Although large scale informatics studies on introns can be useful in making broad inferences concerning patterns of intron gain and loss, more specific questions about intron evolution at a finer scale can be addressed using a gene family where structure and function are well known. Genome wide surveys of tetraspanins from a broad array of organisms with fully sequenced genomes are an excellent means to understand specifics of intron evolution. Our approach incorporated several new fully sequenced genomes that cover the major lineages of the animal kingdom as well as plants, protists and fungi. The analysis of exon/intron gene structure in such an evolutionary broad set of genomes allowed us to identify ancestral intron structure in tetraspanins throughout the eukaryotic tree of life.We performed a phylogenomic analysis of the intron/exon structure of the tetraspanin protein family. In addition, to the already characterized tetraspanin introns numbered 1 through 6 found in animals, three additional ancient, phase 0 introns we call 4a, 4b and 4c were found. These three novel introns in combination with the ancestral introns 1 to 6, define three basic tetraspanin gene structures which have been conserved throughout the animal kingdom. Our phylogenomic approach also allows the estimation of the time at which the introns of the 33 human tetraspanin paralogs appeared, which in many cases coincides with the concomitant acquisition of new introns. On the other hand, we observed that new introns  were not randomly inserted into the tetraspanin gene structure. The region of tetraspanin genes corresponding to the small extracellular loop (SEL) accounts for only 10.5% of the total sequence length but had 46% of the new animal intron insertions.Our results indicate that tests of intron evolution are strengthened by the phylogenomic approach with specific gene families like tetraspanins. These tests add to our understanding of genomic innovation coupled to major evolutionary divergence events, functional constraints and the timing of the appearance of evolutionary novelty. Eukaryotic protein coding genes are interspersed with non coding sequences called introns that are removed from the corresponding transcripts by the spliceosome, a complex RNA-protein assemblage. Introns and sequences of proteins from the splicing machinery have been found in all eukaryotic species with fully sequenced genomes Studies on the evolution of spliceosomal introns mainly use broad genomic data sets of conserved homologous genes from diverse eukaryotic organisms DrosophilaWe suggest that suitable gene families to apply the phylogenomic approach to examine intron/exon structure would be ones with many members, several introns in each paralog and a broad phylogenetic distribution. The tetraspanin superfamily of proteins meets all three of these important requirements. This large family has 33 paralogs in the human genome and at least 37 members in Drosophila; the CD9 and CD81 tetraspanins, which are involved in mammalian sperm: oocyte fusion The biochemical functions of tetraspanins, a broadly expressed superfamily of transmembrane proteins, are based upon their ability to form large integrated signalling complexes or tetraspanin-enriched microdomains by their primary associations with multiple transmembrane and intracellular signaling/cytoskeletal proteins and secondary associations with themselves The typical intron/exon structure of most tetraspanins has been reported, in bilaterian animals, to consist of six introns in positions that do not break the reading frame  that we have named introns 1 to 6  [40].In the present study we add to our recent analyses of tetraspanin relationships With a broad taxonomic sampling of tetraspanin genes and precise description of their exon/intron structure we can test several hypotheses relevant to the evolution of tetraspanin introns. First, we can examine whether the tetraspanins corroborate already well-established patterns of exon/intron loss in other genes. Because there are several introns in the tetraspanins this allows us to test hypotheses of intron gain/loss very precisely. Second, we hypothesize that major structural changes in tetraspanin genes with respect to exon/intron structure are associated with major cladogenetic events in the eukaryotic tree of life. This hypothesis stems from ideas about major radiations of organisms being accompanied by similar major adaptations. In order to test this hypothesis, we reconstruct the gain/loss of introns on a well corroborated eukaryotic tree and examine where on the tree the gain/loss events occur. Third, we hypothesize that substantial exon/intron structural changes that occur in tetraspanins are associated with major functional changes in these proteins. This hypothesis can be tested by examining whether any other physical aspects of tetraspanins change concurrently with exon/intron alterations. Specifically, we examine if changes in the cysteine motifs in tetraspanins are coincidental or correlated with exon/intron changes.Oscarella carmella ESTs, designed primers to obtain introns and determined the exon/intron structure by sequencing O. carmella genomic DNA. All six ancestral intron positions were present in one of the five sequences of O. carmella tetraspanins that we obtained  for tetraspanins revealed the presence of all six introns in the tetraspanins of these diploblastic animals. We found ancient intron 4 also in Fungi, Plants and introns 1 and 4 in Amoebozoa  and the unicellular choanoflagellate  to mammals (Sponges are often considered the most primitive diploblastic (two tissue layers) animals. Since fully sequenced sponge genomes are not yet available, we searched the NCBI database for sponge tetraspanins in the expressed sequence tag (ESTs) database. We identified several tetraspanins from the sponge obtained . While wmoebozoa . In addi mammals  and S3. Ciona intesinallis) and vertebrates and has been conserved since  divide animal tetraspanins into three groups; we call them CD63-like (CD63L) , TSPAN15-like (TSPAN15L)  and TSPAN13-like (TSPAN13L)  , and S3.ed since . In animed since Monosiga brevicolis, a unicellular organism, which is considered the closest relative to multicellular animals M. brevicollis has four additional specific introns .Ciona) throughout vertebrates. Another example is that of the CD37/CD82 subgroup which has accrued a new intron (intron 1c) in the ancestor of vertebrates , in contrast to the ancestral nine phase 0 well-conserved introns (introns 1 through 6). These phase 1 or 2 introns are found in tetraspanin groups: CD9, Tsp2, CD37 (intron 1a); Tsp8, (intron 1b); CD82, CD37, (intron 1c); Tsp 12 (intron 1d); Tsp32, (intron 1e); ROM, RDS, (intron 4d); and Tsp10, (intron 4e). For example, the gene structure of the tetraspanin subgroup Tsp2/CD81/CD9 shows a new intron  into a matrix C. elegans (42 gains), with ten percent occurring in the sea squirt (11 gains) and over five percent occurring in Drosophila (6 gains) and choanoflagellate (5 gains). The rest of the gains are dispersed across the other taxa. The four losses occur in the common ancestor of protosotomes  and in the sea squirt (1).Using the mapped presence/absence of introns on the phylogenetic tree we calculated there are 105 intron gain events and only four intron loss events. More than one fourth of these gains (27 gains) are in hypothetical ancestors in the tree. The majority of the gains occur in a single species, in the nematode C. elegans tetraspanins, which show a very high frequency of intron gain  . In thisth intron in mouse CD81 gene is shifted one amino acid codon relative to that in the same gene in Danio rerio  were almost always found in the same position and phase in the aligned sequences of orthologous tetraspanins genes. However, in a few tetraspanins some intron positions are shifted in position by one to three amino acid residues  and S6. io rerio . Anotherse Tsp14 . A closese Tsp14 .C. elegans and D. melanogaster).Our phylogenomic approach using tetraspanins allows us to specifically address the general observation from genome wide informatics studies that intron gain is more prevalent than intron loss D. melanogaster and C. elegans). Most tetraspanins (20 of 33 human tetraspanins) originated in the common ancestor of vertebrates Homo sapiens, and hence few orthologous intron acquisitions. Because we only used a limited taxonomic sampling for our comparisons, the suggestion that major intron loss and gains coincide with the divergence of major organismal groups needs to be tested with the acquisition of more whole genomes. Nevertheless, our data are consistent with the notion that intron structure changes are major evolutionary events that coincide with adaptive or anatomical divergence.Early studies of intron gain/loss indicated that intron gain is prevalent in eukaryotic genomes The generation of functional diversity in gene families is another interesting aspect that might be correlated with intron gain/loss events. In order to examine this possibility, we utilize the interesting result that three combinations of the nine ancient intron positions divide all the animal tetraspanins into three groups CD63L, TSPAN15L and TSPAN13L . All aniEach of these three groups have a distinct scaffold of disulfide bonds in the LEL variable region suggesting that each group could perform different functions as these particular structures are in is the region of tetraspanins that is involved in protein-protein recognition Utilizing a phylogenomic analysis of tetraspanin protein sequences, we previously suggested that tetraspanins can be divided into four major groups - the CD family, the CD63 family, the uroplakin family and the RDS family Finally, the TSP15L group includes all the RDS family tetraspanins  except for the RDS/ROM and TSPAN10 (oculospanin). These 8 cysteine pattern proteins are close to the base of the tree in Garcia-Espana et al Interestingly, 36.7% of the derived newly gained 79 tetraspanin introns are found in the SEL which make up only 10.5% of the total sequence length. A correlation of some intron positions in the DNA with protein structural features has been observed, but how or why this correlation occurs is not fully understood. It has been reported that a non-random tendency exists for introns to be located in interdomain regions of proteins The regions in tetraspanins where introns are less frequently found (mainly the transmembrane domains) have distinct and conserved secondary structures mainly formed by α-helices. These α-helices shape the transmembrane domains and form a tree trunk like structure in the LEL constant domain from which the LEL variable domain extends That specific