This gene is missing from the plastid genomes of two species in the parasitic plant genus Cuscuta but is found in all other published land plant and streptophyte algal plastid genomes, including that of the nonphotosynthetic angiosperm Epifagus virginiana and two other species of Cuscuta. By examining matK and plastid intron distribution in Cuscuta, we add support to the hypothesis that its normal role is in splicing seven of the eight group IIA introns in the genome. We also analyze matK nucleotide sequences from Cuscuta species and relatives that retain matK to test whether changes in selective pressure in the maturase are associated with intron deletion. Stepwise loss of most group IIA introns from the plastid genome results in substantial change in selective pressure within the hypothetical RNA-binding domain of matK in both Cuscuta and Epifagus, either through evolution from a generalist to a specialist intron splicer or due to loss of a particular intron responsible for most of the constraint on the binding region. The possibility of intron-specific specialization in the X-domain is implicated by evidence of positive selection on the lineage leading to C. nitida in association with the loss of six of seven introns putatively spliced by matK. Moreover, transfer RNA gene deletion facilitated by parasitism combined with an unusually high rate of intron loss from remaining functional plastid genes created a unique circumstance on the lineage leading to Cuscuta subgenus Grammica that allowed elimination of matK in the most species-rich lineage of Cuscuta.Plastid genome content and arrangement are highly conserved across most land plants and their closest relatives, streptophyte algae, with nearly all plastid introns having invaded the genome in their common ancestor at least 450 million years ago. One such intron, within the transfer RNA  Unlike those in eukaryotic nuclear genes, they do not rely on spliceosomes for excision from RNA transcripts, and unlike the similarly structured self-splicing introns of prokaryotes, they typically require other trans-acting factors for efficient splicing matK, consistently found within trnK-UUU. Although matK has been shown to be an essential factor for the splicing of the trnK intron within which it is containedEpifagus encodes only four proteins not involved in transcription or translation and lacks a functional trnK genetrnK pseudogene retains a complete open reading frame for matK which is evolving under selective constraint, indicating matK is essential for other functions beyond splicing the trnK intron in that speciestrnK loss with retention of matK is seen in the photosynthetic streptophyte alga Zygnema circumcarinatumcrs2, for splicingclpP intron 2, is present in the chloroplast genomes of most land plants but was not examined in those studies because it is not present in grasses. Excision of the only group I intron, trnL-UAA, is unaffected by any of these factors, as is splicing of the second of two group IIB introns found within ycf3matK in splicing introns other than the trnK intron within which it resides.Only one transcribed open reading frame has been identified within any plastid intron, the presumed intron maturase Epifagus (Orobanchaceae), members of the genus Cuscuta (Convolvulaceae) are parasitic plants that have undergone substantial gene loss from their plastid genomes Cuscuta species were reported more than a decade ago trans-spliced rps12 gene was shown to be polymorphic in the genusCuscuta, two from subgenus Monogyna and two from subgenus Grammica, shows that intron content between the two subgenera differs greatly; specifically, both matK and all group IIA introns except the second intron of clpP are lost from the plastid genomes of the closely related members of subgenus GrammicaclpP, acquired in the common ancestor of land plants millions of years after matK and the other seven plastid group IIA introns Cuscuta gronovii in the absence of plastid matKLike Cuscuta in order to ascertain the distribution of matK and plastid introns in the genus. For those taxa that still contain matK, we investigated whether or not significant changes in selective constraint occurred on branches where intron loss has occurred. Finally, we conducted similar branchXsite tests on an equal sample size of the variously parasitic family Orobanchaceae, where loss of most plastid introns is known to have occurred at least in Epifagus.In this study we sampled across the taxonomic range of matK at the trnK-UUU locus along with all known group IIA introns and three group IIB introns (one in trnG-UCC and two within ycf3) from a variety of Cuscuta species representing all three currently recognized subgenera , five group IIB introns, and the trnL-UAA group I intron within otherwise normal genes, corroborating prior results that resident plastid matK is not necessary for the splicing of these introns Although the Cuscuta that still possess matK also possess at least four group IIA introns with the exception of Cuscuta nitida, which retains only the 3′ rps12 intron . Using outgroup sequences from available plastid genomes, a well-supported phylogeny was constructed that agrees fully with published relationships within Convolvulaceae and CuscutaIpomoea (tribe Convolvuleae) was strongly supported as sister to Cuscuta, although alternative hypotheses at this node could not be rejected in a previous study studyCuscuta is sparse, we conservatively chose to collapse this node as a polytomy for analyses of selective constraint. We were especially interested in changes in selective constraint within domain X, the portion of matK that has been identified as the putative RNA binding domain Nd/Sd = ω) were constrained across the phylogeny, domain X was found to be evolving under stronger purifying selection (ω = 0.21) than the remainder of the gene  conserved across land plants and charophytesmatK remains functional among all Cuscuta that still possess it.All sampled species of 2 intron , Fig. 1ANd/Sd (M0) versus models with two or three rate ratio classes  without switching across the tree  with the exception of the one contained in the 3′ portion of rps12. We used branchXsites models implemented in codemla priori hypothesis that the pattern of variation in constraint among sites was different on the branch leading to C. nitida (specified as the foreground branch) than the pattern of among-site variation across the rest of the tree (background branches). This approach is analogous to the switching test implemented in fitmodel, but in this case we have an a priori hypothesis that switches among rate ratio classes are concentrated on a single branch. The alternative hypothesis for branchXsites tests of Yang, Nielson and colleagues (implemented in codeml) have fewer parameters than the unconstrained switching model implemented in fitmodel, and thus this approach may have more statistical power when one has well defined hypothesis for the branch on which switching is expected to have occurred. In fact, the branchXsites model fits the domain X data significantly better than the rates across sites model (M3) when the C. nitida branch was specified as the foreground  and neutral (ω1 = 1) evolution, with a positive selection branchXsites model that includes a third, positive rate ratio class (ω2>1) for a fraction of sites evolving on the foreground branch. The second, more stringent test compares a branchXsites null model with ω2 = 1 on the foreground branch to the positive selection branchXsites model (i.e. ω2>1 on the foreground branch). In addition, codemla posteriori Bayes empirical Bayes (BEB) estimation of the probability that each site on the foreground branch is evolving under positive selection (ω2>1). The likelihood for the branchXsites positive selection model was significantly better than for the nearly neutral model, and adaptive evolution on the branch leading to C. nitida was strongly supported for one site, position 16 in the domain X alignment  and a fully autotrophic sister-group to the parasites (Lindenbergia philippinensis) were also included in the analysis, and the same outgroups were used as for the Convolvulaceae tests. The phylogeny obtained for these species  was lower for domain X than for the rest of the gene (0.28 vs. 0.47), and branchXsites models with the Epifagus lineage set as the foreground were significantly better than the rates across sites models  is primarily responsible for constraint of domain X across streptophytes, that intron may be lost on the branch leading to Epifagus as well. As we saw with the Cuscuta analysis, the Maximum Likelihood estimations of ω were >4.0 for some codons in domain X; however, we are not able to reject the hypothesis that these sites are evolving under neutrality  trees were estimated in PAUP*4.0b10 Cuscuta nitida and Epifagus. Likelihood ratio tests were applied to compare a series of nested models including equal constraint (M0 N/dSd = ω), variation in ω across sites  and distinct patterns of variation across sites on foreground and background branches (branchXsites models). Model parameter and likelihood values (codeml within the PAML package v.3.15 http://abacus.gene.ucl.ac.uk/software/paml.html). Foreground branches were specified as those leading to Cuscuta nitida or Epifagus in separate analyses. Sites with Bayes empirical Bayes posterior probabilities >0.95 for ω2>1.0 were estimated in codemlSeparate d values  and 3 wefitmodel v0.5.2 program www.cebl.auckland.ac.nz/~sguindon/fitmodel.html]. Unlike codeml, fitmodel does not specify foreground and background branches.We also checked for switching among ω rate ratio classes across the Convolvulaceae and Orobanchaceae trees using


