover, the neem oil with both fungicidal and bactericidal properties could treat these infections caused from the bite of ticks. The oil is easily available for its use as an inexpensive herbal medicine. This result will be very much useful for the farmers all over the world.


The enrichment of specific intronic splicing enhancers upstream of weak PY tracts suggests a novel mechanism for intron recognition that compensates for a weakened canonical pre-mRNA splicing motif. While the current model of pre-mRNA splicing is based on the recognition of four canonical intronic motifs  tract and 3' splice site), it is becoming increasingly clear that splicing is regulated by both canonical and non-canonical splicing signals located in the RNA sequence of introns and exons that act to recruit the spliceosome and associated splicing factors. The diversity of human intronic sequences suggests the existence of novel recognition pathways for non-canonical introns. This study addresses the recognition and splicing of human introns that lack a canonical PY tract. The PY tract is a uridine-rich region at the 3' end of introns that acts as a binding site for U2AF65, a key factor in splicing machinery recruitment.In vivo splicing studies show that C-rich and G-rich motifs function as intronic splicing enhancers in a combinatorial manner to compensate for weak PY tracts.Human introns were classified computationally into low- and high-scoring PY tracts by scoring the likely U2AF65 binding site strength. Biochemical studies confirmed that low-scoring PY tracts are weak U2AF65 binding sites while high-scoring PY tracts are strong U2AF65 binding sites. A large population of human introns contains weak PY tracts. Computational analysis revealed many families of motifs, including C-rich and G-rich motifs, that are enriched upstream of weak PY tracts. The enrichment of specific intronic splicing enhancers upstream of weak PY tracts suggests that a novel mechanism for intron recognition exists, which compensates for a weakened canonical pre-mRNA splicing motif. Pre-mRNA splicing is an essential processing step where non-coding intervening sequences (introns) are removed from the initial RNA transcript and coding sequences (exons) are ligated together to produce mature mRNA. Pre-mRNA splicing is mediated by the spliceosome, a multi-component complex composed of small nuclear ribonucleoproteins (snRNPs) and over 100 accessory proteins ,3. Pre-mSeveral canonical intronic sequences define an intron and recruit the spliceosome to the pre-mRNA: the 5' splice site , the branchpoint sequence (CURAY), the polypyrimidine (PY) tract (a run of polypyrimidines located between the 3' splice site and the branchpoint), and the 3' splice site . These four canonical intronic sequences are recognized by specific components of the spliceosome or associated splicing factors. In the initial stage of splicing, when the decision to remove an intron is made, the U1 snRNP recognizes the 5'ss ,9, splicAlthough canonical splice elements are located within the intron, the exon is generally considered to be the unit that is first recognized and defined by the spliceosome. This is known as exon definition and is thought to be a dominant mode of recognition in human genes where the exons are small and the introns are large . In the cis-elements appear to be essential for accurate identification of many splice sites, and various cis-splicing elements have been identified in both exonic and intronic regions. Based upon their locations and effects upon splicing, these have been categorized as exonic and intronic splicing enhancers  or exonic and intronic splicing silencers  .In vitro selection studies have determined that U2AF65 binds with highest affinity to continuous runs of uridines interrupted by cytidines [We are interested in the question of how introns that lack a canonical splice element are recognized and spliced. We have focused on introns that lack a canonical PY tract. In humans, U2AF65 binding to the PY tract is believed to be critical for intron recognition and splicing. ytidines . This agin vitro for some substrates [in vivo [cis-elements present in the intron could alleviate the need for strong U2AF65-RNA interactions. These cis-elements could include the branchpoint sequence and 3'ss, which recruit SF1 and U2AF35, respectively, both of which can bind U2AF65 cooperatively through protein-protein interactions [cis-elements such as ESEs and ISEs could function in the recognition of introns containing weak PY tracts. Previous studies have indicated that ESEs located in the downstream exon are able to compensate for weak PY tracts [One model predicts that U2AF65 is not essential for the splicing of these introns. Several human introns have been shown to be spliced when U2AF65 levels are significantly reduced by RNA interference . U2AF65 bstrates . There i[in vivo . Alternaractions ,31,32. AY tracts ,34. In tY tracts . We propWe have used a computational approach to classify human introns in terms of their U2AF65 binding site strength. We conclude that a significant population