intron junctions sometimes differ among species by only a few nucleotides is a well known although rare phenomena The tetraspanin family is particularly well suited to addressing questions about protein function and evolution in a phylogenomic context. Because the structure and function of many of the members of the gene family are well known, these attributes of tretraspanins can be examined for correlation with intron change and organismal evolution. Because the number of introns in the tetraspanin family is reasonably large, and the phylogenetic distribution of genomes that can be used for such studies covers all of eukaryotes, specific evolutionary questions about introns can be addressed. Using tetraspanins, we were able to examine several aspects of intron evolution in a very precise manner. First, we examined the overall phylogenetic distribution of intron change and found that much of the intron gain/loss occurs in major phylogenetic branching events, specifically in the origin of the vertebrates. Second, we show that a proportionately larger number of intron gain/loss events occur in the large and small extracellular loops (LEL and SEL) than anywhere else in the protein structure. Finally, we examine the dynamics of intron gain/loss in the context of reading phase shifts. Close examination of tetraspanins allows for very precise statements to be made about these phenomena related to intron evolution.www.ncbi.nlm.nih.gov) and manual supervision of the splice consensus signals. Information about the position of introns was gathered from different sources such as: Ensmbl, (www.ensembl.org).Intron-exon borders  were detTaeniopygia guttata was prepared with the RNeasy Midi Kit  and DNA from the sponge Oscarella carmela was kindly donated by Scott A. Nichols . The genomic fragments of the Oscarella and Taeniopygia tetraspanin genes were amplified by PCR using the AccuPrime Taq DNA polymerase System .Genomic DNA from the bird www.ebi.ac.uk/) or at NPS Web server (http://npsa-pbil.ibcp.fr). Tetraspanin splicing transcripts were searched in the NCBI AceView database (http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/index.html) and published data An intron was considered to be shared if it was found in the same aminoacid position and the same codon position (the same intron phase) in sequences the alignments of the FASTA-formatted files containing the protein. We used introns positions in the tetraspanins reading frame between intron 1 and 6 and exclude the divergent ends of tetraspanin proteins. To compare intron positions we used several alignments with Clustal W Click here for additional data file.Figure S1Intron junction analysis of the CD63L group. Alignment of full length representative CD63 tetraspanin from human (Hs), mouse (Mm), and zebrafish (Dr) with Sea anemone and Trychoplax CD63-like tetraspanins. The presence of an intron is shown within the amino acid sequences by a number, shaded in red, indicating the intron phase . Ancestral introns 1–6 are numbered above the alignment. Cysteines in the variable subdomain in the LEL loop are shaded in yellow.(0.25 MB TIF)Click here for additional data file.Figure S2Intron junction analysis of the TSPAN15L group. Alignment of Deuterostomes  and Protostomes (Dm) TSPAN15L proteines with Sea anemone, Trychoplax and Sponge tetraspanins of the TSPAN15L group. Ancestral introns 1–4, and 6 plus introns 4b and 4c characteristics of this group of tetraspanins are numbered above the aligned sequences. Cysteines in the variable LEL loop domain and introns positions are marked yellow as in (0.34 MB TIF)Click here for additional data file.Figure S3Intron junction analysis of TSPAN13L group. Alignment of Deuterostomes  and Protostomes (Dm) TSPAN13L proteines with Sea anemone, Trychoplax and Monosiga tetraspanins of the TSPAN15L group. Characteristic introns of this group  are numbered above the aligned sequences. Cysteines in the LEL loop and introns positions are marked as in (0.37 MB TIF)Click here for additional data file.Figure S4Evolution of the Intron structure in tetraspanins supports several nodes in animal phylogeny. Intron structures are represented above the tetraspanins from the cluster we called CD group in Garcia-Espana et al (0.16 MB TIF)Click here for additional data file.Figure S5Junction analysis of the CD81 group. (A) Protein alignment of the full exon protein sequences between introns 4 and 6 of several CD81 tetraspanins. Cysteines are shaded in yellow. (B) Clustal W DNA alignment of the above exons 5 and 6 in blue letters plus the intron sequence between them in black letters of CD81 tetraspanins from Aves, chicken (Gg) and zebra finch (Tg). Other species: Hs, human; Pt, chimpanzee; Cf, dog; Rn, rat; Mm, mouse; Md, opossum; Ac, green anole lizard; Xt and XL, frogs; Dr, zebrafish; Fr and Tn, puffer fish; Ol, medaka; Ga, stickleback. * indicates indentical bases in both sequences.(0.24 MB TIF)Click here for additional data file.Figure S6Junction analysis of theTSPAN14 group. (A) Protein alignment of the exon protein sequences between introns 4b and 6 of several TSPAN14 tetraspanins. Intron/exon junctions are indicated with arrowheads. (B) Clustal W DNA alignment of exons sequences in blue and partial intron sequences in black of the TSPAN14 sequences from fish stickleback and medaka. (C) Alignment of sequences from zebrafish, fugu, medaka and stickleback.Dashes indicate intron sequence not shown. Species are designated as in S5.(0.24 MB TIF)Click here for additional data file.


Ostreococcus, which exhibit highly constrained intron sequences through most of the intron-poor genome, but not in one much more intron-dense genomic region. Second, we find evidence that ancestral genomes contained highly variable branch point sequences, similar to more complex modern intron-rich eukaryotic lineages. In addition, ancestral structures are likely to have included polyT tails similar to those in metazoans and plants, which we found in a variety of protist lineages. Intriguingly, intron structure evolution appears to be quite different across lineages experiencing different types of genome reduction: whereas lineages with very few introns tend towards highly regular intronic sequences, lineages with very short introns tend towards highly degenerate sequences. Together, these results attest to the complex nature of ancestral eukaryotic splicing, the qualitatively different evolutionary forces acting on intron structures across modern lineages, and the impressive evolutionary malleability of eukaryotic gene structures.The presence of spliceosomal introns in eukaryotes raises a range of questions about genomic evolution. Along with the fundamental mysteries of introns' initial proliferation and persistence, the evolutionary forces acting on intron sequences remain largely mysterious. Intron number varies across species from a few introns per genome to several introns per gene, and the elements of intron sequences directly implicated in splicing vary from degenerate to strict consensus motifs. We report a 50-species comparative genomic study of intron sequences across most eukaryotic groups. We find two broad and striking patterns. First, we find that some highly intron-poor lineages have undergone evolutionary convergence to strong 3′ consensus intron structures. This finding holds for both branch point sequence and distance between the branch point and the 3′ splice site. Interestingly, this difference appears to exist within the genomes of green alga of the genus  Ostreococcus, in which the single intron-rich genomic region shows far more variable intron sequences than in the otherwise intron-poor genome. We suggest that different concentrations of spliceosomal complexes may explain these differences. In addition, we report the existence of 3′ polyT tails in diverse eukaryotic protists, suggesting that this structure is ancestral. Together, these results underscore the complexity of ancestral eukaryotic splicing, the qualitatively different evolutionary forces acting on intron sequences in modern eukaryotes, and the impressive evolutionary malleability of eukaryotic genes.The spliceosomal introns that interrupt eukaryotic genes show great number and sequence variation across species, from the rare, highly uniform yeast introns to the ubiquitous and highly variable vertebrate intron sequences. The causes of these differences remain mysterious. We studied sequences of intron branch points and 3′ termini in 50 eukaryotic species. All intron-rich species exhibit variable 3′ sequences. However, intron-poor species range from variable sequences, to uniform branch point motifs, to uniform branch point motifs in uniform positions along the intronic sequence. This is a more complex pattern than the clear relationship between intron number and 5′ intron sequence uniformity found previously. The correspondence of sequence uniformity and intron number extends to species of the green algal genus  Bigelowiella natans nucleomorph Spliceosomal introns are genomically-encoded sequences that are removed from RNA transcripts by the spliceosome, a massive RNA-protein complex. The spliceosome and spliceosomal introns are common and ancestral to eukaryotes Cryptosporidium, 84% of introns begin with the most common sixmer GTAAGT, enabling close complementary base pairing with the U1 RNA of the spliceosome , a terminal 3′ AG (the “acceptor”), and a degenerate few nt “branch point” site, lying somewhere within the intron. In other species, sequences are more conserved. For instance, in the apicomplexan parasite Saccharomyces cerevisiae) to several lineages in which previous studies have failed to find branch points Clear differences are also observed in other structures. For instance, branch points vary across species from a highly conserved seven-mer (TACTAAC) in hemiascomycetous yeasts  is found in all fully-sequenced hemiascomycetous yeasts, with 66–100% of introns in each species containing this motif. Extended branch points of up to eight bases were found in some species  probable introns  contain CTRACA. Finally, excluding two questionable predicted introns . Thus all lineages with strong branch points conformed to a general WCTRAYN consensus.We first studied intron branch points in available genomes from intron-poor species . Many of species . We alsorons see  in the frons see , all 13 Cryptosporidium parvum failed to reveal a potential branch point site. The case for the few introns of the Guillardia theta nucleomorph is less clear. 