Arabidopsis to an even greater extent than the positions of the major, U2 introns. The U12 introns, especially, conserved ones are concentrated in 5'-portions of plant and animal genes, where the U12 to U2 conversions occurs preferentially in the 3'-portions of genes. These results are compatible with the hypothesis that the high level of conservation of U12 intron positions and their persistence in genomes despite the unidirectional U12 to U2 conversion are explained by the role of the slowly excised U12 introns in down-regulation of gene expression.We report that the positions of minor, U12 introns are conserved in orthologous genes from human and This article was reviewed by John Logsdon and Manyuan Long. For the full reviews, please go to the Reviewers' Reports section. Most of the eukaryotic protein-coding sequences are interrupted by multiple non-coding sequences, introns, which are excised from the transcript through the action of a complex molecular machine, the spliceosome, which consists of snRNP  and hnRNPs (heterogeneous nuclear ribonucleoprotein particles), and is conserved throughout the eukaryotic world -3. ThereThe atypical U12 introns have been recognized through their unusual splice junction structure, namely, |AT at the donor splice site and AC| at the acceptor splice site ,8. A cloComparative analyses of the gene structures in orthologous genes from diverse eukaryotes have shown that up to 30% of U2 intron positions are conserved between animals and plants ,16. Comb+/H+ antiporters [Arabidopsis thaliana genomes in order to systematically compare their conservation with that of U2 introns.The positions of U12 introns tend to be conserved among vertebrates ,19, and iporters . Howeverbona fide evolutionary conservation. Moreover, and unexpectedly, the fraction of conserved U12 intron positions in the analyzed set of human/Arabidopsis orthologs was significantly greater than the fraction of conserved U2 introns  is likely to reflect intensive U12 to U2 conversion in plant evolution which might be the reason behind the small number of U12 introns in Arabidopsis compared to humans.Among the "mixed" positions, 15 contain U12 introns in the human genes opposite a U2 intron in Arabidopsis, and only 5 contains a U2 intron in the human gene opposite U12 in Arabidopsis. This significantly asymmetric distribution of the U12-U2 mixed sites . Here, the authors focus on Homo-Arabidopsis comparisons; since the animal-plant split represents a deep divergence among eukaryotes, this should allow for inferences about early eukaryotic gene evolution. Previous comparisons of intron conservation between animal and plant genes have indicated that high fractions of intron positions are conserved. Since the fraction of U12 introns is less than 1% of all introns, these previous studies were necessarily focused on U2 introns (even though the introns were not explicitly classified as such). Since the types of introns can now be classified, the authors wished to explicitly compare levels and patterns of conservation among both U2 and U12 introns in Homo and Arabidopsis genes.This brief contribution provides an interesting assessment of the conservation of spliceosomal introns comprising the major (U2) and minor (U12) classes. The analysis takes advantage of a recent comprehensive classification of introns into these two types . In any case, the key here is that there was a significant statistical excess of shared U12 introns, much higher than the fraction of shared U2 introns. Furthermore, of the "mixed" U2–U12 shared positions, a considerable majority are U12 type in Arabidopsis. Finally, the within-gene distribution of all U2 and U12 introns (not just the few shared ones), suggest a preference for U12 introns in 5' ends of genes; but when considering the shared U12 introns, the 5' bias is particularly prevalent in the shared U12 introns. The potential functional relationship between 5' introns and regulation makes this latter observation particularly interesting.Of the 133 homologous Homo and Arabidopsis). These and other authors have defended the hypothesis that the (high fraction of) similarly-identified animal-plant shared introns are mostly homologous, and that is a starting premise of this paper. However, such inferences remain untested by addition and consideration of many other intervening taxa . Interestingly the shared U12 introns almost certainly represent homologous introns that can be traced to the animal-plant common ancestor; their rarity makes parallel insertion a highly improbable explanation. Perhaps this argument is similar for "mixed" introns, but the likelihood of parallel gain seems reasonably tenable. Overall, the paper is appropriate for publication in Biology Direct.One weakness of this study is the fact that it relied solely on pairwise comparisons between two distantly related taxa (This manuscript reported a statistical analysis of the position conservation of two types of introns, U2 and U12. The U2 type has been found in 99% of eukaryotic introns. However, in the shared intron positions between plants and animals, this paper reports more conservation of U12 types, an unexpected and interesting asymmetric distribution of two types of introns. The interpretation for this observation is not a straightforward thing because of lack of outgroup to assign ancestral states. However, the authors proposed that this could be caused by the conversion of U12 to U2 introns in plant lineages. Their explanation is not unreasonable, because biologically the U12 splicing is not so efficient as U2 types so there could be a selective pressure against U2 to U12 conversion. Nevertheless, when more plant genomes are sequenced, the hypothesis that plant lineages are subject to higher rate of the U12 to U2 conversion may have opportunity to be tested by looking at the distribution of the turnover rates by comparing various branches of plants.alignments of protein sequences of orthologous proteins from Arabidopsis and human employed for this analysis.Click here for file


Positions of spliceosomal introns are often conserved between remotely related genes. Introns that reside in non-conserved positions are either novel or remnants of frequent losses of introns in some evolutionary lineages. A recent gain of such introns is difficult to prove. However, introns verified as novel are needed to evaluate contemporary processes of intron gain.We identified 25 unambiguous cases of novel intron positions in 31 Drosophila genes that exhibit near intron pairs (NIPs). Here, a NIP consists of an ancient and a novel intron position that are separated by less than 32 nt. Within a single gene, such closely-spaced introns are very unlikely to have coexisted. In most cases, therefore, the ancient intron position must have disappeared in favour of the novel one. A survey for NIPs among 12 Drosophila genomes identifies intron sliding (migration) as one of the more frequent causes of novel intron positions. Other novel introns seem to have been gained by regional tandem duplications of coding sequences containing a proto-splice site.Recent intron gains sometimes appear to have arisen by duplication of exonic sequences and subsequent intronization of one of the copies. Intron migration and exon duplication together may account for a significant amount of novel intron positions in conserved coding sequences. Daphnia populations [Comparative studies of spliceosomal intron densities have suggested relatively high rates of intron gain during eukaryote evolution ,2. The eulations  suggesteThe analysis of near intron pairs  allows aHere we use the relatively recently diverged genomes of 12 Drosophila species  to identTheir distribution supports the known species phylogeny. Both introns of a NIP were evaluated for sequence similarity to introns, neighboring exons and transposable elements and screened for repetitiveness and potentially meaningful secondary structures, using the ancient, plesiomorphic intron of the NIP as control. In addition, we looked for cryptic splice signals in the adjacent exonic and intronic sequences. There is evidence for intron sliding in 9 of the 31 Drosophila NIPs, while 5 other cases probably arose by tandem duplications within the ORF. In contrast, we found no evidence for any intron insertion mechanism based on alien sequences in our data set.We started with 12386 sets of orthologous protein-coding genes of Drosophila species (Methods). After an automatic NIP extraction alignment procedure, we obtained 122 NIP regions containing two or more near introns (NIP distance < 50 nt). These were manually inspected for splice site, alignment and conservation validity, resulting in 40 regions comprising 41 NIPs. Based on the frequency of short exons and NIP distances within Drosophila (data not shown), we decided to decrease the maximal allowed intron distance to 31 nt, which resulted in 35 alignment regions comprising 36 NIPs  intron position that could not be clearly excluded by splice site sequence analysis, we evaluated seven NIP candidates experimentally. As no EST data supported these NIPs, we performed RT-PCR or genomic PCR experiments in some crucial species  contradicts the established tree of the 12 Drosophila species. This NIP is clearly associated with a sliding event (see below). The local nature of sliding makes independent changes of intron position with identical results much more likely than any of the other mechanisms, which require independent targeting of the same genomic position. We conclude that NIPs could be reliable phylogenetic markers also for recent radiations. However, short branch lengths, as observed between Drosophila species, could critically limit the number of available characters. Intron sliding accounts for a low but detectable level of homoplasy in this type of character.Next, we evaluated possible evolutionary mechanisms that had produced introns at novel positions. For all the introns of the 31 NIPs, we could not find any relevant sequence conservation to other introns, to transposons, or to exons (data not shown). To control whether some of the intron position changes resulted from intron sliding, we evaluated all introns of the NIPs concerning properties that would be expected if a coordinated migration of both splice sites occurred in relation to the former intron and the CDS. These properties are: (1) There is no intermediate, intron-less state in the tree; (2) NIP distance is a multiple of 3 allowing a stepwise intron shift; (3) cryptic splice sites occur at NIP distances and are supported by amino acid conservation Figure ; (4) oneD. yakuba and D. melanogaster) [D. ananassae and D. melanogaster). No footprint of internal sequence conservation across the two intron positions has been retained in any of these cases.In another 6 NIPs, at least three of the other four criteria are fulfilled. While the first two conditions are also consistent with mechanisms of intron gain (see below), cryptic splice sites at the corresponding distance and one-sided shifts of intron borders in some species are specific requirements for intron sliding Figures  and 3B. ogaster)  and was,In summary, 9 out of 31 NIPs most likely originated by intron sliding. This is surprising since earlier studies concluded that intron sliding is a very rare event . On the Donor and acceptor site consensus sequences of spliceosomal splicing also include exonic parts. At the donor (5') splice site, the last two exonic nucleotides  could bind to the U1 snRNA  and the Concomitantly, such a duplication will allow differential splice patterns (intron retentions) as a temporary or permanent alternative to the establishment of a constitutive intron if the duplication size is a multiple of three. Alternatively, unspliced mRNA variants may be degraded by nonsense-mediated decay (NMD) . Thus, ND. melanogaster, but well above the splice site scores of typical exonic sequences surrounding Drosophila introns , the proto-splice sites are conserved also in Drosophila species having no intron there, and should thus already have been present at the time of intron origin. The potential functionality of these sites both as donor and acceptor sites is supported by their splice site scores  that