The objective of this article is to study the problem of pedestrian classification across different light spectrum domains (visible and far-infrared (FIR)) and modalities . In recent years, there has been a number of approaches for classifying and detecting pedestrians in both FIR and visible images, but the methods are difficult to compare, because either the datasets are not publicly available or they do not offer a comparison between the two domains. Our two primary contributions are the following: (1) we propose a public dataset, named RIFIR , containing both FIR and visible images collected in an urban environment from a moving vehicle during daytime; and (2) we compare the state-of-the-art features in a multi-modality setup: intensity, depth and flow, in far-infrared over visible domains. The experiments show that features families, intensity self-similarity (ISS), local binary patterns (LBP), local gradient patterns (LGP) and histogram of oriented gradients (HOG), computed from FIR and visible domains are highly complementary, but their relative performance varies across different modalities. In our experiments, the FIR domain has proven superior to the visible one for the task of pedestrian classification, but the overall best results are obtained by a multi-domain multi-modality multi-feature fusion. In this context, pedestrian protection systems have become a necessity. However, passive components, like airbags, are not sufficient: active safety technology assisting the driver in the prevention of a crash is vital. Therefore, a system for pedestrian detection plays a fundamental role. Motivated by the importance of such systems, there exists an extensive amount of work done in connection with this field in the last fifty years, but an efficient, robust and real-time solution does not exist yet.Depending on the sensors used to acquire the road scene and the environment information, there exist different systems for pedestrian detection. Although active sensors, like Light Detection And Ranging (LiDAR) –3, work A pedestrian detection involves the following steps: hypothesis generation ; hypothesis verification, either by heuristics ,5) or, m or, m5])A pedestrian detection system can be evaluated using either a per-image measure , as in , or a peThe objective of this article is to study the problem of pedestrian classification across different light spectrum domains (visible and far-infrared (FIR)) and modalities .This article is organized as follows. In Section 2 is presented related work in visible and far-infrared domains in a multi-modality context. Section 3 overviews our main contributions. In Section 4, we present existing datasets for pedestrian detection and classification and also introduce a new dataset that we have acquired and annotated using both a visible and an FIR camera, which we call RIFIR. In Section 5, we evaluate different features over two domains and three modalities, followed by a discussion in Section 6. We conclude in Section 7.2.There is a significant amount of research work in the domain of pedestrian detection and/or classification. In , a revieA few surveys make a direct comparison of different systems at the feature and classifier levels on visible images. For instance, in , the autRecently, a new direction of research for pedestrian classification and detection is represented by the combination of different features and modalities, extracted from the visible domain, such as intensity, motion information from optical flow  and dept2.1.Most of the existing research works use depth and motion for hypothesis generation, by constructing a model of the road scene geometry. For example, in , stereovRather than just using depth and motion as cues for hypothesis generation, a few research works began integrating features extracted from these modalities directly into the classification algorithm. For instance, in , the useThe first extensive analysis was made in , where t2.2.In addition to multi-modality fusion  in the visible domain, several studies use stereovision in the far-infrared domain. For example, in , a four-In ,27, the Overall, there exists an important amount of work around multi-modality in the visible domain, and only a few articles that treat this problem in the FIR one; however, to our knowledge, there does not exist yet a study that integrates both multi-domain and multi-modality aspects.3.In this article, our objective is to analyse the relative performance of the state-of-the-art features, including a feature representation the we proposed in our previous work , called (Q1) Is the FIR domain better than the visible one for the pedestrian classification in the daytime?(Q2) Do the state-of-the-art feature families in the visible domain have the same relative performance when tested in FIR?versus FIR domains?