14/16 predicted G. theta introns contain a YAAY branch point-like sequence between 2 and 6 nucleotides from the acceptor AG (compared to only 2/16 that contain such a motif within the next 10 positions (7 to 17)). Intriguingly, a second YAAY signal exists further upstream – 8/16 introns have a YAAY 24–28 nt from the 3′ terminus. The single known intron in each of the sequenced Trypanosomatid genomes does not show a clear branch point structure; however it is of note that despite having few cis-spliced introns, these species do have large numbers of 3′ splicing boundaries due to the ubiquity of spliceosome-mediated spliced leader trans-splicing However, in at least one intron-poor lineage we failed to find such clear branch point sequences. Visual inspection and computational analysis (see below) of the intron-poor apicomplexan parasite AY or CTRAYN with varying levels of two-fold degeneracy . For instance, the ACTAAC hexamer common to all C. merolae introns has no degeneracy, whereas the CTRACA motif of G. lamblia has one degenerate site, and the general consensus WCTRAY contains three two-fold degenerate sites. As with the other sites, either one or two nucleotides at a time were allowed at the final “N” site .We next studied conservation of branch points in 33 more intron-rich species. We studied the occurrence of motifs conforming to the consensus branch point WCTRFor each species, and for each level of degeneracy (zero to three degenerate sites), we calculated the fraction of introns that contain the same motif within the last 200 nt of the intron see . Then, fAT) in Drosophila melanogaster or 10.97% (ACTGAC) in Aspergillus fumigatus), and less than 36% allowing one degenerate site. This relation between intron numbers and branch point consensus strength is underscored in G. thetha NM and C. parvum (red asterisks)): intron-rich/weak branch point consensus and intron-poor/strong branch consensus . The pattern becomes even more striking when intron sequences are divided along the lines of the previously reported genomic heterogeneity of O. lucimarinus, predicted introns in O. tauri show far lower conservation of branch points , show strikingly similar patterns  in T. vaginalis and G. lamblia; Next, we studied acceptor sequence conservation within genomes. The 3′ sequences of spliceosomal introns generally show more similarity across species, with most species showing a terminal YAG, sometimes preceded by a poly-pyrimidine tract , althougpatterns . The thrO. lucimarinus introns show a preference for CGCAG, though notably weaker than that found in the in the other three species . It is interesting then that both O. lucimarinus and the constrained BP-AG distance species prefer a C at position –5 and a R at position –4. We were unable to find an explanation involving snRNA sequences for this preference. Finally, C. elegans introns also show stronger 3′ consensus, matching TTTCAG with nucleotides -6 and -5 significantly more conserved  showing large variations in BP-AG length and five species showing clear BP-AG constraint  showed a strong sequence consensus at the 3′ end of the sequence, with 80.0% having a G at position -4 , and 50.0% having an A at position -5 . However no clear general trend towards stronger boundaries for short BP-AG introns was observed: introns with a BP-AG distance of 7 nt did now show a stronger 3′ consensus than introns with larger BP-AG distances. Y. lipolytica did not show differences in strength of 3′ sequence consensus for different BP-AG distances.Intriguingly, in Y. lipolytica, the most common BP-AG distance is 2 nt, compared to 3 in C. guilliermondii, 4–5 for C. lusitaniae, 6–8 for E. gossypii and 7–8 for D. hanseii.Mapping of BP-AG distances across hemiascomyces shows a complex phylogenetic pattern . The fivIt seems unlikely that a species with a strong preference for a certain BP-AG distance would convert to a different distance, since this would require indels of a very specific length occurring in dozens of already constrained introns. It seems more likely that this condition reflects ancestrally relatively unconstrained BP-AG distance, and convergent evolution of constrained BP-AG distances in different lineages.Toxoplasma gondii, H. sapiens, S. pombe, A. fumigatus, and A. thaliana), we compared 5′ splice site strength and branch point conservation between introns putatively orthologous to introns retained in at least one intron-poor species and the total set of introns in these species. Significant differences for both intron structures were found for A. fumigatus and T. gondii introns  sequences and change of retained intron sequences to consensus boundaries. However, the relative impact of these two factors is unknown. We attempted to address this issue by identifying introns in intron-rich species that were present at the exact homologous position to introns in any available intron-poor species, and thus are likely to be ancestral to both species. If strong consensus sequences in intron-poor species are due to differential retention of introns with conserved ancestral sequences, it is possible that orthologous introns in intron-rich species could retain some of this signal. For each intron-rich species (the apicomplexan  introns .T. gondii-C. parvum and A. fumigatus-hemiascomycetes diverged both many hundred million years ago) and associated large amounts of sequence change. Therefore, the finding of a positive signal for any of these comparisons is surprising and intriguing. To further test whether the observed stronger intron consensus sequence signal in intron-poor organisms could truly reflect retained greater boundary strengths from ancestor, we further divided the orthologous set into those introns shared with a more closely related intron-poor species  and those shared only with distantly related species. Whereas the former could conceivably retain some specific ancestral signal due to lack of change, the second set represent divergences dating back upwards of a billion years ago to early eukaryotic evolution, seemingly precluding similarities in trends across individual intron boundary strengths representing lack of sequence change since that time. Unexpectedly, we observed that this second subset of introns, shared only with older relatives, show stronger signal than those introns shared with the closest intron-poor ancestor in T. gondii (A. fumigatus distantly-related group for comparison).Despite this analysis being perhaps the most direct way to test the hypothesis of preferential intron retention available, it is deeply undermined by the great phylogenetic distances between intron-poor species and even their closest relatives , they do not more closely resemble Y. lipolytica boundaries (consensus GTGAGT). Indeed, we observe the opposite trend: only 15.2% (26/171) of the A. fumigatus introns shared with Y. lipolytica have a G in position +3, whereas in the whole set of A. fumigatus introns, 21.4% have a G in that position.Another argument also argues that these intron subsets' stronger boundaries reflects not retention of ancestral boundary strength, but something else: Finally, we studied the distribution of characteristic intronic polyT motifs along intron length. For each species, we calculated frequencies of intronic minimal “polyT motifs”  polyT motifs concentrate near the intron terminus  polyTs are concentrated in the last ∼10 nt, and are underrepresented 10–15 nt from the terminus. This pattern could suggest a more 3′ branch point position, although branch points in these species are difficult to determine Plasmodiophora brassicae seems consistent with this broad pattern; however, the small number of available introns renders confident conclusions difficult.Almost all species conformed to one of 3 broad patterns, which tend to be conserved within large phylogenetic groups . For theterminus . The 5′ C. parvum as well as the more moderate T-rich introns of hemiascomycete yeasts  and in the intron-poor fungus Ustilago maydis , suggests that the ancestral intronic structure had polyT motifs concentrated between the BP and the terminal AG, and that broader polyT distributions evolved early in fungal evolution.We report convergent evolution of strong branch point consensus sequences and constrained branch points positions in eukaryotic lineages ranging from fungi to plants to protists. These observations join our previous findings of convergent evolution to strong 5′ splice site boundaries However, different intron sequence characteristics show different degrees of co-evolution with intron number. Whereas strong splice sites show a one-to-one correspondence with low intron number across species S. cerevisiae, shows considerable alterations in the mechanisms and protein components of its spliceosome Difference in levels of intron structure constraint likely is associated with  changes in the spliceosomal machinery that have led to increased requirements for adherence to consensus sequences Notably, we failed to find intermediate stages. 5′ splice site strength shows a clear gap between intron-poor lineages (at least 5 bits of information content), and intron-rich (1–4 bits) For 5′ splice sites, this lack of intermediates is consistent with some qualitative difference in the selective regimes acting within intron-poor and intron-rich species, leading to a lack of intermediate strengths. For branch point sequences and positions the case is more subtle. Do weak branch points in some intron-poor lineages reflect an ongoing process, or are these lineages somehow refractory 3′ intron convergence? Repeated evolution of constraint in hemiascomycetes may suggest an ongoing process, however in this case we might expect to observe intermediate stages. Possibly, once put in motion, intron structure constraint proceeds rapidly, which could explain the lack of observed intermediates.Paramecium tetraurelia, the nucleomorph of B. natans, and Dicyemids, so-called mesozoans), whereas introns in very intron-poor species are not particularly short  or simple (with short intergenic regions flanking a modest complement of nearly intronless genes), many genomes defy such straight-forward characterization. Intron-exon structures provide a clear example: the three genomes with the shortest known intron structures each have relatively high intron densities (ly short .Dicyemid introns give the weakest known score for 5′ intron boundaries (0.5 bits) and 5′ splice sites of P. tetraurelia and B. natans are largely restricted to GT(A). These three lineages also show no signature of branch points (E. cuniculi introns (35.8 nt on average) and many T. vaginalis introns (∼25 nt) are short, yet both show conserved 5′ splice site and branch point sequences . This pattern underscores the importance of intron number, and not simply genome reduction, in driving the emergence of strong consensus sequences.Interestingly, these two classes of reduced