of human introns does not contain a strong U2AF65 binding site in the PY tract region. This classification of human PY tract strength enabled us to computationally identify intronic motifs over-represented upstream of weak PY tracts. We propose that these over-represented motifs are putative ISEs that are important for the splicing of introns containing weak PY tracts.UGAC), is known to result in intron retention, causing familial LCAT deficiency (complete deficiency) or fish-eye disease , which can lead to premature atherosclerosis [LCAT (lecithin cholesterol acyltransferase) intron 4 is a short (83 nucleotide) constitutively spliced intron with a weak PY tract. Mutation of the branchpoint sequence U to C  experiments using human U2AF65 did not reveal a well defined consensus motif shared by high affinity RNAs [65 score. The S65score, for a given intron, is the average degree to which all pentamers (using a sliding window) found in the PY tract region (-30 to -3 relative to the acceptor splice-junction) are themselves enriched within the SELEX derived sequences .In order to carry out this analysis, we first needed to correlate the composition of the PY tract of introns with likely affinities towards U2AF65. Several theoretical models have been presented that describe the relationship between binding site composition and the ΔG of binding between nucleic acids and nucleic acid binding proteins ,45. Thesity RNAs ,39. Seveity RNAs ,47. ThouFor this analysis, the PY tract was defined as the region from -30 to -3 (relative to the acceptor splice junction). This region is highly enriched in the pentamers that are most abundant within the U2AF65 selected sequences Figure  is consi65 scores for the SELEX RNAs appear to be normally distributed with a mean of 1.5  relative to the scores for the U2AF65 selected RNAs, suggesting that a large portion of human PY tracts represent weaker than optimal U2AF65 binding sites.The S5 Figure . In contWe chose to classify PY tracts that score below the median of 0.811 as 'weak' PY tracts and those above 0.811 as 'strong' PY tracts or likely to have high affinity U2AF65 binding sites. Using this designation, only a single SELEX-derived sequence scores as 'weak'. We are therefore asking whether there are statistically significant differences in the composition of the -80 to -30 region of two types of introns: ones that contain a PY tract with affinities similar to those derived using SELEX, and those with PY tracts with lower affinities.65 score and observed U2AF65 binding affinities, we evaluated the binding of recombinant human U2AF65 to several human PY tracts of varying S65 scores using gel-shift mobility assays . This PY tract is interrupted by several purines that are expected to impair U2AF65 binding. We also evaluated three other low-scoring PY tracts with scores closer to the median, and, therefore, correspond to the more 'typical' human PY tract: BRUNOL4 intron 9 (S65 = 0.3602), ITGB4 intron 31 (S65 = 0.3608), and LCAT intron 4 (S65 = 0.5068). All three of these are cytidine-enriched. In addition, we tested three high-scoring PY tracts that had scores spanning the higher range of the distribution: INSR intron 10 (S65 = 0.9593), U2AF2 intron 6, (S65 = 1.1787), and SR140 intron 9 (S65 = 1.8434), and an altered version of the LCAT intron 4 in which the central region was modified to contain an eight nucleotide poly-uridine run (LCATmut with a S65 of 1.2060). All four of these high-scoring sequences are uridine-enriched. Binding data were also obtained using two sequences derived from the PY tract of the adenovirus major late (ADML) pre-mRNA, similar to previously studied ADML PY tracts [In order to asses the relationship between the SY tracts ,49.65 scores demonstrated moderate affinities in the binding assay. In contrast, three of the uridine-rich sequences (with high S65 scores) bound with high affinity. An interesting exception was the INSR-derived sequence, which bound U2AF65 more weakly than the more cytidine-rich LCAT-derived sequence. Importantly, for both LCAT and ADML, the binding of the mutant versions correlates well with the predicted affinities based upon the S65 score.We expected the MBNL1 intron 6 PY tract to represent the weakest U2AF65 binding target and observed no detectable levels of U2AF65 binding at the protein concentrations tested , this is consistent with the supposition that S65 is linearly related to ΔG. Linear regression of the observed affinities and S65 scores demonstrates that these values are strongly correlated . Therefore, the S65 score is a better predictor of binding affinity than uridine content alone and suggests that U2AF65 is recognizing sequence features more complex than the simple presence or absence of contiguous runs of uridines.Overall, there is a good agreement between the observed binding affinities for U2AF65 and the predicted affinities based upon the Se Figure . Since Δ65 scores less than 0.811; see Materials and methods). We were specifically interested in identifying sequences located in the region upstream of the branchpoint itself. Since