(Q3) Is the intensity self-similarity (ISS) feature family appropriate for pedestrian classification in the considered modalities  in the visible (Q4) What is the relative gain of multi-domain/multi-modality fusion?Our intention is to provide an extensive benchmark framework, which should allow us to answer the following questions:4.There exist several datasets that are publicly available and commonly used for pedestrian classification and detection in the visible domain. In what follows, we briefly present a few widely-used datasets. A more detailed comparison is made in . INRIA  is a welCaltech  is one oAlthough there exists a reasonable number of challenging public benchmark datasets for pedestrian detection in the visible domain, in the case of FIR images, most of the datasets are not publicly available. In Recently in , was prohttp://vislab.it/). Firstly, we have extended the annotations on the dataset proposed by [www.vision.roboslang.org). Moreover, for the two datasets, we have identified and labelled the occluded pedestrians  of the stereovision cameras was of 0.44 × 0.34 radians, although the FOV of the FIR cameras was 0.30 × 0.24 radians. The scene registered with the visible camera is thus larger compared with the one registered with the FIR camera, as i.e., at the data or pixel level).The Parma Tetravisionsystem has been the subject of a strong calibration process, which is essential to obtaining pertinent information about the registered scene. When the system is moving on the road, especially when the road trajectory is uneven or after long distances are reached, it is possible that the system may suffer a miss-calibration. When this issue becomes significant, it could be solved by a manual calibration of the system. Nevertheless, the multi-domain fusion scheme we propose in this paper, at the multi-modal feature level, is less susceptible to the non-calibrations of on-board cameras compared to the ones performed at the lowest level of information (4.2.We have acquired the proposed RIFIR dataset with two cameras: one visible domain camera (colour) with a resolution of 720 × 480 and an FIR camera with a resolution of 640 × 480. We have acquired two sequences in two different image acquisition sessions in similar weather conditions; thus, one of them was used in training and the other one in testing.As for the Parma Tetravision dataset, because of differences in camera optics and position, we have had to annotate the pedestrian independently in the visible and FIR images. In Following the same methodology as in the case of the Parma-Tetravision dataset, for the constructed classification dataset, we have only considered those pedestrians with a height above 32 pixels, which are visible in both cameras, and do not present major occlusions. We have chosen this threshold height due to the fact that a pedestrian with a height of 32 pixels in infrared images is much smaller (with approximately 10 pixels) in the visible spectrum because of the difference in camera optics. In , the reaIn 5.5.1.vice versa.In our experiments, we considered, for the visible domain, the state-of-the-art features: HOG , LBP 4040 and LGHOG features are computed on cells of 8 × 8 pixels, accumulated on overlapping 16 × 16 pixel blocks, with a spatial shift of 8 pixels. This results in 1980 features.LBP and LGP features are computed using cells of 8 × 8 pixels, and a maximum number of 0 – 1 transitions of 2. This results in a number of 4248 features.ISS features are computed on cells of 8 × 8 pixels, a histogram size of 16 pixels and histogram difference. This results in 5944 features.For all considered databases , in order to be consistent in the classification process, we have resized the annotated BBs to a size of 48 pixels in width and 96 pixels in height:These features are fed to a linear SVM classifier. For this, we have used the library LIBLINEAR .vs. detection rate), considering as a reference point the false positive rate obtained for a classification rate of 90%.All of the results in this section are reported in terms of the ROC curve  in the visible domain using the Daimler Multi-Cue dataset. Next, we have computed the relative performance between the considered features  on the visible domain from the Parma-Tetravision and RIFIR datasets. The same methodology is followed in the FIR domain, with the difference being that the state-of-the-art features are evaluated individually on the OlmedaFIR-Classif dataset. Then, the relative performance of these features is considered again on both multi-domain databases. Moreover, multi-modality  over multi-domain  feature comparison and their fusion are performed on the Parma-Tetravision dataset. If on the Daimler Multi-Cue dataset, the disparity map (depth information) is already computed, for the Parma Tetravision dataset, we have used the algorithm proposed in  for effi5.2.We started by analysing the performance of the considered features in the visible domain on the Daimler Multi-Cue dataset.vision.roboslang.org).In In , the autIt is to be noted that our HOG and LBP feature implementation reaches the same performance as the results presented in . We deciThus, while ISS features manage to be more robust for the RIFIR dataset (in the context of noise), HOG and LGP perform better for the Parma-Tetravision dataset . In fact, RIFIR colour images have more noise than the grayscale images from Parma-Tetravision. This has a direct impact on the performance of features based on the gradient: HOG and LGP.This first set of experiments on the visible domain allows us to emphasis the pertinence of ISS features, especially for the depth modality, to demonstrate that the considered features are complementary and that their relative performances are correlated with the characteristics of the images for a given image dataset.5.3.Having established the relative performance of the considered features in the visible domain for the intensity modality, we evaluate next their performance for the intensity modality, this time in the