lineages appear to show opposed patterns of intron evolution. Whereas intron-poor lineages tend towards highly-constrained intron sequence elements, short-intron lineages seem to show very weak sequence constraint. Available h points . This doThe finding of a general inverse correspondence between intron number and splicing signals' strength is unexpected and remains unexplained. Previously, we suggested that in intron-poor species, selection against aberrant splicing of cryptic splice sites would drive changes in the spliceosome towards stricter splicing requirements, which would in turn drive sequence change in (or loss of) non-conforming introns Ostreococcus lucimarinus provides a rare natural experiment to test this hypothesis. While genes in the majority of the genome exhibit very low intron densities, the genes spanning roughly half of chromosome 2 show a much higher density, well within that of “intron-rich” species The genome of the ultra-small green alga O. tauriOstreococcus 5′ splice sites and branch points (GTGCGTG and GACTGACG in O. lucimarinus) could have thwarted their identification in the previous study, and this possibility is worth exploring. Alternatively (though more difficult to test), a single core RNA splicing machinery could associate with different sets of protein components in distinct spliceosomes with different splicing activities.One possibility is that the two intron sets are serviced by different spliceosomes (as is the case of U2 and U12 in different eukaryotic lineages). However, a computational search turned up only single copies of spliceosomal RNA components in congenitor O. lucimarinus contains a single spliceosome, strongly suggesting that the constrained introns through most of the genome, as well as those of intron-poor species that they so closely resemble, do not reflect inherent changes in the spliceosomal machinery. A simpler possibility is that differences in local (in O. lucimarinus) or cellular (in other species) concentrations of spliceosomal complexes is the driving factor. It seems possible or even likely that spliceosomal complexes in intron-poor species are downregulated. Such downregulation could reflect either selection to reduce incorrect splicing of truly exonic sequence , or could be favored by reducing energetic costs associated with transcription, processing, and translation of spliceosomal components. If so, the lowered concentration of spliceosomal components would require stronger binding affinity of individual splice sites to corresponding snRNAs for efficient splicing, which would in turn drive the evolution of stronger boundaries (or intron loss). Differential local concentrations across genomic regions in Ostreococcus could be maintained if spliceosomes were preferentially recruited to the intron-rich genomic region. This scenario is similar to our previous hypothesis in invoking a tradeoff between the costs of efficient splicing of weak boundaries (maintenance of high spliceosome concentration) and the costs of mis-splicing, which we argue would likely be different in intron-rich and -poor species. This hypothesis makes the testable prediction that spliceosomal components should show reduced expression in intron-poor species relative to intron-rich species.More likely, however, Another hypothesis concerning the concentration of spliceosomal complexes, suggested to us by Tony Russell, sees a very different role for selection. A predicted consequence of increasing the length of snRNA-intron element base-pairing interactions is a reduction in the overall rate of splicing, simply due to a tighter association between intron and spliceosome. Such a decrease in splicing rate will be tolerated in intron-poor species if spliceosomal components are in excess relative to the number of introns. However, in intron-rich species spliceosomal components may not be in excess, in which case stronger base-pairing between intron and snRNAs could be disfavored. Notably, these two hypotheses make qualitatively different predictions. Whereas the latter hypothesis predicts that strong boundaries would be disfavored in intron-rich species, the former predicts that they would as or more fit than weaker boundaries. Comparative analysis of closely related species to test these predictions is underway.Two factors could drive evolutionary convergence to strong intron boundaries: sequence changes in existing intron sequences to consensus sequences, and preferential loss of non-consensus introns. The relative contributions of these factors may depend on the precise evolutionary pathway from  genomes with many introns with weak boundaries and relatively lax splicing requirements to fewer introns with strong boundaries and stricter requirements.−9 per year, comparable to some estimated mutation rates First, widespread (mostly random) intron loss could lead to selective conditions favoring the evolution of a spliceosome with stricter sequence requirements for splicing , the three will proceed in tandem. Differences in the relative contributions of the three phenomena will depend on mutation rates and selective coefficients for different kinds of changes , which may vary considerably across times and lineages.The convergent U2 intron structures observed here and elsewhere in some intron poor species – strong 5′ and branch point consensus sequences, constrained BP-AG length – are strikingly reminiscent of the U12-type intron structures found across a wide variety of lineages We find different patterns of polyT motif distribution along the introns in different lineages, which likely reflect differences in polyT functionality and in how polyT-binding factors regulate splicing in these lineages. Indeed, the differential conservation and evolution of spliceosomal proteins binding polyT tracts  in each species is likely to determine the position of polyT motifs along introns and what role these motifs play in splicing regulation.Among these locations, we find that the so-called “polyT tail”, spanning from a position likely corresponding to the branch point to the 3′ intron terminus, is common to a wide variety of groups ranging from plants to animals to various, widely diverged, protists, strongly suggesting that the existence of a polyT tail may be ancestral. Furthermore, we show that, as in the case of 5′ splice sites, strong branch point site consensus have evolved independently in only intron-poor species, whereas all intron-rich species have weak branch point sites. Since a wide variety of studies have shown that eukaryotic ancestors have harbored relatively high intron numbers et al. These conclusions extend an excellent recent study from Schwartz The presence of these intronic features, polyT tail and weak branch point sites, in the eukaryotic ancestors adds to the developing picture of the spliceosomal system in early eukaryotes – with highly developed spliceosome, weak 5′ and branch point sequences, a polyT tail and complex splicing patterns One limitation of the data deserves comment. We analyze annotated intron sequences, making our analysis subject to the quality of available annotations. Problems with the annotations may very directly influence the characteristics studied here, since for instance more consensus-like sequences are more likely to be identified as introns. Such concerns may also affect comparative analysis if the annotation efforts for different species are differentially sensitive to different kinds of introns.However, it is very unlikely that such limitations are likely to drive the qualitative differences we see here. For a “weak” species with for instance 30% of its true introns exhibiting the same branch point to be incorrectly identified as a “strong” species  would require that the vast majority of its non-consensus introns have gone unannotated. For the reverse to occur, it would be required that there were so many falsely predicted introns that it had drowned out the signal almost entirely. Thus, while it is important to point out that our results are likely not accurate to the second decimal place due to problems with annotations, such problems are very unlikely to be driving the large qualitative differences observed.E. cuniculi may not in fact be introns at all: both are a multiple of 3 basepairs and lack inframe stop codons; these two introns have the weakest 5′ boundaries (matching the consensus (GT)AAGT at 1 and 2 out of 4 positions, compared to at least 3 matches for the other 13 introns); and one intron has similar sequences at the two boundaries, suggesting that this intron prediction could reflect a reverse transcriptase artifact in EST preparation E. cuniculi represented the only intron-poor species lacking very clear strong boundaries. Excluding these two questionable introns, E. cuniculi has a donor site information content of 6.2 bits, comparable to the other intron-poor lineages.Possibly, the most important impact of annotation errors could be to reduce the signal in very intron-poor species. For instance, further scrutiny suggested that 2 of the 15 predicted introns in These results attest to plasticity of spliceosomal intron structures through the history of eukaryotes. The availability of large numbers of eukaryotic genomes now allows comparative analysis of an increasing diversity of genomic structures. Present and previous works have provided an increasingly detailed picture of the patterns and determinants of intron-exon structures, one of the hallmarks of eukaryotic genome organization. Definitive identification of the causes of highly regular 3′ intron structures awaits the identification of additional lineages exhibiting this pattern.http://www.ncbi.nlm.nih.gov) or Ensembl database (http://www.ensembl.org) for 7 metazoa: human (Homo sapiens (NCBI 36 Ensembl 38.36)), zebra fish (Danio rerio (Zv5 Ensembl 38.35e)), Strongylocentrotus purpuratus (AAGJ00000000.2), Drosophila melanogaster (release 4.1), mosquito (Anopheles gambiae (AgamP3 Vectorbase 38.3a)), Caenorhabditis elegans (WS150 Wormbase 38.150a), Ciona intestinalis (CINT1.95); 16 fungi: Aspergillus fumigatus Af293 (AAHF00000000.1), Aspergillus nidulans FGSC A4 (AACD00000000.1), Neurospora crassa OR74 A (AABX00000000.1), Gibberella zeae PH-1 (AACM00000000.1), Yarrowia lipolytica CLIB122 (CR382127-31.1), Saccharomyces cerevisiae YJM789 (AAFW00000000.1), Candida glabrata CBS138 (provided by J. E. Stajich), Candida albicans SC5314 (provided by J. E. Stajich), Kluyveromyces lactis NCYC 2644 (AADM00000000.1), Eremothecium gossypii ATCC 10895 (AE016814-20.1), Debaryomyces hansenii CBS767 (CR382133-9.1), Schizosaccharomyces pombe 972h (AL672256-8.1), Ustilago maydis 521 (AACP00000000.1), Cryptococcus neoformans B3501-A , Encephalitozoon