reside within the variability of functional splice sites of s Figure , Table 2The concept of the proto-splice site was proposed more than 20 years ago . DespiteD. melanogaster and detected 1901 duplications, mostly in tandem, with a median size of 367 nt in at least one line. Irrespective of the strong evidence for purifying selection against this type of mutations, 624 of these duplications included some exonic sequences, but not a whole gene. Given this significant amount of function-challenging mutations, the gain of an intron may compensate for a duplication simply if a proto-splice site is included.In contrast, tandem duplications frequently occur in natural populations. Emerson et al.  comparedIn order to further support the hypothesis that the 7 TD introns arose by tandem duplication rather than by reverse splicing, we evaluated their proto-splice sites. If splice sites had emerged directly by duplication of proto-splice sites, (1) proto-splice and splice sites should be similar to each other and (2) both should be functional splice sites. If intronic splice sites stemmed from a reverse-spliced intron, proto-splice sites would not need to be similar to splice sites but should have provided a preferential binding site for the spliceosome that has inserted the intron. The sequence specificity of spliceosome-binding during forward splicing is mediated by the binding of the U1 snRNP to the donor site . HoweverTo distinguish between the two alternative pathways, we evaluated the splice sites, the proto-splice sites and the relative location of the new introns in more detail:(1) We determined scores for reference splice sites and proto-splice sites excluding the central AG/GY motif. These scores are based on the nts +3 to +6 (donor site) and the nts -13 to -3 (acceptor site). We found that such partial proto-splice acceptor sites of TD introns show intermediate scores between reference splice sites (RS) on the one hand, and potential proto-splice sites surrounding reference introns (RP) and intron-less AGGY proto-splice site motifs of exons (IS) Figure  on the o(2) If the supposedly duplicated sequences of both intron/exon borders of one intron are compared under exclusion of the central AG/GY motif, these regions of TD introns are significantly more similar to each other compared to other NIP introns (ON) or reference introns (RS/RP)  The location of the TD introns within the genes does not support an origin by reverse splicing. A reverse-transcription mediated pathway would imply a 3' biased distribution of novel introns as the reverse transcriptase should start primarily at the 3' end of the mRNA . ApomorpD. melanogaster lineage after divergence from the D. erecta lineage and thus appears to be between 5 and 10 million years old . Accordingly, the footprint of duplication might have been lost.The above suggested cases of exon duplications are not supported by sequence conservation of (surrounding) exonic and unconstrained intronic sequences. This could be due to the relatively large evolutionary age of all cases of exon duplications reported here. The youngest intron that may have originated by duplication has evolved in the Drosophila and Sophophora. Alternatively, there might have been an ancient intron that was lost before the duplication (FBgn0050101). In the remaining two cases, the other intron seems to be novel, too, but has emerged in another lineage by an unknown mechanism  are reliable phylogenetic markers. Furthermore, our results support that intron sliding (migration) is one of the causes of recently emerged intron positions within conserved protein-coding Drosophila genes. We also found evidence for the rise of novel introns by tandem duplication of exonic DNA. The origin for 17 out of 31 identified NIPs remains unknown  . For eacCDS were compiled according to the FlyBase peptide annotations and aligned at the protein level. The translated multiple alignments  were performed using a slightly modified version of the TransAlign utility . Our modD. melanogaster) are available, the annotation and reference protein were selected according to highest sequence similarity to all other species' translations. Intron positions within the multiple alignments were named according to their absolute positions within the Dme reference peptide sequence by using translated BLAT [By mapping the various intron positions onto the multiple translated alignment, orthologous and nearby intron positions become apparent. In cases where several translations (isoforms) for the reference species (ted BLAT . In a laWe obtained 4044 putative NIP regions each containing at least two intron positions. We excluded NIPs from this set that contained genes with both introns of a pair and such which failed to show the splice rule consensus for both donor (GYR) and acceptor (HAG). In addition, we required at least 6 non-gap characters at both exon borders. This resulted in 959 candidate NIP regions, which we ordered according to a conservation score collected from the most similar pair of amino acid sequences representing different intron positions of the local multiple alignment. The conservation score was calculated as the average of relative sum-of-pair scores utilizing the BLOSUM45 substitution matrix of Clustal W . By requDmo to FBgn0002526), 8 sequences were removed (e.g. Dgr from FBgn0015572), and one obvious misannotation/sequence error could be corrected (Der in FBgn0046689) , to obtain a more reliable data set, based on a comparison of the abundances of short exons and NIP distances (data not shown) within Drosophila. The manual analysis included the search for orthologous outgroup sequences to infer the plesiomorphic and apomorphic intron positions where possible. Additionally, 2 Drosophila sequences were manually added to the alignments  that were originally used for whole genome sequencing [We performed PCR to confirm the intron positions of 7 NIPs that were only weakly supported. For this purpose, we isolated female whole body RNA and genomic DNA from adults of the appropriate Drosophila species. In case of quencing . The totFrom the 31 remaining NIPs, an intron pair matrix was manually created and analyzed in MacClade 4.0   [D. melanogaster transcripts) were sampled that contain the AGGY proto-splice site motif and whose potential intron positions would be located more than 31 nt from the exon borders. Position weight matrices (PWMs) for splice site scoring were generated from the reference splice sites (RS). For each (proto-) splice site the score was calculated according to [Reference data sets of splice sites were compiled to compare the splice site strengths of potential proto-splice sites. The reference set of in total 16766 splice sites (RS) was created from introns of all transcripts of  points) . A seconrding to , i.e. usDan: Drosophila ananassae; Der: Drosophila erecta; Dgr: Drosophila grimshawi; Dme: Drosophila melanogaster; Dmo: Drosophila mojavensis; Dpe: Drosophila persimilis, Dps: Drosophila pseudoobscura; Dse: Drosophila sechellia; Dsi: Drosophila simulans; Dvi: Drosophila virilis; Dwi: Drosophila willistoni; Dya: Drosophila yakuba; FBgn: FlyBase identifier number for genes; NIP: near intron pair; nt: nucleotide.CDS: coding sequence; JL carried out the bioinformatic acquisition and data analysis, performed statistical analyses, participated in the design of the study and helped to draft the manuscript. CE performed the PCR analysis and helped to draft the manuscript. PFS participated in the design of the study and helped to draft the manuscript. VK conceived the study, participated in its design and analysis and drafted the manuscript. All authors read and approved the final manuscript.Table of NIP data. This file contains data about plesiomorphic and apomorphic intron positions, their support by ESTs, intron-less species, RT-PCR, CG number and regional amino acid conservation (PDF format).Click here for fileNIP alignments. Alignments of all Drosophila NIP regions (Text format).Click here for fileTable of PCR analysis results of selected NIP candidates. This file contains the PCR primer sequences and validation results for the selected NIP candidates that were in doubt concerning the reliability of the shifted intron position (PDF format).Click here for fileIntron pair matrix. This file contains the coded intron position data of 31 NIPs. It was used to build the tree of Figure Click here for file


Brain-Derived Neurotrophic Factor (BDNF) is a central nervous system modulator of nociception. In animal models of chronic pain, BDNF exerts its effects on nociceptive processing by binding to the full-length receptor tropomyosin-related kinase B (trkB.FL) and transducing intracellular signaling to produce nocifensive behaviors. In addition to trkB.FL, the trkB locus also produces a widely-expressed alternatively-spliced truncated isoform, trkB.T1. TrkB.T1 binds BDNF with high affinity; however the unique 11 amino acid intracellular cytoplasmic tail lacks the kinase domain of trkB.FL. Recently, trkB.T1 was shown to be specifically up-regulated in a model of HIV-associated neuropathic pain, potentially implicating trkB.T1 as a modulator of nociception. Here, we report that trkB.T1 mRNA and protein is up-regulated in the spinal dorsal horn at times following antiretroviral drug treatment and hind paw inflammation in which nocifensive behaviors develop. While genetic depletion of trkB.T1 did not affect baseline mechanical and thermal thresholds, the absence of trkB.T1 resulted in significant attenuation of inflammation- and antiretroviral-induced nocifensive behaviors. Our results suggest that trkB.T1 up-regulation following antiretroviral treatment and tissue inflammation participates in the development and maintenance of nocifensive behavior and may represent a novel therapeutic target for pain treatment. C and D. Quantification of trkB.FL mRNA expression in the dorsal horn of trkB.T1 wildtype mice (C) and trkB.T1 knockout mice (D) after receiving a saline hind paw injection  or a hind paw injection of CFA . E and F. TrkB.FL protein expression in the dorsal horn of trkB.T1 wildtype (E) and trkB.T1 knockout (F) mice in the antiretroviral model was assayed via western blot. The top panels shows a representative western blot of trkB.FL following saline or d4T injection at 1d and 3d. Blots were stripped and re-probed with anti-actin to control for protein loading (shown in lower panel). Below the panel the results from saline treated  and 1d  or 3d  after d4T treatment are quantified. G and H. E and F. TrkB.FL protein expression in the dorsal horn in trkB.T1 wildtype (G) and trkB.T1 knockout (H) mice in the CFA model was assayed via western blot. The top panel shows a representative western blot of trkB.FL following a saline or CFA hind paw injection at 3d. Blots were stripped and re-probed with anti-actin to control for protein loading (shown in lower panel). Below the panel the results from saline treated  and 3d CFA  are quantified.Click here for file