FIR domain. In For the RIFIR and OlmedaFIR-Classif datasets, the best performing feature is LBP, followed closely by LGP. For the Parma-Tetravision dataset, the best performing feature is LGP, followed closely by LBP. For the Parma-Tetravision and OlmedaFIR-Classif datasets, HOG features perform better than ISS, while on the RIFIR dataset, the situation is reversed.In our opinion, the difference in performance between features comes from the fact that even if all three datasets were obtain using FIR cameras, there is a difference in sensors, road scenes and some environmental conditions, like outside temperature. It seems that the LBP and LGP features are more adapted than HOG and ISS features for the task of pedestrian classification in FIR images, taking advantage of local changes in intensity values. Nevertheless, because we consider that those are complementary, we will test this hypothesis with a fusion of features in Section 5.5.The first set of experiments performed on the visible domain has shown that the relative performance between the feature families  varies from one modality to another among the considered modalities . This second set of experiments done in the FIR domain allows us to answer Q2, since the results have shown clearly that the feature families do not have the same relative performances in the visible over the FIR domain. In the following section, we will attempt to answer Q1, if the FIR domain is better than the visible one for pedestrian classification.5.4.Having the performance of different features on both the The performance difference for the RIFIR dataset between FIR and visible is quite large for LGP and LBP with a factor of approximatively 30. HOG and ISS features computed on the intensity from the FIR domain result in a smaller number of false positives than the intensity from visible domains, with a factor of two, on both datasets.5.5.In Section 5.4, we have shown that on the two visible-FIR datasets considered, for the task of pedestrian classification, features computed on FIR images performed better than the counterpart computed on visible ones.By fusing both spectra, as seen in Classification on HOG features computed on the visible and FIR improves by a factor of two for the results, in comparison with just computing those features on the FIR domain, for the RIFIR dataset, and by a factor of five for Parma-Tetravision. For the RIFIR dataset, the same factor of approximately two is obtained for LBP, LGP and ISS features, while on Parma-Tetravision, the factor will be usually equal to or larger than five.5.6.In The best performing feature family for the visible domain, that of LBP with a factor of two less false positives than HOG features, is quite different than the results obtained on the Daimler Multi-Cue dataset, where HOG features had the best performance.For the infrared domain, the best performing feature family is LGP, followed closely by LBP. The HOG and ISS features have, for the infrared domain, quite similar performance, but those features have a larger error rate, in comparison with the LGP and LBP ones. Thus, classification with LGP features has a factor of five less false positives than with HOG features.For the depth modality, the best performing feature family is LBP, followed this time by HOG features. Even if for Daimler Multi-Cue dataset, the ISS features have had the best results for depth, for the Parma-Tetravision, those features are not very robust, having a factor of two more false positives than the LBP features.As concerns the motion modality, in comparison with the experiments performed on the Daimler Multi-Cue dataset where LGP features gave the best results, on these images, the best performing feature family was HOG. We believe that this variation could be due to the difference in quality of the dense optical flow images among these datasets.In conclusion, the relative performances of features in a multi-modality multi-domain context vary depending on the quality of sensors, the environment and image processing. Therefore, we decided to fuse all of them, with the exception of features for the flow modality, which, in our case, were not discriminative enough, in order to take advantage of their complementarity.5.7.We compare for each feature family different fusion schemes: intensity in the visible domain with the infrared one, intensity in the visible domain with the depth modality, the infrared domain with the depth modality, along with the multi-modal multi-domain scheme in the visible, depth and infrared domains. The results obtained on the Parma-Tetravision dataset are given in Fusing the infrared domain with the depth modality lowers the false positive rate in comparison with that obtained for the infrared domain. The relative improvement of performance varies from one feature family to another, being approximately 4 for the HOG features, 1.6 for ISS features, 3.3 for LBP and with a staggering factor of 96 for LGP features.The fusion of the visible and infrared domains with the depth modality provides the overall best results for all feature families on the Parma-Tetravision dataset. This fusion scheme allows similar improvement for the HOG and ISS features. However, the family of local binary features is much more robust, since the fusion of LBP features computed for visible, depth and IR has a factor of nine less false positives than with a classifier trained on the HOG features