cuniculi GB-M1 , Rhizopus oryzae RA 99-880 (AACW00000000.2); 2 amoebae: Dictyostelium discoideum AX4 (AAFI00000000.1), Entamoeba histolytica HM-1:IMSS (AAFB00000000.1); 2 plants: Arabidopsis thaliana , Oryza sativa (Build 2.1); 3 green algae: Chlamydomonas reinhardtii (JGI Chlamy 3.0), Ostreococcus lucimarinus CCE9901 , Ostreococcus tauri OTH95 ; 6 aplicomplexans: Plasmodium falciparum HB3 (AANS00000000.1), Plasmodium yoelii yoelii (AABL00000000.1), Plasmodium chabaudi (CAAJ00000000.1), Theileria parva Muguga (AAGK00000000.1), Cryptosporidium parvum Iowa (AAEE00000000.1) and Toxoplasma gondii RH (AAQM00000000.1); and 2 heterokonts: Phytophthora ramorum strain Pr102 (AAQX00000000.1) and Phytophthora sojae strain P6497 (AAQY00000000.1).GenBank fully-sequence genome annotations were downloaded from NCBI webpage : Brassica oleracea (1096 introns in 314 genes). Introns for Paramecium tetraurelia were extracted from nucleotide links of NCBI taxonomy (1082 introns in 401 genes). Introns for Candida guilliermondii (13 introns), Candida lusitaniae (10 introns) and Candida tropicalis (34 introns) were provided by J. E. Stajich.Available gene sequences for unpublished or incomplete genomes were downloaded one by one as CDS annotations from NCBI web page  shows a clear correspondence between the measure of strength as the percentage of introns with the most common sequence motif  and as information content, used broadly in the literature, with a coefficient of correlation between both variables for the species included in this study is r = 0.96.http://weblogo.berkeley.edu/logo.cgi). To better characterize the evolution of BP-AG constraint in Y. lipolytica, we further studied BP-AG distance in the 9 hemiascomycetes species. In all hemiascomycetes species, the vast majority of introns contained a single TACTAAC sequence, used as the branch point. The BP-AG distance was defined as the number of base pairs (Nn) for 5′TACTAAC|Nn|AG-exon3′.We aligned the final 20 nt of each intron for each species using WebLogo (http://genes.mit.edu/pictogram.html).For 5′ splice sites we used a similar methodology as described in O. lucimarinus from NCBI on April 4th, and performed standalone BLASTN searches for each predicted intron-containing O. lucimarinus gene against the ESTs. Preliminary confirmed introns were identified as those in which an EST hit with >60 bits and with >90% sequence identity spanned the intron position (reaching at least 3 nt on each side of the intron position). Each of these introns was then analyzed by eye to exclude non-canonical intron positions as well as those for which sequence similarity between the regions spanning the 5′ and 3′ predicted splice boundaries were consistent with a template-switching artifact during the reverse transcription step of the EST library preparation We downloaded available EST sequences for C. parvum for T. gondii; Y. lipolytica (chosen as the representative hemiascomycete due to its greater intron density (3 times higher than S. cerevisiae), in order to increase sample size) for S. pombe and A. fumigatus; and the C. merolae and the G. theta nuclemorph for A. thaliana. Due to the lack of related intron-poor species, H. sapiens introns were divided into only two groups.For each species considered, databases of intron/exon structures of predicted gene transcripts were prepared from the genome annotations. Homologs were identified by one-way BLASTP searches of intron-containing genes from intron-poor species against predicted proteomes from intron-rich species. Putatively orthologous introns were identified as those present at identical alignment positions (position and phase) in both species. Related species were defined as: We used the minimal definition for polyT motif, defined as six consecutive nucleotides containing at least 3 T's and no A's


Advances in the science of toxicogenomics have opened the door to major advances in our understanding of the molecular basis of environmental pathogenesis and the role of environmental factors in human disease. This report summarizes major findings in the laboratory defining the molecular basis of L1 retroelement activation in mammalian cells and the architecture of gene regulatory networks involved in phenotypic control. Mammalian retroelements are ubiquitous genetic elements that amplify themselves in the mammalian genome via reverse transcriptase and that modify the biology of cells by reinsertion and possibly by modulation of RNA and microRNA biology. Eukaryotic retroelements rely on reverse transcriptase to transpose via an RNA intermediate to a different location. The general structure of a "typical" retroelement includes two open reading frames and 5' and 3' untranslated regions involved in regulation of transcriptional activity and stability.In mammals, up to 42% of the genome is believed to be comprised of retrotransposons; this translates into millions of elements most of which are not able of self replication or so called "dead". Of particular interest are members of the LINE-1 (L1) family, abundant elements in the human and mouse genomes believed to have contributed extensively to genomic evolution and to participate in reprogramming of genetic programs during the course of development and pathogenesis. L1s are targeted for epigenetic silencing during early embryonic development and remain inactive in most cells and tissues upon completion of cellular differentiation. Understanding how retrotransposons and their host genomes have coevolved and the molecular mechanisms that help to optimize mutual survival is still in its infancy. The ENCODE Project shows that protein coding DNA makes up barely 2% of the overall genome, yet 80% of 30 million bases analyzed to date show evidence of being expressed) [Within the context of environmental disease, research in my laboratory over the past 15 years has identified retroelements as molecular targets of environmental carcinogens . The abiEarlier studies focused on the transfection of HeLa cells with a human L1 element tagged with a Neo cassette in the 3' untranslated region in order to evaluate the L1 mobilization and reinsertion upon challenge with low doses of benzo(a)pyrene, a widespread environmental carcinogen and atherogen. The construct used has two promoters; P1 and P2 drive transcription in opposite directions, a β-globin intron inserted into Neo cassette in opposite orientation, splicing donor and acceptor sites and G418 resistance gene . The speLittle is understood about the complex biology of L1 elements. To begin to elucidate genetic interactions of L1 computational biology studies have been initiated to elucidate the structure of L1 gene networks and to define biological interactions of relevance to the regulation of cellular phenotypes. Given the abundance of retroelements in the mammalian genome and the extensive redundancy associated with elements that are no longer active, our initial studies focused on the study of an L1 element previously identified in the mouse genome and localized to chromosome 4 as a "unique" element . The disIn these experiments, AHR was downregulated using a specific silencing (si)RNA specific and the relative expression of genes within the network was measured by RT-PCR in cells treated with BaP to reactivate L1. siRNA targeting the AHR blocked L1 inducibility and modified the expression of other genes within the predicted biological network. These findings established a role for members of the PAS superfamily of proteins in the regulation of retroelement reactivation profiles in mammalian cells. The specificity of these interactions was confirmed in subsequent experiments showing that genetic or pharmacological targeting of the AHR blocks L1 inducibility in multiple cell types . In otheGene transcription is controlled via sequence elements that are recognized and bound by transcription factors and by chromatin modifications at the DNA and histone levels. Transcriptional regulation is often combinatorial in nature and therefore a major goal in studies of genetic regulation is the identification of combinatorial interactions that cooperate in the regulation of gene expression and that constitute a recurrent regulation motif for environmental interference. Work by others established that global hypomethylation of CpG islands leads to L1 reactivation, chromosomal instability and elevated mutation rates and that the E2F/Rb macromolecular complex binds CpG islands to regulate gene expression. Of interest to the regulation of L1 is that the E2F/RB complex associates with histone methyltransferases and histone deacetylases to regulate gene expression. On the basis of these important biological relationships we have hypothesized that L1 reactivation by environmental stress is associated with modulation of DNA and histone methylation and differential recruitment of chromatin modifying complexes onto the L1 DNA template. Preliminary studies support this hypothesis and shown enrichment of L1 DNA amplification in mouse and human cells using E2F1 and E2F4 antisera compared to non-specific antibody. In fact, Rb-deficient cells appear to exhibit exacerbated L1 expression upon chemical stress.Studies of L1 biology using toxicogenomics approaches have shed light into the complex biology of retroelements within the human and murine genomes. These studies are helping to bridge the gap by taking advantage of mathematical models to achieve a formal and unified description of biophysical phenomena. Our approach also emphasizes the importance of biological validation of theory and computation and the need for experimentation to rely upon, and be guided by, theory and computation.The CoD algorithm has been successfully used to define structural and functional determinants of a genetic regulatory network of L1. RNA Pol II and a per homolog were identified computationally as primary attractors within the L1 network, and AHR found to predictably regulate genes within the L1 regulatory network. E2F/Rb complexes may participate in epigenetic regulation of L1 via nucleosomal histone modifications and recruitment of HDACs 1 and 2. As such, L1 reactivation may be due to failure of co-repressor protein recruitment by Rb reflecting loss of histone epigenetic marks and histone acetylation.Functional genomics approaches can help unravel the complexity of biological systems. The integration of technological advances in the fields of genomics, computational biology and mathematics has led to emergence of systems biology as a means of mapping and managing the complex interactions that govern biological systems and the discovery of novel biomarkers with diagnostic and clinical applications.The author declares that they have no competing interests.