most branchpoints are located between -17 and -30  for introns with PY tracts categorized as 'weak' relative to the set of all introns  in the AT-rich class, and 349 n-mers were determined to be significantly enriched in the GC-rich class. For comparison, we drew random samples of the same size as the corresponding weak PY tract class for both the AT-rich and GC-rich introns, and determined enrichment using the same method as above. The average number of n-mers (for to seven nucleotides) that were determined to be significantly enriched in the randomly drawn samples was ten for the AT-rich and zero for the GC-rich class. Therefore, the enrichment measured appears to be strongly correlated with the composition of the PY tract as measured by the S65 score.Enrichment of n-mers in the -80 to -30 region for introns with weak PY tracts versus all GC or AT-rich introns was determined . The entire list of enriched n-mers used in this study is available in Additional data files 2 and 3. According to this analysis, 99 n-mers were determined to be significantly enriched  introns may differ from those governing splicing of longer introns . TherefoTo determine motifs, the enriched n-mers were clustered using the graph clustering method and software presented by Voelker and Berglund . ClusterMotifs containing three to four contiguous guanidines are greatly enriched upstream of weak PY tracts for both AT-rich and GC-rich introns  are enriched upstream of weak GC-rich PY tracts . We refer to this value as the percent coverage. As an example, 80% coverage indicates that 80% of the -80 to -30 region (or 40 of the 50 nucleotides) matches one or more of the enriched n-mers. This analysis  revealed that most introns have at least one match to an enriched n-mer. This is not surprising considering that the n-mers are only four to seven nucleotides in length, and, therefore, are expected to occur by chance with fairly high frequency. However, this analysis also revealed that introns with weak PY tracts are likely to have a greater coverage than introns with strong PY tracts. This is especially true of the GC-rich class of introns. For instance, while only 10% of GC-rich introns with strong PY tracts have 80-100% coverage, 23% of introns with weak PY tracts have this level of coverage . A smaller difference in coverage is seen between AT-rich introns with strong and weak PY tracts; however, the overall trend is the same . In both cases, the enriched n-mers tend to make up a greater portion of the -80 to -30 region for introns with weak PY tracts. Together, these observations indicate that the sequences represented by the enriched n-mers are rather common but they tend to cluster in introns with weak PY tracts.LCAT intron 4 contains both C-rich and G-rich motifs upstream of the PY tract similar to those we identified computationally that are also highly conserved. The PY tract of LCAT intron 4 is a low-scoring PY tract and is not well conserved. To investigate the role of C-rich and G-rich motifs present in LCAT intron 4, we used a mini-gene system. We created a mini-gene that contains the last 50 nucleotides of LCAT intron 3, LCAT exon 4, LCAT intron 4, LCAT exon 5 and the first 50 nucleotides of LCAT intron 5. We included the downstream and upstream flanking introns in order to allow exon definition to occur, although short introns are often observed to function by intron definition .We examined the role of two G-rich motifs (G-rich motif (GRM)1 and GRM2) present upstream of the PY tract of LCAT intron 4 Figure . The wilTo determine whether the C-rich motifs function as ISEs, we mutated two C-rich motifs: C-rich motif (CRM)1 and CRM2 Figure , which aWe hypothesized that the G-rich motifs and C-rich motifs could be functioning together in the recognition of LCAT intron 4. We have observed that there are many examples of introns where the G-rich and C-rich motifs are both present (data not shown). Mutation of both GRM1 and CRM1 . In order to test the function of the three C-rich motifs, we created a mini-gene containing exon 2, intron 2 and exon 3. The WT GNPTG intron 2 mini-gene splices such that 29 ± 6% pre-mRNA is observed  short intron containing multiple C-rich motifs upstream of a low scoring PY tract  [65 score is generally able to distinguish between sequences displaying strong and weak interactions with U2AF65, and it is more accurate than using simple uridine content alone.Our U2AF65 binding studies using various human intron PY tracts . Although LCAT intron 4 is constitutively spliced, expressed sequence tag data suggest that GNPTG intron 2 is alternatively spliced, with some expressed sequence tags containing a retained intron 2. We expect to find examples where these motifs may play important roles in both constitutive and alternative splicing for both short and long introns.G-rich motifs have been shown to be enriched in short mammalian introns ,65. The Our results also show that C-rich motifs can act as ISEs like the G-rich motifs, but that the C-rich motifs may play more of an ancillary role to the G-rich motifs, at least in the