The systems for mRNA surveillance, capping, and cleavage/polyadenylation are proposed to play pivotal roles in the physical establishment and distribution of spliceosomal introns along a transcript. Saccharomyces cerevisiae) [Schizosaccharomyces pombe) and reaches ~38,000 and ~140,000 in the genomes of the fruit fly Drosophila melanogaster and Homo sapiens, respectively [In eukaryotes (and viruses), genes may be organized into coding and noncoding regions, called exons and  introns, respectively . Both tyevisiae) , but thiectively . Explainectively .Spliceosomal introns are noncoding intervening sequences of eukaryotic and viral genes that are removed during the process of pre-mRNA maturation, leaving only coding sequence (exons) to be part of the messenger RNA. Three additional classes of introns are known—the group-I, group-II, and group-III introns, all of which are capable of self-splicing. Spliceosomal introns cannot self-splice but are removed by a dynamic nuclear apparatus, the spliceosome. Such machinery typically consists of five uridylate-rich small nuclear RNAs  and a large number of associated proteins.Although the evidence is circumstantial, it is widely thought that spliceosomal introns originated from group-II introns—self-splicing introns that are widely found in fungi, plants, protists, and bacteria—which invaded the uninterrupted nuclear genes of an early eukaryote and subsequently lost the ability to self-splice, as the host genome took over this function . HoweverRecent studies on vertebrate genome evolution have shown that numerous exons have emerged from pre-existing noncoding sequences  ,16. The The process of translation normally ends when the ribosome reaches a nonsense (or stop) codon at the end of the coding mRNA. A nonsense codon that is positioned upstream of the true stop can lead to premature translation termination and thus is called a premature termination codon (PTC). PTCs found in eukaryotic coding sequences are sometimes excluded from the mature mRNA , for exaWe suggest that the cell's capacity to filter out aberrant transcripts, in concert with imperfect splice-site recognition ,35, may In this model, we propose that protein-coding sequences that fortuitously contain the minimal requisite sequence information for spliceosome-mediated recognition  a short length, thereby minimizing the number of lost codons; and (2) a sequence length that is a multiple of three, so as to preserve the ORF. Also, as the emergence of introns in small exons would lead to the creation of two even smaller flanking exons, whose correct splicing might be compromised , we expeAs it is unlikely that the process of intronization is instantaneous, we predict the existence of a transient phase during which gene regions are neither fully exonic nor fully intronic, essentially exhibiting the features of DNA sequences undergoing alternative splicing. Alternative splicing is a nuclear process that leads to the incorporation of noncoding regions and/or the exclusion of coding regions from mature mRNAs , events Several findings support the hypothesized gradual conversion from exonic to intronic sequences. Specifically, across vertebrates, constitutively spliced introns in one species often align to homologous alternatively spliced coding sequences of orthologous genes from another species —suggestin in size when located within the coding region [The gradual conversion that we envision for the process of intronization has been reported also for the process of exonization . In partg region ; unusualg region ,50; and g region ,52; theyg region .The extensive network of interactions between mRNA-associated processes  suggestsSeveral connections have been found between the processes of cleavage/polyadenylation and splicing , and splWe propose that CPFs regularly access U-rich tracts along the mRNA during transcription, but are antagonized (or interfered with) by SFs when the U-rich regions are located within an intron. Notably, these two sets of factors are also known to antagonize/interfere in exons, as the U1 small nuclear ribonucleoprotein (an SF) inhibits 3′-end processing when bound to the 3′-end of a pre-mRNA in the vicinity of the cleavage-polyadenylation site . Under oThe CBC also influences splicing, acting as a splicing enhancer by increasing the population of SFs local to the 5′ end , thus faFinally, the antagonistic interactions between SFs and CPFs are likely mediated by two other major classes of competing proteins , namely Central to our hypothesis is the observation that the more favorable splicing at the 5′ end of a gene parallels the spatial pattern of the efficiency of NMD degradation of aberrant transcripts. Specifically, NMD effectiveness is maximal when a PTC is proximal to the 5′ end of the mRNA and minimal when it resides in the most 3′ exon, close to the 3′ end of the transcript  Figure . Thus, nThe distribution of introns over the length of the coding sequence is consistent with the idea that NMD, as well as the interactions between CPFs and SFs, cooperatively guides the successful colonization by introns. In particular, the NMD pathway appears to have been lost in eukaryotic lineages that have no or nearly no introns . While in introns in the ciliate Paramecium tetraurelia were revealed to be about twice as frequent compared to PTC-containing introns of the two other size classes [2 = 23.26, p = 1.41 × 10−6) but less frequent toward the 3′ end , consistent with the idea that splicing-eliciting PTCs arise more frequently in the 5′ ends of genes .Two possible explanations for the existence of intronless genes are: (1) that introns can simply be lost, so that a subset of intron-free genes is to be expected; and (2) that some intronless genes may be derived retrogenes, i.e., mature mRNAs that are reverse transcribed into DNA copies and inserted into the genome . HoweverWe have proposed a novel hypothesis for the origin of spliceosomal introns, invoking endogenous production within translatable sequences (at least in the case of protein-coding genes), facilitated by the activity of cellular surveillance mechanisms. Despite the mutational hazard associated with intron presence and proliferation , we argu


The identification of TDP-43 as the major component of the pathologic inclusions in most forms of sporadic and familial frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS) resolved a long-standing enigma concerning the nature of the ubiquitinated disease protein under these conditions. Anti-TDP-43 immunohistochemistry and the recent development of novel tools, such as phosphorylation-specific TDP-43 antibodies, have increased our knowledge about the spectrum of pathological changes associated with FTLD-U and ALS and moreover, facilitated the neuropathological routine diagnosis of these conditions. This review summarizes the recent advances in our understanding on the molecular neuropathology and pathobiology of TDP-43 in FTLD and ALS. Frontotemporal lobar degeneration (FTLD), the second most common form of presenile dementia, refers to a clinically, genetically and neuropathologically heterogeneous group of neurodegenerative disorders. Clinically, FTLD is characterized by behavioural and/or language dysfunction . In addiMAPT) gene in familial forms of FTLD [While the designation FTLD reflects the prominent frontal and temporal lobe degeneration, the characteristic lesions in the majority of FTLD brains are abnormal ubiquitinated protein inclusions. The protein tau has been identified as the protein building block in the inclusions in about 40% of FTLD cases, and its role in the pathogenesis of neurodegenerative diseases is well established especially after identification of mutation in the microtubule-associated protein tau  in FTLD-U, they did not prove to be informative. Characterization of UBIs was complicated by the relative low abundance of UBIs, the uneven distribution of UBIs among different FTLD-U cases, and the non-amyloidogenic nature of UBIs as demonstrated by absence of staining with amyloid-binding dyes such as thioflavin S, Congo red, or silver stains. Therefore, an alternative immunologic approach was performed to identify the protein components in the ubiquitinated inclusions in FTLD-U , 12. BriMost importantly, several disease-associated TDP-43 alterations with potential functional implications have been observed . Thus, TTARDBP encoding for TDP-43 in ALS (see below) provides further strong evidence for a direct link between TDP-43 dysfunction and neurodegeneration.The subsequent identification of 20 mutations to date in the gene GRN) gene, valosin-containing protein (VCP) gene and those with linkage to chromosome 9p [As demonstrated in the initial reports and rapidly confirmed in numerous follow-up studies, TDP-43 is a specific and sensitive marker to detect the characteristic inclusions , dystrophic neurites (DN) and neuronal intranuclear inclusions (NII)) in the majority of sporadic and familial FTLD-U cases, including familial FTLD-U forms with mutations in the progranulin  of TDP-43 further improve and facilitate the neuropathological assessment of TDP-43 pathology, as they only label abnormal TDP-43 in inclusions, but not the often strong physiological nuclear TDP-43 .et al [While UBIs in FTLD-U are generally found in the frontal and temporal cortex as well as in dentate granule cells of the hippocampus in most FTLD-U cases, heterogeneity of FTLD-U pathology with respect to morphology, laminar distribution of ubiquitin and TDP-43 positive inclusions and relative proportion of DNs versus NCIs have been described in two independent studies, leading to the description of four distinct histological FTLD-U subtypes (subtypes 1—4) , 15, 21.et al . The chaBriefly, subtype 1 histology  is charIn subtype 2 cases  the preGRN mutations exclusively show subtype 3 pathology [The abundance of small neuritic profiles and NCIs, often ring-shaped, predominantly in the superficial cortical layers characterizes subtype 3 histology . Especiathology , 22.VCP mutations and vice versa.The characteristic neuropathological feature of subtype 4 pathology is the abundance of ubiquitin and TDP-43 positive NIIs and DNs with only few NCIs in affected cortical regions and the absence of inclusions in the hippocampal dentate granule cells , 23. So The relevance of the heterogeneity of TDP-43 pathology among FTLD-U cases with respect to pathogenesis remains to be determined. However, the striking correlation of distinct histological subtypes with different genetic forms of familial FTLD-U  and its CHMP2B) gene do not contain TDP-43 [While the initial data suggested that all FTLD-U cases are indeed characterized by TDP-43 pathology, recent follow-up papers on large series of FTLD-U cohorts have shown that there are some important exceptions. Thus, the ubiquitin-positive inclusions in the brains of family members from familial FTLD caused by a mutation in the charged