Prior studies demonstrated that human ORF1p, L1 RNA, and an ORF2p-encoded reverse transcriptase activity are present in ribonucleoprotein (RNP) complexes. However, the inability to physically detect ORF2p from engineered human L1 constructs has remained a technical challenge in the field. Here, we have employed an epitope/RNA tagging strategy with engineered human L1 retrotransposons to identify ORF1p, ORF2p, and L1 RNA in a RNP complex. We next used this system to assess how mutations in ORF1p and/or ORF2p impact RNP formation. Importantly, we demonstrate that mutations in the coiled-coil domain and RNA recognition motif of ORF1p, as well as the cysteine-rich domain of ORF2p, reduce the levels of ORF1p and/or ORF2p in L1 RNPs. Finally, we used this tagging strategy to localize the L1–encoded proteins and L1 RNA to cytoplasmic foci that often were associated with stress granules. Thus, we conclude that a precise interplay among ORF1p, ORF2p, and L1 RNA is critical for L1 RNP assembly, function, and L1 retrotransposition.The average human genome contains a small cohort of active L1 retrotransposons that encode two proteins (ORF1p and ORF2p) required for their mobility ( Long Interspersed Element-1 (LINE-1 or L1) sequences are the predominant class of autonomous retrotransposons in the human genome and comprise an astounding 17% of human DNA. Although the majority of L1s are considered to be “dead,” an average human genome contains ∼80–100 active L1s. Active L1s encode two proteins (ORF1p and ORF2p) that are required for mobility (retrotransposition) by a “copy and paste” mechanism termed target-site primed reverse transcription. Prior experiments suggested that ORF1p, ORF2p reverse transcriptase activity, and L1 mRNA associate in ribonucleoprotein (RNP) particles and that RNP formation is a necessary step in L1 retrotransposition. However, the difficulty in detecting ORF2p from engineered human L1s has prevented a thorough understanding of its role in L1 retrotransposition. Here, we have exploited epitope and/or RNA–tagging strategies to detect and characterize a “basal” RNP complex from engineered human L1s. We also expanded on previous studies and characterized how mutations in conserved functional domains of ORF1p and ORF2p can adversely affect L1 RNP formation/function. Finally, our strategy allowed us to detect the L1–encoded proteins and L1 RNA in cytoplasmic foci. Thus, we have developed and employed a system to gain greater understanding of LINE-1 retrotransposition at the molecular level. The amino acid sequence of the ORF1p amino-terminus is poorly conserved among mammalian L1s, but it is predicted to form a coiled-coil or α-helical domain that is important for ORF1p multimerization 261–262 in human L1) can decrease ORF1p RNA binding or mouse ORF1p nucleic acid chaperone activity 261–262, and YPAKLS282–287, respectively) both compromise the ability of ORF1p to localize to RNPs and severely reduce L1 retrotransposition efficiency The carboxyl-terminus of ORF1p contains amino acid residues that are conserved among mammalian L1s Biochemical and genetic studies have revealed that human and mouse ORF2 are translated by an unconventional mechanism Epitope-tagging systems and enzymatic assays have been developed to facilitate detection of L1 ORF1p and ORF2p RT activity from engineered wild-type and mutant human L1s Previous studies have examined the co-localization of L1 ORF1p and L1 RNA in RNPs derived from cells transfected with epitope-tagged wild-type or mutant human L1 expression constructs mneoI), subcloned into a pCEP4 episomal expression vector, and were assayed for retrotransposition in cultured human HeLa cells vs. pJM101/L1.3). Similarly, the inclusion of the MS2 stem loop sequences into the L1 3′UTR did not dramatically affect L1 retrotransposition efficiency (vs. pJM101/L1.3), although we did observe an approximate 2.7 fold reduction in L1 retrotransposition efficiency from a construct containing both the protein and MS2 tags (vs. pJM101/L1.3). As a negative control, we demonstrated that a construct containing a missense mutation in the putative L1 RT active site  was defective for retrotransposition , which most likely is due to the additional amino acids imparted by the T7 epitope tag. Controls revealed that ORF1p was not detected from a construct that lacks ORF1 . The ∼170 kDa product corresponds to the predicted size of ORF2p (∼150 kDa) plus the predicted size of the TAP tag (∼19 kDa) vs. pJM101/L1.3). Consistent with previous genetic studies, TAP-tagged ORF2p expression was greatly diminished by introducing a stop codon in ORF1 , we transfected HeLa cells with pAD2TE1, selected for transfected cells, and isolated RNPs by ultracentrifugation see  [16], , , [2activity  [17]. Th230A). We also observed a severe reduction of ORF2p, as it was only detected upon longer film exposures (data not shown), and a strong decrease of LEAP activity in RNPs derived from the tested cysteine-rich domain mutant . Finally, the leucine zipper/C-domain double mutant  displayed both a reduction of ORF1p in RNPs and a concomitant decrease in LEAP activity  and/or stress granules. Consistent with previous analyses, ORF1p and ORF2p associated with an Ago2-GFP fusion protein that localizes both to P-bodies and stress granules  and carboxyl-terminal RNA binding domain  led to a reduction in the number of L1 cytoplasmic foci (235–238AAAA), an additional carboxyl-terminal domain mutant , and the putative leucine zipper domain/carboxyl-terminal RNA binding domain double mutant . By comparison, mutations in the putative ORF1p LZ domain  or mutations that affect the nucleic acid chaperone activity of mouse ORF1p  had little effect on L1 cytoplasmic foci formation   .trans-complementation experiments in cis, and that this association allows L1 cytoplasmic foci formation in the absence of ORF2p.The above data suggest that the ability of ORF1p to bind L1 RNA is critical for L1 cytoplasmic foci formation . Consistin vivoORF2p has been notoriously difficult to detect from engineered human L1s in cultured cells. It has been hypothesized that human ORF2p is translated at low levels when compared to ORF1p and/or may be an unstable protein, which might help explain why it has evaded detection Why our approach allows the ready detection of ORF2p expressed from engineered human L1 constructs requires further study. Experiments conducted with anti-TAP antibodies consistently yielded more robust detection of ORF2p when compared to anti-HA or anti-ORF2p antibodies. Thus, the inclusion of a large carboxyl-terminal tag, such as the TAP-tag, might stabilize ORF2p. However, since engineered L1 constructs containing either the TAP or HA epitope tags on the carboxyl-terminus of ORF2p remain retrotransposition-competent, the strategy described here allows a way to both directly study the expression of ORF1p and ORF2p from a bicistronic transcript and establishes an experimental platform to determine how each protein interacts with L1 RNA. Furthermore, this strategy now allows a comprehensive means to assess how mutations in ORF1p and/or ORF2p affect L1 RNP biogenesis and/or L1 retrotransposition.261–262AA) markedly reduced ORF1p levels in RNPs, but did not noticeably affect ORF2p accumulation or LEAP activity  or RRM  domains led to a reduced amount of ORF2p in the RNP fraction, as well as a decrease in LEAP activity. The L93,100,107,114V mutations reside in the N-terminal coiled-coil domain of ORF1p and could potentially alter the structure of the protein. Similarly, the NLR157–159ALA mutations reside near coiled-coil domain/RRM junction and structural studies indicate that a hydrogen bond between N157 and D252 is important for correct folding of the RRM domain 93,100,107,114V, L93,100,107,114V/RR261–262AA, and NLR157–159ALA mutants significantly affect ORF2p translation, since our preliminary data indicate that each mutant can serve as a “driver” in a genetic-based trans-complementation assay .Our studies further suggest that an interplay exists between ORF1p, ORF2p and L1 RNA that is critical for proper L1 RNP formation/function . For exa230A) had no discernable affect on the ability of ORF1p to accumulate in RNPs when compared to a wild-type control construct. However, this mutation consistently led to a slightly reduced amount of ORF2p in RNPs, which correlated with a lower LEAP activity 230A mutant consistently exhibited lower levels of endonuclease-independent L1 retrotransposition in Chinese Hamster Ovary cells that are deficient in the non-homologous end-joining pathway of DNA repair when compared to a D205A endonuclease domain mutation As expected, a mutation in the endonuclease domain of ORF2p  did not have a major effect on the ability of ORF1p to accumulate in the RNP fraction. However, these mutations led to a reduced amount of ORF2p in the RNP fraction and a concomitant decrease in LEAP activity when compared to a wild-type control construct. How mutations in the C-domain affect ORF2p accumulation in RNPs requires further study; however, it is possible that these mutations alter the ability of ORF2p to interact with L1 RNA and/or host factors that are important for the biogenesis of L1 RNPs  or carboxyl-terminal domain of ORF1p  reduced L1 cytoplasmic foci formation 93,100,107,114V) or mutations analogous to those that adversely affect the nucleic acid chaperone activity of mouse ORF1p , which are not predicted to inhibit L1 RNA binding, or mutations in ORF2p had little effect on L1 cytoplasmic foci formation A second assay allowed us to determine how mutations in the L1-encoded proteins affect L1 protein expression and cytoplasmic foci formation shortly after transfection. First, we observed that ORF1p and ORF2p can be detected when transiently expressed from the wild-type and mutant L1 constructs