case of LCAT intron 4 Figure . C-rich Interestingly, the C-rich and G-rich motifs together function additively Figure . This suWe have also observed that the C-rich motifs can only partially compensate in the place of G-rich motifs in LCAT intron 4 splicing, while G-rich motifs appear to fully compensate in the place of C-rich motifs Figure . It may An examination of the primary sequence and predicted secondary structure (using mfold ) of LCATin vivo [There are multiple candidate proteins that could be recognizing the G-rich and C-rich motifs present in LCAT intron 4. A G-rich motif trans-factor, hnRNP H, has been identified and shown to bind G-rich sequences and regulate splicing both positively and negatively -56. Sevein vivo .in vitro [cis-elements, including G-rich ISEs, have been shown to act as both splicing enhancers and silencers [hnRNP K and the α-CP proteins are the major poly-C-binding proteins identified in mammalian cells ,71. Bothin vitro . This isilencers -56,72. TOur results suggest a model where ISEs present upstream of a weak PY tract compensate for a weakened U2AF65-RNA interaction Figure . In the An alternative model to weakened U2AF65-RNA interactions is that a splicing factor other than U2AF65 binds the weakened PY tracts. Recent work has shown that PUF60 plays a role in splicing by interacting with the PY tract . Our obscis-elements, in this case the G-rich and C-rich motifs, appears to be crucial for the recognition of non-canonical introns. In the future we plan to explore additional ISEs identified by this study to gain a broader picture of how the splicing machinery functions in the recognition of introns with weak PY tracts. While we have focused our attention here on a single key canonical splicing element, the PY tract, we plan on extending our analyses and expect to find that similar strategies exist for the recognition of other classes of non-canonical pre-mRNAs.The pool of introns containing low-scoring U2AF65 binding sites represents a significant class of human introns lacking a canonical splicing element. The ISEs identified and validated here suggest that novel mechanisms exist in the cell for coping with weakened U2AF65-RNA interactions. Specifically, we have observed that the interplay of multiple The March 2006 human reference sequence (NCBI Build 36.1) in conjunction with the UCSC KnownGenes (hg18) annotation database (Release 8 April 2007)  was usedin vitro SELEX experiments using human U2AF65. In particular, if we let the frequency of occurrence of an n-mer n of length k within the SELEX sequences be represented by fn, then for a subject sequence of length L the S65 score is determined according to:In order to score PY tracts according to their likely affinity towards U2AF65, we developed a score that reflects the level of similarity of the PY tract sequence to the sequences that were enriched in RNAs derived from fn represents the frequency (within the SELEX population) of the n-mer found at position i in the subject sequence.where k. For this study we chose the n-mer length to be five and the SELEX data were those reported in Singh et al. [et al. [in vitro SELEX had S65 scores above this value.The term h et al.  and both [et al. . The freIn order to avoid biases due to long interspersed repetitive elements (LINEs) and short interspersed repetitive elements (SINEs), repetitive elements in the intronic sequence database (obtained as described above) were masked using the masking coordinates associated with the UCSC hg18 annotation database (Release 8 April 2007) . HoweverP < 0.01), and 349 n-mers were determined to be significantly enriched for the GC-rich class. Enriched n-mers and corresponding counts and statistics are available in Additional data files 2 and 3. Enriched n-mers were used to construct motifs as in Voelker and Berglund [For each of these data sets, the occurrence of all n-mers (4-7 nucleotides) in the 50 nucleotide region from -80 to -30 (relative to the acceptor splice-junction) were determined using a sliding window. These counts were used to determine the background expectations for each n-mer. The occurrence of each 4-7 nucleotide n-mer within the equivalent region for all introns possessing 'weak' PY tracts (defined as above) was determined using a sliding window. From these values, n-mers that are enriched upstream of the branchpoint region for introns possessing weak PY tracts was determined using the binomial confidence interval method described in Voelker and Berglund . For theBerglund . All of 32P ATP using T4 polynucleotide kinase  for 30 minutes at 37°C. The RNAs were then gel purified using an 8% denaturing gel, eluted from the gel in 0.3M Na acetate and ethanol precipitated. The resulting pellet was resuspended in nanopure water and purified with a Bio-spin 6 column  equilibrated with nanopure water. The radioactivity level of the purified RNA solution was determined by scintillation. Gel-shift binding assays were performed using varying concentrations of recombinant human U2AF65 with constant amounts of radiolabeled RNA oligonucleotides as previously