multivesicular body protein  remain to be identified .ALS, the most common form of adult-onset motor neuron disease, is characterized by a loss of upper and lower motor neurons, that results in progressive weakness, muscular wasting, and spasticity. About ~10 % of ALS cases are familial (fALS) and mutations in the Cu/Zn superoxide dismutase (SOD1) gene are thought to be the most common ones accounting for ~20% of fALS .After the initial report that the characteristic lesions obtained in remaining neurons in sporadic ALS (sALS), typically filamentous skeins or compact round inclusions, are composed of TDP-43, the role of TDP-43 in sALS versus fALS was further evaluated. Remarkably, while TDP-43 pathology is a consistent feature in all sALS and non-SOD1-fALS, there is neither histological nor biochemical evidence for TDP-43 pathology in SOD1-fALS cases , 30. TogIn addition to neuronal inclusions, GCIs are a consistent feature in sALS and non-SOD1-fALS . TDP-43 TARDBP, the gene encoding TDP-43 a promising candidate for genetic screening. Initial screens in sporadic FTD and ALS, as well as familial FTD patients failed to identify mutations and no genetic variations could be identified as risk factor for developing FTD or ALS [TARDBP mutations are missense mutations mostly affecting highly conserved amino residues in the C-terminal region of TDP-43.The presence of TDP-43 pathology in ALS made D or ALS –36. HoweD or ALS –44. TwelTARDBP mutation, showing characteristic TDP-43 positive neuronal and glial inclusions in addition to motor neuron loss and presence of Bunina bodies.Neuropathological data are so far available for the G294A , G298S , and Q34TARDBP mutations are unknown and need to be investigated in more detail in future studies.Thus far, the functional consequences of Co-occurrence of distinct neurodegenerative disease lesions in the brains of patients with neurodegenerative disorders is an emerging theme in research in these conditions, as exemplified by the high frequency of Lewy body pathology in Alzheimer’s disease (AD) that was only recognized after the routine use of antibodies against α-synuclein. Analogous, TDP-43 immunohistochemistry now also enables the investigation of the co-occurrence of FTLD-U type TDP-43 pathology in the setting of other neurodegenerative disorders and indeed some degree of TDP-43 pathology has now been reported in a variety of other neurodegenerative diseases besides FTLD and ALS.Thus, TDP-43 pathology is a highly consistent finding in most cases of ALS-Parkinsonism-dementia complex of Guam , 47. ConTARDBP gene on chromosome 1. It was first cloned as a human protein capable of binding to the transactive response DNA of human immunodeficiency virus type 1 [TDP-43 is a 414 amino acid protein encoded by the s type 1 , and lats type 1  and the s type 1 . TDP-43 s type 1 .In addition to its well characterized role in transcription and splicing regulation, more recent studies suggest that TDP might be involved in other cellular processes such as in microRNA biogenesis, apoptosis, cell division, mRNA stabilization and regulation of neuronal plasticity by acting as neuronal activity response factor –61. FinaThe mechanistic aspects leading to accumulation of pathologic TDP-43 in cytoplasmic, neuritic, and nuclear inclusions and the functional consequences of TDP-43 accumulation are currently not well understood. The dramatic change in subcellular distribution of TDP-43 from the nucleus to the cytoplasm in affected cells in FTLD and ALS suggests that maybe loss of physiological nuclear TDP-43 function in transcription and mRNA processing might play a pathogenic role. Alternatively, generation and sequestration of abnormal TDP-43 species such as C-terminal fragments and hyperphopshorylated TDP-43 enriched in inclusions might induce a toxic gain of function. Recent studies have shown that TDP-43 continuously shuttles between the nucleus and cytoplasm, a process partially regulated by nuclear localization signal (NLS) and nuclear export signal (NES) motifs ,64. RestTARDBP mutations will provide us with important insights also into the disease mechanisms underlying sporadic ALS and FTLD-U. Currently discussed hypotheses are that TARDBP mutations might interfere with protein-protein interaction, which might affect its nuclear transport or well-known transcriptional and splicing activities; or that TARDBP mutations might increase the aggregation tendency of TDP-43 perhaps due to increased phosphorylation [It is expected that elucidating the functional consequences of ALS-associated rylation –43.TDP-43 is the major disease protein in most forms of FTLD-U and ALS, thereby providing strong evidence that both conditions are part of a clinico-pathological spectrum with common underlying pathomechanisms. The use of anti-TDP-43 antibodies have dramatically improved our knowledge about the spectrum of pathological changes underlying FTLD and ALS and it is highly recommended to implement immunohistochemistry for TDP-43 as routine stain in the neuropathological diagnostics of neurodegenerative diseases. Future research approaches, including development of novel cell culture and animal models, have to determine whether loss of function, toxic gain of function or a combination of both mechanisms contributes to cell death in TDP-43 proteinopathies.


The origin and importance of exon-intron architecture comprises one of the remaining mysteries of gene evolution. Several studies have investigated the variations of intron length, GC content, ordinal position in a gene and divergence. However, there is little study about the structural variation of exons and introns.P < 0.001 in most cases): an ordinal reduction of length and divergence in both exon and intron, a co-variation between exon and its flanking introns in their length, GC content and divergence, and a decrease of average exon (or intron) length, GC content and divergence as the total exon numbers of a gene increased. In addition, we observed that the shorter introns had either low or high GC content, and the GC content of long introns was intermediate.We investigated the length, GC content, ordinal position and divergence in both exons and introns of 13 eukaryotic genomes, representing plant and animal. Our analyses revealed that three basic patterns of exon-intron variation were present in nearly all analyzed genomes  exist and may shape both exons and introns; the ordinal reduction patterns may reflect a time-orderly evolution; and the larger first and last exons may be splicing-required. These clues provide a framework for elucidating mechanisms involved in the organization of eukaryotic genomes and particularly in building exon-intron structures. The major function of exons to present mRNA and to code proteins was discovered around 40 years ago . During Recent studies have produced data that shed light on the pattern of intron properties, e.g., the variations of length, GC content, ordinal position in a gene  and divergence of intron sequences. Various factors are revealed to influence the intron size ,12. For Different from introns, there is little data about the patterns of exon properties or variations of exon-intron architecture. Only a few studies included the basic statistical analyses, such as the distribution of exon length, the average number of exons per gene from eukaryotic model organisms , and theOur data revealed three consistent patterns, which present in almost all of the genomes we analyzed. Elucidation of these common patterns provides a basis for understanding the factors responsible for organization of the eukaryotic genomes, and for describing the exon-intron architectures.The complex relationships between intron length and GC content were revealed in human and fly genome ,18. A siInterestingly, the complicated relationships between intron length and GC content could be characterized by a curve when the introns were sorted by their GC content from low to high, then the average length of every 1000 introns were plotted against their average GC content Fig. . The conP < 0.001, Additional file P < 0.001) is present in the rice genome as well as in chicken and fly genomes. In mammalian genomes, the GC content of the first intron is significantly higher than the others, and thereafter no perceptible difference was seen. In the other genomes, e.g., worm and zebrafish, no clear variation was found. The results of the systematic investigation on many genomes suggested the intron length was not randomly distributed in the ordinal positions. However, while this supposition may be partly true for the distribution of intron GC content in the ordinal positions, no clear pattern was obtained for the genomes analyzed.In addition, intron length is negatively correlated with ordinal position in a gene across all genomes analyzed . In general, there appears to be a negative tendency between the length and ordinal position of exons. However, a positive correlation was present in worm.When the last exon, which is the longest one , was excP < 0.001; Fig P < 0.001.The similar pattern in the distribution of length and GC content in both introns and exons suggests the existence of co-variation in some of these traits. To test this hypothesis, we calculated all possible correlations among these specific traits between introns and exons, and found that some of traits were correlated. A close positive correlation was found between intron GC content and its flanking exon GC content , and this was present in all analyzed genomes  of the fewer-intron genes were shorter than the more-intron genes , genes with fewer introns had relatively larger average intron length  length, GC content and divergence as the increase of total exon numbers in a gene. Although the first few introns  with a generally consistent variation pattern. In addition, a strong complicated correlation  exists between GC content and the length of introns (or exons), and this correlation is present in all genomes analyzed. Although more significant correlations were observed , only these four patterns were consistent among species.Our investigation of the variation patterns of exon-intron comparisons in 13 eukaryotic genomes revealed three basic patterns: an ordinal reduction of length and divergence in both exon and intron Fig.  and 4A–Dvs. chimpanzee and rice Nipponbare vs. 93-11) as examples to assess the consistency  for the pattern of ordinal reduction in introns and exons. In other words, we calculated the two-intron, three-intron... 