used in the study. We next measured the ability of these proteins to form cytoplasmic foci. Consistent with previous studies, retrotransposition-defective L1s containing mutations in either the RRM : 5′-CACATTCCACAGCTGATCGATACC-3′Neo8161S: 5′-GGGTTCGAAATCGATAAGCTTGGATCCAGAC-3′L1 3′end: 5′-CAAACCACAACTAGAATGCAGTG-3′LEAP-86: 5′-GTGAAATTTGTGATGCTATTGC-3′LEAP-46: mneoI indicator cassette The following plasmids are based on the previously described pJM101/L1.3 and pDK101 constructs pADO2Tt contains a Tandem Affinity Purification epitope tag (TAP tag) pAD2TE1 is derived from pDK101 (L1.3) gene 10 epitope tag on the carboxyl-terminus of ORF1p and a TAP tag on the carboxyl-terminus of ORF2p.pAD2TE1-Δ2 is derived from pAD2TE1, but lacks CMV promoter and SV40 polyadenylation signal present in the original pCEP4 vector.pAD2TE1-NT is identical to pAD2TE1, but lacks the mneoI indicator cassette.pES2TE1 is identical to pAD2TE1, but contains a tandem affinity FLAG-HA tag on the carboxyl-terminus ORF2p pAD500 is derived from L1.3ΔORF1NN pADL1MT is derived from pJM101/L1.3 and contains 24 repeats of the MS2 stem-loop (MS2 tag) upstream of the mneoI indicator cassette in the L1 3′UTR. The MS2 repeats were subcloned from the pTRIP vector pAD3TE1 is identical to pAD2TE1, but contains the MS2 tag in the 3′UTR (at the same position as in pADL1MT).pADO1S is identical to pAD2TE1, but contains three stop codons in ORF1. The first two stop codons  were generated by introducing a thymidine at nucleotide position 928 to create a frameshift mutation and by mutating an A to a T at nucleotide position 930. The third stop codon is from the construct pJM108/L1.3 carrying the mutation S119Stop pADLZC is identical to pAD2TE1, but contains four leucine to valine mutations  in the ORF1p putative leucine zipper domain.pAD102 is identical to pAD2TE1, but contains the REKG235–238AAAA mutations in the ORF1p RRM domain pAD105 is identical to pAD2TE1, but contains the RR261–262AA mutations in the ORF1p C-terminal domain pAD106 is identical to pAD2TE1, but contains the RR261–262KK mutations in the ORF1p C-terminal domain pAD107 is identical to pAD2TE1, but contains the RR261–262KR mutation in the ORF1p C-terminal domain pAD113 is identical to pAD2TE1, but contains the NLR157–159ALA mutations in the ORF1p RRM domain pAD116 is identical to pAD2TE1, but contains the YPAKLS282–287AAAALA substitution in the ORF1p C-terminal domain pAD135 is identical to pAD2TE1, but contains the D702A mutation in the putative ORF2p RT active site pAD136 is identical to pAD2TE1, but contains the H230A mutation in the ORF2p EN domain pAD162 is identical to pAD2TE1, but contains the CWWDC1143–1147SWWDS mutations in the ORF2p C-domain pADL/R is identical to pAD2TE1, but contains a putative leucine zipper domain as well as a C-terminal domain mutant  in ORF1p.pADL/C is identical to pAD2TE1, but contains a putative leucine zipper domain mutation  in ORF1p as well as a C-domain mutation (CWWDC1143–1147SWWDS) in ORF2p.LZC is derived from pDK101 and contains four leucine to valine mutations  in the ORF1p putative leucine zipper domain.LZ1/2 is derived from pDK101 and contains two leucine to valine mutations  in the ORF1p putative leucine zipper domain.LZ2/3 is derived from pDK101 and contains two leucine to valine mutations  in the ORF1p putative leucine zipper domain.LZ3/4 is derived from pDK101 and contains two leucine to valine mutations  in the ORF1p putative leucine zipper domain.pDK101,pDK102,pDK105,pDK106,pDK107,pDK108,pDK116,pDK135,and pDK500 were described previously pMS2-GFP-nls, pMS2-CFP, and pTRIP were generous gifts from Edouard Bertrand pAgo2-GFP and pDCP1α-GFP were generous gifts from Gregory Hannon pG3BP-GFP was a generous gift from Jamal Tazi 2 level) in high glucose Dulbecco's modified Eagle medium (DMEM) without pyruvate (GIBCO), supplemented with 10% fetal bovine calf serum and 1X Penicillin-Streptomycin-Glutamine (GIBCO) as described previously Cell lines were maintained in a tissue culture incubator ) using FuGene-6 transfection reagent (Roche). Three days post-transfection, cells were grown in the presence of G418 (400 µg/mL) to select for retrotransposition events. The media was changed daily. After ∼12 days of selection, the resultant cells were washed with 1X Phosphate-Buffered Saline (PBS), fixed, and stained with crystal violet to visualize colonies. In parallel, HeLa cells were plated in 6 well dishes and transfected with 0.5 µg of the same plasmids and hrGFP (Stratagene). Three days post-transfection cells were subjected to flow cytometry and the transfection efficiency was determined based on the number of GFP positive cells by FACS. In some experiments, 2×105 HeLa cells/well were transfected to monitor L1 retrotransposition.The cultured cell retrotransposition assay was conducted as described previously 2, 5 mM Tris-HCl, pH 7.5, 1% deoxycholic acid, 1% Triton X-100, 1X Complete Mini EDTA-free Protease Inhibitor Cocktail (Roche Applied Science). The cells were resuspended by gentle pipetting and incubated on ice for 10 minutes. The lysate was cleared by centrifugation at 3,000 g for 5 minutes at 4°C. Untransfected HeLa cell samples were obtained three days after plating. The Bradford reagent (Bio-Rad) was used to determine the protein concentrations HeLa cells were transfected with a given L1 expression construct in T-25, T-75, or T-175 tissue culture flasks. Whole cell lysates then were prepared after 9 days of hygromycin selection as described previously RNA isolation was performed with the RNeasy Kit (QIAGEN) coupled to an on-column DNase treatment (QIAGEN). Whole cell lysates (10–50 µL) were used as starting material. The isolated RNAs were resuspended in Ultrapure distilled water (GIBCO) and quantified using a Nanodrop spectrophotometer (Thermo Scientific). For the LEAP assay controls, RNA was isolated from a 50 µL RNP sample (1.5 mg/mL). RT-PCR was performed on 0.5 µg total RNA, using the 3′RACE adapter primer (0.4 µM) and M-MLV reverse transcriptase (200U) (Promega). The resultant cDNA products then were amplified by PCR using HotStart Pfu Turbo polymerase (Stratagene) with one primer specific to the transfected L1 constructs (L1 3′ end) or GAPDH (GAPDH 3′ end) and the 3′RACE outer primer, as described previously 6 cells/flask in T-175 flasks, and transfected within 24 hours with 30 µg plasmid DNA (Midiprep Plasmid DNA Kit (QIAGEN)) using FuGene-6 transfection reagent (Roche). HeLa cells were grown in the presence of hygromycin from days 3 to 9 post-transfection (200 µg/mL) to select for episome-containing cells. HeLa cells grown for three days in the absence of hygromycin served as an untransfected (naïve) control. On day 9, transfected cells and naïve HeLa cells were harvested, lysed, and the cleared whole cell lysates were centrifuged through an 8.5%/17% (w/v) sucrose cushion at 178,000 g for 2 hours. The resultant pellet was resuspended with 100 µL dH2O +1X Complete EDTA-free protease inhibitor cocktail (Roche). Bradford reagent (Bio-Rad) was used to determine protein concentration and this RNP sample was diluted to a final concentration of 1.5 mg/mL. An aliquot (1.5 µg) of the RNP sample was added to 49 µL of LEAP assay master mix (50 mM Tris-HCL (pH = 7.5), 50 mM KCl, 5 mM MgCl2, 10 mM DTT, 0.4 µM 3′RACE adapter primer, 20U RNasin (Promega), 0.2 mM dNTPs, and 0.05% (v/v) Tween 20) and was incubated at 37°C for 1 hour. LEAP cDNA products (1 µL) were amplified in a standard 50 µL PCR reaction containing 0.4 µM of the 3′RACE outer primer and 0.4 µM of one of the following forward primers: L1 3′ end; Neo promoter sense; Neo8161S; LEAP-86; LEAP-46, using HotStart Pfu Turbo polymerase (Stratagene) according to the manufacturer's protocol  were equilibrated in 0.1M Glycine (pH 2.2) (5 µL for 100 µL of beads) for 5 minutes at room temperature. After addition of Tris-HCl (pH 8.0) (10 µL for 100 µL of beads), the beads were spun down for 3 minutes at 3000 rpm and then washed 3 times with IP FLAG buffer (mentioned above). For each condition, 3 mg of protein extract (input) was then incubated on a rotating wheel overnight at 4°C with 20 µL of the pre-equilibrated anti-FLAG beads. The next day, the beads were washed 5 times with 1 mL of IP FLAG Buffer for 10 minutes at 4°C. The beads were incubated 1 hour (at 4°C on the wheel) with 200 µL of IP FLAG buffer containing 200 µg/mL of 3X FLAG peptide (Sigma). The elution fraction then was collected and analyzed alongside the corresponding input fraction by western blotting (as described above in the dedicated section). The femto ECL substrate (Pierce) was used in the detection of both T7-tagged ORF1p and FLAG-HA-tagged ORF2p in this experiment. An aliquot (1 µL) of the input and elution samples then were used to perform the LEAP assay (see previous section for detailed protocol).Quantitative PCR was performed on LEAP cDNA samples or M-MLV RT-PCR products using the 7300 Real Time PCR system (Applied Biosystems). For analysis, 1 µL of LEAP or M-MLV RT products was added to 19 µL of master mix (1X SYBR Green PCR Master Mix (Applied Biosystems), 500 nM L1 3′ end primer, and 500 nM L1 Reverse primer), and amplified in a standard Q-PCR run of 45 cycles. The average cycle threshold (Ct) value for each experimental or control sample was calculated from three independent reactions within a Q-PCR run. The ‘absolute quantitation by standard curve’ method was used to determine the number of cDNA molecules in each LEAP RNP or RNA sample. A standard curve was generated using dilutions of a L1 LEAP product cloned into a plasmid, and a best fit line (log(molecules) versus average Ct value) for these standards was generated by linear regression. For each wild-type or mutant L1, RNA levels from three independent RNP samples were examined by at least one RT reaction and two Q-PCR runs. The level of LEAP