described [RNA oligonucleotides . The forward primer included a 5 cells were plated in 6-well plates and transfected 18-20 h later at approximately 70% confluency. Plasmid (1 μg) was transfected into each well of cells using 5 μl of Lipofectin  and 10 μl of Plus reagent (Invitrogen) according to the manufacturer's protocols. For the GNPTG splicing experiments, 2 × 105 cells were plated in 6-well plates and transfected with 1 μg plasmid 18-20 h later using 5 μl of Lipofectamine 2000. Cells were harvested 24 h (LCAT experiments) or 16 h (GNPTG experiments) after transfection using TriplE (GIBCO) and then pelleted by centrifugation. RNA was isolated from the cell pellets using an RNeasy kit .HeLa cells were grown in monolayers in DMEM with GLUTAMAX  and supplemented with 10% fetal bovine serum (GIBCO). For the LCAT splicing experiments 1.5 (± 0.2) × 10Isolated RNA (500 ng) was incubated with 1 unit of RQI DNase  in a 10 μl reaction for 2 h (LCAT experiments) or 1 h (GNPTG experiments) according to the manufacturer's protocol. DNAsed RNA (2 μl (100 ng)) was reverse transcribed in a 10 μl reaction (1:5 dilution) using Superscript II and an LCAT-specific reverse primer or a reverse primer to the pCDNA3 SP6 sequence for the GNPTG experiments, according to manufacturer's protocols with the exception that we used half the recommended amount of Superscript II . For the LCAT splicing experiments, 2 μl of the reverse transcription reaction was subjected to 20 rounds of PCR amplification in a 20 μl reaction (1:10 dilution) using LCAT specific primers spiked with a kinased LCAT forward primer (0.4 nM). Twenty rounds of PCR were found to be within the linear range for this PCR experiment (data not shown). The resulting PCR products were run on an 8% (19:1) polyacrylamide native gel. For the GNPTG splicing experiments, 2 μl of the reverse transcription reaction was subjected to 27 rounds of PCR amplification in a 20 μl reaction (1:10 dilution) using primers specific to the T7 (forward) and SP6 (reverse) sequences of the pcDNA3 plasmid spiked with kinased T7 forward primer. Twenty-seven rounds of PCR were found to be within the linear range for this PCR experiment (data not shown). The resulting PCR products were run on a 10% (19:1) polyacrylamide gel. The gels were dried and exposed overnight to a phosphorimager screen. Quantification of the radioactive bands was performed using ImageQuant software . The percent pre-mRNA was calculated by dividing the amount of the pre-mRNA band by the total amount of the pre-mRNA and mRNA bands and multiplying by 100%.65 score, U2AF65 binding site score; SF, splicing factor; snRNP, small nuclear ribonucleoprotein; ss, splice site; U2AF, U2 snRNP auxilliary factor; WT, wild-type.ADML, adenovirus major late; CRM, C-rich motif; ESE, exonic splicing enhancer; ESS, exonic splicing silencer; GNPTG, N-acetylglucosamine-1-phosphotransferase gamma subunit; GRM, G-rich motif; hnRNP, heterogeneous nuclear ribonucleoproteins; ISE, intronic splicing enhancer; ISS, intronic splicing silencer; LCAT, lecithin cholesterol acyltransferase; PY tract, polypyrimidine tract; SJIM and RBV designed experiments, performed experiments, analyzed data and wrote the paper. KLH and MBW performed experiments and analyzed data. JAB designed experiments, analyzed data and wrote the paper.The following additional data files are available with the online version of this paper. Additional data file et al. [et al. [Table listing the count and the probability of occurrence (using a sliding window) for all pentamers found in the sequences reported in Singh et al.  and both [et al. .Click here for fileAssociated statistics and listing of n-mers (4-7 nucleotides) determined to be enriched in the 50 nucleotide region upstream of weak PY tracts from GC-rich introns.Click here for fileAssociated statistics and listing of n-mers (4-7 nucleotides) determined to be enriched in the 50 nucleotide region upstream of weak PY tracts from AT-rich introns.Click here for fileListing of all clusters derived from n-mers enriched in the 50 nucleotide region upstream of weak PY tracts from GC-rich introns. Included are the individual n-mers and associated statistics used to produce each motif.Click here for fileListing of all clusters derived from n-mers enriched in the 50 nucleotide region upstream of weak PY tracts from AT-rich introns. Included are the individual n-mers and associated statistics used to produce each motif.Click here for file65 scores for short (<200 nucleotide) introns is plotted versus long (≥ 200 nucleotide) introns. (a) Data for GC-rich introns. (b) Data for AT-rich introns.The Z-scores for enrichment of all 4-7 nucleotide n-mers in the intronic region upstream (-80 to -30 relative to the acceptor splice-junction) of PY tracts with low SClick here for fileThe portion of the sequence corresponding to the -80 to -30 region matching one or more of the n-mers enriched in the same region for introns with weak PY tracts  was determined. These values (referred to as the percent coverage) were binned as indicated along the x-axis.Click here for file