9-intron and > 9-intron (or -exon) genes separately, to compare the genes with an equal number of introns or exons. We chose human and rice  that cause these patterns remain unclear, our results provide some clues to such a phenomenon. Firstly, the co-variation pattern of exon-intron in their GC content, length and divergence suggests that the basic variation patterns are caused by factor(s) common to either exons or introns or to both. Given that there are common factors, they are likely to affect exons and functional elements of introns because of their importance. Therefore, the functional association between exon and intron, e.g., the splicing elements, may be the basis of co-variation patterns. Secondly, the monotonic reduction of length, GC content and divergence as the ordinal variation or as the total number increase in introns or exons indicates that this pattern may be the key to decipher the major factors which shape these patterns. The decay-like curves for these orderly reductions suggest that the ordinal patterns may be a reflection of time-related order . Thirdly, the first and the last exons are generally the longest exons in a gene, indicating the necessity of maintaining a larger exon in the beginning and in the end of a coding sequence. The selectively maintained first and last larger exons may be transcription-, splicing- or translation-required. Finally, the first intron generally has the highest GC content and thereafter there is almost no regular ordinal-pattern, which suggests that the first intron may evolve differently from the other introns.In fact, the enrichment of regulatory elements in the first intron was most often used to explain the evolutionary pattern of the intron . For exaet al. [The exon-intron architecture was also revealed to be important in the determination of splicing phenotype, and the efficient recognition of exons was influenced by the length of flanking introns . The splet al.  suggesteObviously, the ordinal (or orderly) reduction patterns of exon-intron architecture constitute an important puzzle with theories about the determinants of genome evolution. The ordinal patterns may reflect a time-orderly evolution. If the amount of exons or introns follows an increasing trend (during the process of genome-size expansion), the first exon and intron are older than the next ones; the older introns have more time to be inserted and become longer; the inserted sequences in introns generally have a lower GC content; and the later occurring introns cut the coding sequences into shorter ones except for the first and the last exon, which are required by splicing-related factors; the subsequent recruited exons (or parts of them), compared with the first exons, have a higher possibility of coming from intron sequences and therefore have a lower GC content. The timely-ordered model could describe the consistent decrease of length, GC content and divergence observed in this study.Some expectations could be tested by comparing the side (the first or the last) and the inner exons. Based on the UniProt database , the proThe exon-increase model involves more in the variation of existed exons/introns, e.g., their length, divergence and GC content, but less in their gain and loss. Under this model, the preferential gain of introns in 3'-portions of genes is expected. This is more consistent with the introns-late hypothesis, in which the introns appeared later and at random in early eukaryotic genomes, and gained an adaptive role in gene evolution following insertion . SverdloWe showed that there were three basic patterns of exon-intron variation in eukaryotic genomes, and these patterns were consistently present in almost all 13 genomes analyzed. The first pattern is the ordinal reduction of length and divergence in both exons and introns. The decay-curve-like variation and the orderly reduction suggest that this pattern may reflect a time-sequential evolution . The second pattern is the co-variation of length, GC content and divergence between exons and flanking introns. The closely-correlated co-variation between exons and introns indicates common factor(s) exist in shaping both exons and introns. The third pattern is the decrease of average length, GC content and divergence in exons and introns as the total number of exons in a gene increased. This phenomenon may also be a reflection of chronological variation. In addition, a significant non-linear correlation was observed between GC content and the length of introns or exons. All these patterns of exon-intron variation revealed by our investigation provide a framework for elucidating mechanisms involved in the organization of eukaryotic genomes and particularly in building exon-intron structures.Homo sapiens), chimpanzee (Pan troglodytes), mouse (Mus musculus), rat (Rattus norvegicus), Cow (Bos taurus), dog (Canis familiaris), chicken , zebrafish (Danio rerio), pufferfish (Tetraodon nigroviridis), fruit fly (Drosophila melanogaster), worm (Caenorhabditis elegans), Arabidopsis thaliana and rice (Oryza Sativa). The genome sequences and annotations of two rice lines (Oryza sativa L. var. Nipponbare vs. var. 93-11) were obtained from GRAMENE [Thirteen full sequenced genomes were selected Table , includi GRAMENE . All gen GRAMENE . Ensembl GRAMENE  were useTo investigate the relationships among length, GC content, ordinal position and nucleotide divergence in both introns and exons, each trait was first sorted by order, and then partitioned into bins containing 1000 introns or 1000 exons each . Then another trait (y axis) was calculated for each of these bins and plotted against the first trait. For example, when examining the relationship between intron length and GC content, all introns in a genome were first sorted either by length or GC content into bins, then the average of GC content or length of every bin was calculated for each bin and plotted against length or GC content in figures. Only those introns within coding sequences and exons without UTR (untranslated regions) were used for analyses in all figures. In addition, the short introns (< 20 bp) were excluded to avoid systematic errors, which were reported in the introns by using automated annotation methods [The pairwise alignments between human and chimpanzee were downloaded directly from UCSC Genome Browser . PairwisDT and JQC designed the study and drafted the manuscript. LZ and YZ contributed extensively to the bioinformatics analyses. WZ and SY assisted the analyses and manuscript preparation. All authors read and approved the final manuscript.Fig S1-6. The other relationship between the length, GC content, divergence of introns/exons.Click here for fileTable S1-2. The statistical test results.Click here for file


TDP-43 is an evolutionarily conserved RNA-binding protein implicated in the pathogenesis of frontotemporal dementia (FTD), sporadic and familial amyotrophic lateral sclerosis (ALS), and possibly other neurodegenerative diseases. In diseased neurons, TDP-43 is depleted in the nucleus, suggesting a loss-of-function pathogenic mechanism. However, the normal function of TDP-43 in postmitotic neurons is largely unknown.Drosophila TDP-43 (dTDP-43) in vivo significantly increases dendritic branching of sensory neurons in Drosophila larvae. Loss of dTDP-43 function, either in a genetic null mutant or through RNAi knockdown, decreased dendritic branching. Further genetic analysis demonstrated a cell-autonomous role for dTDP-43 in dendrite formation. Moreover, human TDP-43 (hTDP-43) promoted dendritic branching in Drosophila neurons, and this function was attenuated by mutations associated with ALS.Here we demonstrate that overexpression of These findings reveal an essential role for TDP-43 in dendritic structural integrity, supporting the notion that loss of normal TDP-43 function in diseased neurons may compromise neuronal connectivity before neuronal cell loss in FTD and ALS. Frontotemporal lobar degeneration (FTLD) is a devastating age-dependent neurodegenerative condition primarily associated with impairments in cognition and social behaviors, as well as personality changes and other clinical abnormalities . FrontotTDP-43 are responsible for some sporadic and familial amyotrophic lateral sclerosis (ALS) [In addition to pathogenic mutations in the microtubule binding protein tau ,5, rare is (ALS) -16, furtis (ALS) . In healis (ALS) . Moreoveis (ALS) . These fTDP-43 is a ubiquitously expressed RNA-binding protein that contains two RNA recognition motifs, a glycine-rich region, a nuclear localization signal, and a nuclear export signal . It is nDrosophila peripheral nervous system (PNS) as our assay system and performed both gain- and loss-of-function genetic studies. We also examined the functional significance of some genetic mutations in TDP-43 that are associated with ALS. Our findings support the notion that a TDP-43 loss-of-function mechanism may contribute to the pathogenesis of FTD and ALS.TDP-43 is highly conserved at the amino acid level from flies to humans, suggesting an evolutionarily conserved function ,23,26. TdTDP-43 RNAi lines 38377 and 38379 were obtained from the Vienna Drosophila RNAi Center (VDRC). The dTDP-43Q367X mutant allele was identified from the Seattle Drosophila TILLING Project  using specific tilling primers  to generate dTDP-43Q367X/+; Gal4221, UAS-mCD8-GFP/38377, and dTDP-43Q367X/38379; Gal4221, UAS-mCD8-GFP/+ third instar larvae for phenotypic analysis. For transgene overexpression, Gal4221, UAS-mCD8-GFP flies were crossed with UAS-dTDP-43,, UAS-hTDP-43, UAS-hTDP-43-M337V, or UAS-hTDP-43-Q331K transgenic lines. In the above experiments, Gal4221, UAS-mCD8-GFP/+ third larvae served as the control.All flies were raised on standard food medium and kept at 25°C. ansgenes . To visuhTDP-43 cDNA was cloned from HEK293 cells (provided by Dr. J.