activity in each wild-type or mutant L1 was determined from four independent RNP samples. These RNP samples were characterized by at least one and up to three independent LEAP RT reactions and one or two independent Q-PCR runs. For LEAP activity, the negative control RT- (pDK135) gave a background amplification level of ∼15–30 molecules of cDNA due to the presence of the transfected L1 plasmid in the RNP sample. This RT- background control was included in each Q-PCR run and the background amount of molecules was subtracted from each experimental sample in 5 onto sterile glass cover slips in 6 well tissue culture dishes. The following day, cells were transfected using 1 µg of purified plasmid DNA  and 3 µL of FuGene-6 Transfection Reagent (Roche Applied Science). The FISH protocol was adapted from the Robert Singer  lab protocol (available at http://www.singerlab.org/protocols) and was modified to allow protein detection by immunofluorescence. Briefly, 48 h post-transfection, cells were washed twice with 1X PBS and fixed with 4% paraformaldehyde in 1X PBS for 10 minutes at room temperature. The fixed cells then were washed 2 additional times with 1X PBS. The fixed cells were permeabilized by treatment with 70% ethanol overnight at 4°C. The following day, cells were rehydrated with 1X saline-sodium citrate (SSC) and 10% formamide for 5 minutes at room temperature. To prepare the hybridization solution, a first mix containing 40 µg of E.coli tRNA (Sigma), 1X SSC, 10% formamide, and 7.5 ng of MS2-Cy3 probe (generous gift from Dr. Edouard Bertrand) was boiled for 1 minute at 100°C in order to denaturize the probe. The quantities of probe and tRNA are indicated for hybridization of one slide. A second mix was prepared with 10% dextran sulfate, 2 mM vanadyl-ribonucleoside complex (Sigma), and 0.02% RNase free BSA (Roche Applied Science). After probe denaturation, mixes 1 and 2 were combined to form the final hybridization solution. The re-hydrated cells were hybridized overnight at 37°C in 30 µL of this hybridization solution. Cells were then washed twice for 30 minutes at 37°C with 1X SSC, 10% formamide and 3% BSA and then were incubated with primary antibodies for 1 hour at 37°C. The cells were washed three times with 1X PBS and were incubated with secondary antibodies and 0.2 µg/mL 4′,6′-diamidino-2-phenylindole  for 30 minutes at 37°C and washed three times with 1X PBS. The primary and secondary antibodies were diluted in 1X PBS and 3% BSA and are as follows: anti-T7 (Novagen), anti-TAP (Open Biosystems), anti-HA (Roche), rabbit anti-ORF1p (a generous gift from Thomas Fanning) U-2 OS cells were plated at 10The above protocol was used for both RNA and protein detection analyses. In experiments where we only sought to detect RNA, the protocol was stopped after hybridization with the MS2-Cy3 probe and subsequent washes. Cover slides were stained with DAPI and mounted on slides as described above. In experiments where we only sought to detect protein, fixed cells were permeabilized by treatment with anhydrous methanol for 1 minute. After three washes with 1X PBS, the cells were incubated with 3% BSA in 1X PBS for 30 minutes. Antibody incubation and DAPI staining were performed as described above. We verified that the protein A domain contained in the TAP tag of ORF2p did not react with the secondary antibodies (data not shown).In general, L1 cytoplasmic foci formation was measured 48 hours post-transfection. At least two independent series of slides were analyzed. Each analysis corresponds to 100 transfected cells that were quantified in a blinded manner. Cells in which we were able to distinguish a concentrated cytoplasmic signal from a diffuse cytoplasmic signal using a 63x or 100x objective  were considered as L1 cytoplasmic foci.Figure S1Retrotransposition assays with mutant L1 constructs. A. Retrotransposition assays with mutant L1 constructs: 2×104 HeLa cells were transfected with the indicated constructs. pJM101/L1.3 and pAD2TE1 were used as positive controls. All of the pAD-based constructs contain the ORF1p T7 epitope tag and the ORF2p TAP-tag except for pAD500, which lacks ORF1. pAD135 is an RT mutant (D702A), and serves as a negative control. B. Retrotransposition assay with leucine zipper domain mutants: 2×105 HeLa cells were transfected with the indicated pDK101-derived constructs. T7WT (pDK101) is a wild-type L1 (L1.3) that contains the T7 epitope tag on the carboxyl terminus of ORF1p. pDK101 was modified to create LZ1/2 , LZ2/3 , LZ3/4 , and LZC . Each of the mutations abolished L1 retrotransposition.(0.57 MB TIF)Click here for additional data file.Figure S2The effect of ORF1p mutations on LEAP activity. A. Results of western blot analyses: RNPs derived from wild-type (pDK101) and the indicated mutant constructs were subjected to western blot analyses with an anti-T7 antibody (αT7). An ∼40 kDa band indicative of epitope-tagged ORF1p is shown. Untransfected HeLa cells served as a negative control. The ribosomal S6 protein was detected using an anti-S6 (αS6) antibody  and served as a loading control. Molecular weight markers (Invitrogen) are indicated at the left of the gel. B. Results of LEAP assays: Top panel: An aliquot of the above RNPs was used to measure LEAP activity. RNPs derived from wild-type (pDK101) generate strong LEAP products of ∼220–400 bp and served as a positive control. Untransfected HeLa cells and an RT mutant  serve as negative controls. LEAP products generated in ORF1p mutant RNPs are shown. Reactions conducted without template (No Template) or without RNPs (No RNP/No RNA) were used as negative controls. Middle and bottom panels: RT-PCR with M-MLV RT and primers specific to either the transfected L1 constructs or GAPDH confirmed the presence of L1 RNA in RNPs and the integrity of the RNA isolation procedure. DNA size markers (Invitrogen) are indicated at the left of the gel. C. LEAP products derived from the ORF1p RNA binding mutant (RR261-262AA) and putative chaperone mutant (RR261-262KK): Representative LEAP products derived from wild-type (pDK101), an ORF1p RNA binding mutant , and a putative ORF1p nucleic acid chaperone activity mutant  are depicted at the top gel. The middle and bottom gels are RT-PCR reactions conducted with M-MLV RT and primers specific to L1 and GAPDH transcripts, respectively. The black arrow on the middle gel indicates the size of the specific L1 cDNA amplification products. Untransfected HeLa cells and a RT mutant (pDK135) served as negative controls. Additional negative controls include reactions conducted without template (No Template) as well as reactions conducted without RNPs or RNA (No RNP/No RNA). DNA size markers (Invitrogen) are indicated at the left of the gel. D. LZC RNPs have decreased reverse transcriptase activity: Top panel: RNPs derived from T7WT (pDK101) generate strong LEAP products of ∼220–400 bp. By comparison, LZC see  had a le(1.42 MB TIF)Click here for additional data file.Figure S3L1 retrotransposition and L1 cytoplasmic foci formation in U-2 OS cells. A. L1 retrotransposition assays: 2×104 cells were transfected with the indicated L1 constructs. pJM101/L1.3 and pAD2TE1 were used as positive controls. Untransfected cells and pAD135 (RT mutant (D702A)) serve as negative controls. A cartoon of each L1 is shown above the tissue culture dishes. Green rectangle  =  ORF1; Red rectangle  =  ORF2. The relative positions of the T7 and TAP tags also are indicated. All constructs contain the mneoI retrotransposition indicator cassette. B. Time course analyses of L1 cytoplasmic foci formation: Cells were transfected with pAD2TE1. X-axis  =  time after transfection. Y-axis  =  percentage of transfected cells containing L1 cytoplasmic foci. For each time point, 100 transfected cells were analyzed for the presence of ORF1p and ORF2p in L1 cytoplasmic foci. Error bars  =  standard deviation (n = 3). C. Cytoplasmic localization of L1 proteins and RNA: Top panels: Cells were transfected with pAD3TE1. ORF2p was visualized with an anti-ORF2 antibody . ORF1p was visualized with an anti-T7 antibody . L1 RNA was visualized with an MS2-Cy3 FISH probe (red). A merged image is shown in the rightmost column; DAPI (grey) was used to stain nuclear DNA. Middle panels: Cells were transfected with pES2TE1. ORF2p was visualized with an anti-ORF2 antibody  and an anti-HA antibody . ORF1p was visualized with an anti-T7 antibody . A merged image is shown in the rightmost column; DAPI (grey) was used to stain nuclear DNA. Bottom panels: Cells were transfected with pES2TE1. ORF1p was visualized with an anti-ORF1 antibody  and an anti-T7 antibody . ORF2p was visualized with an anti-HA antibody (red). A merged image is shown in the rightmost column; DAPI (grey) was used to stain nuclear DNA. D. Localization of L1 RNA: Top panels: Cells were transfected with pADL1MT. L1 RNA was visualized with an MS2-Cy3 FISH probe . DAPI was used to stain nuclear DNA (gray). A merged image is shown in the rightmost column. Middle panels: Cells were co-transfected with pAD3TE1 and pMS2-GFP-nls. Fluorescence was used to visualize MS2-GFP-nls (green). L1 RNA was visualized with an MS2-Cy3 FISH probe . ORF1p was visualized with an anti-T7 antibody . A merged image is shown in the rightmost column. Bottom panels: Cells were co-transfected with pAD3TE1 and pMS2-CFP. Fluorescence was used to visualize MS2-CFP (green). L1 RNA was visualized with an MS2-Cy3 FISH probe . ORF2p was visualized with an anti-TAP antibody . A merged image is shown in the rightmost column. The use of MS2 binding protein system confirms the cytoplasmic localization of the L1 RNA observed by FISH as well as the co-localization of L1 RNA with ORF1p and ORF2p. The L1 constructs in all panels contain the mneoI retrotransposition indicator cassette.(2.01 MB TIF)Click here for additional data file.