-A. Lee). To generate UAS-hTDP-43, UAS-hTDP-43-M337V, UAS-hTDP-43-Q331K, and UAS-hTDP-43-C-terminal fragment (amino acids 209-414) constructs, the corresponding primers  and cloned into the pUAST vector. These constructs were confirmed by sequencing and microinjected into wild-type (w1118) embryos to generate transgenic lines.Full-length Anti-dTDP-43 polyclonal antibody was generated by immunizing rabbits with a peptide fragment spanning amino acids 179-192 (Thermo Fisher Scientific). For protein expression analysis, adult flies were frozen in ethanol with dry ice and vortexed to remove heads. Approximately 30 heads from each genotype were homogenized in 50 μl of lysis buffer . Homogenate was heated at 65°C for 10 min and centrifuged at 4°C for 10 min at 13,000 rpm. Protein concentrations were determined using Bradford Assay (Bio-Rad).Supernatant containing 10 μg of protein was separated on a 10% acrylamide SDS gel and transferred to a PVDF membrane (Bio-Rad) in a wet transfer system at 4°C for 60 min at 100 V. The membrane was incubated in blocking solution containing 5% milk in TBST  at 4°C overnight, with dTDP-43 antibody (1:1000 in blocking solution) at room temperature for 3 h, and finally with anti-rabbit HRP-conjugated secondary antibody  for 1 h. The signal was visualized with chemiluminescent substrate . For other western blot analyses, the primary antibodies were hTDP-43 antibody , and β-actin antibody .Total RNAs were extracted from adult heads with Trizol (Invitrogen) and used as templates to generate cDNAs with TaqMan reverse transcription reagent (Applied Biosystems). cDNAs were used as templates for qRT-PCR in a final volume of 25 μl. A standard curve was run in each PCR reaction. Individual values were normalized to the value of the gene encoding the ribosomal protein RP-49. Two pairs of primers were designed to detect dTDP-43 transcripts . The significance of differences in dendritic branching complexity was determined with Student's t test.MARCM analysis of sensory neurons in the escribed . First, t test.The dendritic morphology of GFP-labeled dorsal sensory neurons was recorded with a confocal microscope , and dendritic branches of ddaE or ddaF neurons in the A3 dorsal cluster were counted as described . BrieflydTDP-43 transgenic fly lines with insertion sites on different chromosomes. Because dendritic branching patterns are critically important for neuronal function and connectivity and dendritic defects are associated with many neurological disorders [Drosophila larvae as our assay system, which has been useful in uncovering molecular mechanisms of dendritic morphogenesis [221, which targets gene expression in two sensory neurons only in each hemisegment [To examine neuronal functions of dTDP-43, we generated multiple UAS-isorders ,30, we fogenesis . To drivisegment .P < 0.001) in ddaE neurons, and from 22.8 ± 1.0 (n = 14) to 39.9 ± 1.9 (n = 10) (P < 0.001) in ddaF neurons .Ectopic expression of dTDP-43 markedly increased the number of small terminal dendritic branches Figure . For inss Figure . Interess Figure . This efdTDP-43 loss-of-function mutant flies. We used the service from the Seattle Drosophila TILLING Project (Fred Hutchinson Cancer Research Center) and obtained several fly lines in which point mutations were found in dTDP-43. One, a nonsense mutation with a single nucleotide change from G to A, introduced a stop codon at amino acid 367 106/+ heterozygous flies (data not shown). Since this antibody did not detect the putative truncated dTDP-43 protein in mutant flies  driven by tubulin-Gal4 resulted in a similar lethal phenotype. Overexpression of dTDP-43 with the pan-neuronal Gal4C155 driver also led to a similar lethal phenotype, indicating that the proper level of dTDP-43 expression in the nervous system is required for normal viability. Indeed, ectopic expression of dTDP-43 in the eye caused a severe retinal degeneration phenotype (data not shown).Homozygosity for  as well . ExpressGal4221 in ddaE and ddaF neurons in wildtype or dTDP-43Q367X homozygous mutant third instar larvae. Compared with wildtype neurons   Figure . A simil) Figure .dTDP-43Q367X mutants. Therefore, to further confirm that loss of dTDP-43 activity reduces dendritic branching, we used two independent dTDP-43 RNAi lines obtained from VDRC to knock down dTDP-43 expression in these neurons. Again, the number of dendritic ends was decreased in ddaE  vs. 31.3 ± 4.9 (n = 6) (P < 0.001). This result indicates that dTDP-43 functions in a cell-autonomous manner to regulate dendritic branching.In the RNAi experiment described above, dTDP-43 activity was knocked down in only a small number of sensory neurons, suggesting that dTDP-43 functions in a cell-autonomous manner to control dendritic branching. To further confirm this possibility, we used the mosaic analysis with a repressible cell marker (MARCM) technique, which allows one to generate GFP-labeled single neurons containing a mutation of a gene of interest in a mostly wildtype or adult . Using thTDP-43 transgenic flies  also promotes dendritic branching, we generated multiple lines of UAS-s Figure . The C-ts Figure . The levs Figure .Drosophila neurons also promoted dendritic branching   Figure . A simil) Figure . The ect) Figure . These fWT had 55 ± 1.6 (n = 29) dendritic ends but the neurons expressing hTDP-43M337V or hTDP-43Q331K had only 41.1 ± 1.5 (n = 18) or 44.3 ± 2.0 (n = 21) dendritic ends, respectively (P < 0.001)  Figure . A simil) Figure . Thus, AThe pathological role of TDP-43 was first recognized by its presence in ubiquitin-positive but tau-negative inclusions in diseased neurons of FTD and ALS patients ,11. TDP-Drosophila offers a powerful model system to examine the endogenous functions of TDP-43. We obtained dTDP-43 null mutant flies and found that dTDP-43 is required for normal viability, consistent with a study published during the preparation of our manuscript [TDP-43 knockout mice have not been reported yet. Considering the high degree of conservation between dTDP-43 and hTDP-43, it is possible that TDP-43 is also essential for normal development in mammals. At the cellular level, multiple lines of evidence from our current study indicate that TDP-43 promotes dendritic branching in postmitotic neurons. This conclusion was based on overexpression studies, RNAi knockdown, and genetic analysis of a dTDP-43 null allele. TDP-43 seems to also regulate axonal structures at the Drosophila neuromuscular junction (NMJ) [Since TDP-43 is highly conserved at the amino acid sequence level from flies to humans, nuscript . TDP-43 on (NMJ) . These fIn many neurodegenerative diseases, defects in synaptic connections are probably one of the earliest alterations preceding neurodegeneration . Recent Drosophila is also an excellent model system for studying human disease proteins. We found that hTDP-43 also promotes dendritic branching in Drosophila neurons, indicating a functional conservation. More importantly, two point mutations associated with ALS attenuated the dendrite-promoting activity of hTDP-43. Both are located in a C-terminal region that mediates protein-protein interactions [ractions . Thus, tractions . Thus, mDrosophila assay system. RNAi knockdown and genetic analysis of a dTDP-43 null allele revealed a cell-autonomous role for dTDP-43 in promoting dendrite formation. Mutations associated with some forms of ALS reduced the dendrite-promoting activity of hTDP-43, suggesting a loss-of-function pathogenic mechanism. These findings support the notion that loss of normal TDP-43 function may contribute to the pathogenesis of FTD and ALS. The fly model reported here and newly generated relevant reagents will facilitate studies to further elucidate the underlying molecular mechanisms.Overexpression of dTDP-43 or hTDP-43 in vivo significantly increased dendritic branching in a The authors declare that they have no competing interests.YL and FBG designed the experiments, and YL and JF performed the experiments. YL, JF, and FBG analyzed the data, and wrote the paper. All authors read and approved the final version of the manuscript.Primers for different transgenic constructs and sequencing primers.Click here for filedTDP-43 mRNA levels.Primers for qRT-PCR analysis of Click here for file


Sarcoidosis is an idiopathic granulomatous disease with pathologic and immunologic features similar to tuberculosis. Routine histologic staining and culture fail to identify infectious agents. An alternative means for investigating a role of infectious agents in human pathogenesis involves molecular analysis of pathologic tissues for microbial nucleic acids, as well as recognition of microbial antigens by the host immune system. Molecular analysis for superoxide dismutase A (sodA) allows speciation of mycobacteria. SodA is an abundantly secreted virulence factor that generates cellular immune responses in infected hosts. The purpose of this study is to investigate if target antigens of the sarcoidosis immune response can be identified by molecular analysis of sarcoidosis granulomas.M. tuberculosis (MTB) complex, as well as sequences which were genetically divergent. Using peripheral blood mononuclear cells (PBMC) from 12 of the 17 sarcoidosis subjects, we performed enzyme-linked immunospot assay (ELISPOT) to assess for immune recognition of MTB sodA peptides, along with PBMC from 26 PPD- healthy volunteers, and 11 latent tuberculosis subjects.We detected sodA amplicons in 12 of 17 sarcoidosis specimens, compared to 2 of 16 controls , and 3 of 3 tuberculosis specimens (p = 0.54). Analysis of the amplicons revealed sequences identical to Six of 12 sarcoidosis subjects recognized the sodA peptides, compared to one of 26 PPD- controls (p = 0.002), and 6/11 PPD+ subjects (p = .68). Overall, 10 of the 12 sarcoidosis subjects from whom we obtained PBMC and archival tissue possessed molecular or immunologic evidence for sodA.Dual molecular and immunologic analysis increases the ability to find infectious antigens. The detection of Th-1 immune responses to sodA peptides derived from molecular analysis of sarcoidosis granulomas reveals that these are among the target antigens contributing to sarcoidosis granulomatous inflammation. Mycobacterium katG and heat shock proteins by sarcoidosis subjects , or indolent progressive pulmonary decompensation associated with radiographic findings, such as hilar adenopathy, reticulonodular infiltrates, or pulmonary fibrosis); 2) histopathologic features had confluent noncaseating granulomas, well circumscribed within the surrounding tissue with a variable amount of peripheral lymphocytic infiltration ); 3) knoFor the enzyme-linked immunospot assay (ELISPOT), we were able to collect blood on 12 of the 17 sarcoidosis subjects. Due to the Health Insurance Portability and Accountability Act of 1996 (HIPAA) regulations, we could not contact the negative or positive control subjects of whom we had archival tissues. We invited 26 PPD negative (PPD-) healthy volunteers to participate as negative controls for the ELISPOT analysis. The PPD- volunteers had undergone PPD testing by the Vanderbilt employee health services. Subjects with latent tuberculosis (PPD+) were invited as positive controls, and had written documentation of their PPD status with no evidence of active disease at the time of study enrollment. Approximately 60% of the sarcoidosis, PPD- and PPD+ subjects had participated in a previous investigation of immune responses to mycobacterial antigens .For each sarcoidosis and control specimen, the original paraffin-embedded tissue block was retrieved from the archives if available. From each block, we cut a single 5 μm section that was stained with H&E to confirm the pathology and demarcate areas of granulomatous involvement; another ten 10 μm sections were cut, three of which were used for extraction of DNA, and the remaining seven sections were stored for future analysis. The specimens were randomly processed for slide preparation. For each section from patients with sarcoidosis and for the control specimens, granulomata were macrodissected and extracted with disposable surgical blades. For the control specimens without granulomata, all tissue from three 10 μm sections was used for DNA extraction.For the frozen specimens, 25 mg of frozen sarcoidosis or control specimens were initially minced with sterile disposable surgical blades prior to overnight proteinase K digestion. For all specimens, DNA was extracted with the QIAGEN DNAeasy extraction kit  according to the manufacturer's instructions, with the exception of the following changes: 1) 60 μL of proteinase K was used at a concentration of 20 mg/mL; 2) after the overnight digestion, 0.5 mm glass beads  were added to the completely digested tissue, which was subsequently vortexed for 45 seconds; 3) the DNA was eluted with 100 μL of Buffer AE, instead of 200 μL.Tissue dissection and DNA preparation were performed in a dedicated clean room, which was separate from the rooms used for PCR analysis and sequencing. The extracted DNA was stored at -80°C. Groups of tissue specimens from patients with sarcoidosis, tuberculosis, and controls were processed in parallel during all steps of the procedure, including extraction of the DNA, amplification and detection of mycobacterial DNA, and sequence analysis. The clinical diagnosis of each specimen was blinded throughout the entire analysis.Mycobacterium tuberculosis complex sodA (GenBank No. AF061030) were designed using Primer Express software  and are described in Table 4 genomic copies, was used as the positive control, five microliters of irradiated water as the negative control, and five microliters of template DNA from each clinical specimen. Samples whose dissociation curve peaks had a melting temperature within 1°C of TB DNA were selected for further analysis on a 3% TBE gel. Bands that electrophoresed within approximately 10 bp of TB DNA were excised and purified using the QIAquick Gel Extraction Kit . Both strands of the DNA were directly sequenced by GenePass, Inc . Only sequences verified from the positive and negative strand of the amplicon were used for analysis. Alignments of sodA sequences from sarcoidosis patients were performed using the NCBI BLAST program.Six overlapping primer sets that cover the entire ORF for Mycobacterium species were obtained from GenBank. Because there is no genetic variability of sodA among members of MTB complex; sequence alignment was based upon GenBank no. AF061030. In addition to MTB complex and sarcoidosis amplicons, multiple corresponding nucleotide sequence alignments of sodA region 4  from pathogenic and nonpathogenic mycobacteria were created using ClustalX. Phylograms of nucleotide alignments were generated using the Paup 4.0b10 neighbor-joining method, based on HKY85 distances. The resulting phylograms were displayed using midpoint rooting.Sequences of sodA from various PBMC were isolated from blood drawn into tubes containing EDTA, and separated by Ficoll-Hypaque density gradient separation (Amersham Biosciences) according to the manufacturer's instructions. The PBMC were cryopreserved in fetal calf serum with 10% dimethy sulfoxide (DMSO), and stored in liquid nitrogen until time for analysis.AAD15824.1) were generated  and then sodA peptides were added to individual wells in duplicate . Phytohemagluttin A (PHA) or media alone served as positive and negative controls, respectively. The plates were incubated for 18 hours at 37°C in 5% CO2. After washing six times with phosphate buffered saline (PBS) plates were incubated for 2 hours with biotinylated anti-IFN-γ mAb, 7-B6-1 . After additional washes, a 1:2,000 dilution of streptavidin-alkaline phosphatase conjugate was added to each well for 2 hours. The plates were then washed, and IFN-γ producing cells detected after a 10–20 min color reaction using chromogenic alkaline phosphatase substrate . Interferon gamma (IFN-γ) producing cells were counted using an IMMUNOSPOT 3 Analyzer (Cellular Technology Limited (C.T.L); Cleveland, OH). Results were expressed as the number of spot-forming cells (SFC) per 106 PBMC. The mean number of spots in duplicate negative control wells was less than two for all 26 PPD- participants. A positive response was defined as at least 50 SFC/106 PBMC, and at least three times above background. The definition of a positive result was based upon a prior analysis, involving recognition of ESAT-6 and katG in systemic sarcoidosis [ELISPOT assays were performed as previously described . Brieflycoidosis . Assays Comparisons of the distribution of T cell frequencies were performed using the Wilcoxon rank sum test. Categorical comparisons, such as the percentage of individuals with immune reactivity to mycobacterial antigens, were made using Fisher's exact test. All performed comparisons are reported, all p-values are 2-sided, and all analyses were performed using R (Version 2.1.1).M. tuberculosis was cultured from all three. Granulomatous involvement was noted in all 17 sarcoidosis specimens, 10 of 16 control specimens, and all three tuberculosis specimens  were African-American, 953%) were male, and 16(94%) were ≤ 50 years of age ; in Sarcoid 3, regions 2 and 4 were detected. Sequence analysis of the amplicons from 10 specimens revealed 100% positional identity with MTB complex. The primers of regions 1 and 4 were capable of amplifying sodA nucleic acids from nontuberculosis mycobacteria, such as M. kansasii . However, only sequences consistent with MTB complex were detected. In two specimens (Sarcoid 2 and 15), the amplicons from region 4 contained 99% positional identity with MTB complex. The same polymorphism, A302G, was detected in both specimens (GenBank accession no. DQ768096), despite these samples being processed and analyzed three months apart. This nucleotide substitution resulted in a D101G mutation (GenBank accession no. ABG68026) in a previously published amino acid sequence for MTB sodA (GenBank accession no. AAD15824.1)  distinguished them from other members of MTB complex  Table . The sar) Figure . Phylogex Figure .It has been reported that environmental mycobacteria preferentially localize to established tuberculous granulomas . In ordeFrozen and paraffin-embedded specimens sarcoidosis and control specimens were included to determine whether mycobacteria were introduced during tissue fixation. There was not a significant difference in the presence of sodA nucleic acids among the sarcoidosis specimens that were frozen in comparison to those that were formalin-fixed, paraffin-embedded  Table .AAD15824.1), and stimulated PBMC from a subgroup of sarcoidosis, PPD- and PPD+ subjects to identify immunogenic peptides. There was no recognition of any of the 40 sodA peptides among the PPD- control subjects. Among the sarcoidosis and PPD+ subjects, four of the 40 peptides were recognized most frequently: sodA 31, 33, 36 and 38 , and six of 11 subjects with PPD+ infection (p = 1.0) Table . We perfDespite negative culture and histology for mycobacterial infection in sarcoidosis specimens, there was a significant difference in recognition of sodA peptides between the sarcoidosis and PPD- healthy volunteers, but not between sarcoidosis and PPD+ subjects. We also assessed the distribution of the T cell frequencies among the PPD-, sarcoidosis and PPD+ subjects. There was a significant difference between the sarcoidosis and PPD- subjects , but again, not between the sarcoidosis and PPD+ subjects (p = 0.72) Figure .Prior PCR analysis and immune recognition assays for the presence of sodA was significantly distinct between the sarcoidosis and PPD- control subjects. We were interested in the number of sarcoidosis subjects possessing molecular or immunologic evidence for sodA. There were 12 sarcoidosis subjects of whom we obtained archival tissue and blood. Eight of the 12 were positive by PCR analysis, and six positive by the ELISPOT assay. Four subjects  were positive for sodA nucleic acids, as well as recognition of sodA peptides; Sarcoid 11 and 12 were negative by both assays Table . In totaMycobacterium sodA, in systemic sarcoidosis.The absence of histologic and microbiologic evidence of microorganisms in the sarcoidosis granulomas has led to questions about a role for infectious agents in sarcoidosis immunopathogenesis. This report describes molecular and immunologic support for the presence of the virulence factor, Mycobacterium species [AF061030); however, sequence analysis of the amplicons also revealed sequences that were genetically distinct  There is a clear association between the sarcoidosis amplicons and the sequences of members of MTB complex, demonstrating that they share a recent common ancestor, and precluding the possibility that the sarcoidosis sequences are derived from distantly related mycobacterial or Nocardia species. 2) The sequences from Sarcoidosis 2 and 15 form a distinct clade, emphasizing that these sequences may belong to a single mycobacterial species Figure . WhetherB strain . 3) The Histoplasma capsulatum in an individual with rheumatoid arthritis. Prior reports have noted an association with latent tuberculosis in such patients [Superinfecting mycobacteria preferentially home to tuberculous granulomas . This obpatients . The detThe inclusion of sarcoidosis and control specimens that were frozen or paraffin-embedded was designed to address the possibility that mycobacteria are introduced into sarcoidosis specimens during tissue fixation. There are reports of environmental mycobacteria in hospital water systems, and well as fiberoptic bronchoscopes ,21. We cThe dual assessment for mycobacteria in sarcoidosis pathogenesis provides interesting information. Among the 12 subjects, 10 possessed molecular or immunologic evidence for sodA. This illustrates the importance of using complementary approaches to assess for microorganisms in pathologic specimens of unknown etiology. The two sarcoidosis subjects who were negative by both methods suggest that mycobacteria do not have a role in all sarcoidosis specimens, supporting other reports of the inappropriate diagnosis of sarcoidosis in subjects with disease with close phenotypic and pathologic features, such as chronic beryllium disease . Thus saPCR of archival specimens was more sensitive for sodA than ELISPOT analysis of peripheral blood Table . There wThe most interesting finding in this study was the demonstration that molecular analysis of sarcoidosis granulomas for microbial virulence factors can lead to identification of target antigens. The responses detected closely parallel prior reports of immune recognition of secreted proteins in mycobacterial infections ,26. A duThe authors declare that they have no competing interests.SSA carried out the molecular genetic studies, and performed the sequence alignment. WE determined immunogenic sodA epitopes and carried out the immunoassays, along with RH, MN, and JC. DTP performed the phylogenetic analysis and helped draft the manuscript. BES performed the statistical analysis. JEJ confirmed the pathologic diagnosis of all samples. WPD conceived of the study, participated in its design and coordination, and drafted the manuscript. All authors read and approved the final manuscript.This study was approved by the Vanderbilt University Institutional Review Board for human studies, and informed written consent was obtained from the study participant or their surrogates, if required. A copy of the written consent is available for review by the Editor-in-Chief of this journal at any time.