computed over the same three modalities. The multi-modal multi-domain LGP feature fusion has a factor of over 100 less false positives than with the fused HOG features.6.In In Section 3, we have defined four main questions for which this article wants to provide answers (Q1–Q4). We can definitely answer Q1, since on both datasets acquired in daytime conditions, the FIR spectrum gives better results than the visible one for all of the considered features. If we compare a multi-domain fusion of features, we can observe that the relative gain is quite similar, whatever the feature family, but it might be different from one dataset to another.To answer Q2, if the state-of-the-art feature families in the visible domain have the same relative performance when tested in the FIR one, we conclude that this is not always the case. For example, for the RIFIR dataset, for the visible domain, the best performing feature is LBP, followed by ISS, but at a great distance. On the same dataset, for FIR domain, the best performing feature is still LBP, followed this time by LGP.versus FIR domains, we can observe that the ISS features are less robust than LBP or LGP. Despite the good results obtained in our previous work [As concerns question Q3, if the ISS feature family is appropriate for pedestrian classification in the considered modalities  in the visible ous work  and on DTo answer Q4 , we can observe from 7.This paper has studied the problem of pedestrian classification over two domains, visible and far-infrared, and three modalities, intensity, depth and flow. Moreover, we have proposed a new dataset, RIFIR, which is publicly available, in order to benchmark different features for pedestrian classification. This dataset contains both visible and FIR images, along with correlated pedestrian and non-pedestrian bounding boxes in the two spectra.A comparison between features computed for the visible and FIR spectrum was performed. For the two tested datasets that contain both visible and FIR images, RIFIR and Parma-Tetravision, the FIR domain provided more discriminative features. Furthermore, the fusion of the two domains will further decrease the false positive error rate.Various features have different performances across modalities. As a single modality, for the visible domain, intensity has the best performance for all of the features, followed by depth. Even if the fusion of intensity and depth lowers the false positive rate for all features, the intensity values from the FIR domain had consistently lower error rates.Features computed from the FIR and visible domains are highly complementary, and the use of the two spectra will always lower the error rate. Unfortunately, the information fusion is not straightforward, because two different cameras are used, one for FIR and one for the visible domain; therefore, there will always be a difference in point of view. A correlation method between the two domains is necessary. A possible hardware solution is to construct a camera capable of simultaneously capturing information from both light spectra. Furthermore, as future work, we plan to extend the analysis by benchmarking different pedestrian detection algorithms.


Bombyx mori is also a useful model organism for lepidopteran insect. SET-domain-containing proteins belong to a group of enzymes named after a common domain that utilizes the cofactor S-adenosyl-L-methionine (SAM) to achieve methylation of its substrates. Many SET-domain-containing proteins have been shown to display catalytic activity towards particular lysine residues on histones, but emerging evidence also indicates that various nonhistone proteins are specifically targeted by this clade of enzymes. To explore their diverse functions of SET-domain superfamily in insect, we identified, cloned, and analyzed the SET-domains proteins in silkworm, Bombyx mori. Firstly, 24 genes containing SET domain from silkworm genome were characterized and 17 of them belonged to six subfamilies of SUV39, SET1, SET2, SUV4-20, EZ, and SMYD. Secondly, SET domains of silkworm SET-domain family were intraspecifically and interspecifically conserved, especially for the catalytic core “NHSC” motif, substrate binding site, and catalytic site in the SET domain. Lastly, further analyses indicated that silkworm SET-domain gene BmSu(var)3-9 owned different characterization and expression profiles compared to other invertebrates. Overall, our results provide a new insight into the functional and evolutionary features of SET-domain family.As an important economic insect,  SET-domain superfamily includes all but one of the methyltransferases that methylate specific site of histone lysine (K) residues involved in epigenetic regulation. This family is characterized by the highly conserved SET (Su(var)3-9, E(z), Trithorax) domain that consists of approximately 130 amino acids and is responsible for the catalytic activity of these methyltransferases. Through transferring a methyl group from S-adenosyl-L-methionine (AdoMet) to the amino group of a lysine residue on different sites of histone proteins, SET-domain methyltransferases function in making different histone methylation marks. Histone methylation is very important for the chromatin modification and regulation of gene expression , which pBased on the substrate specificity towards histones, SET-domain-containing lysine methyltransferases can be classified into subgroups like KMT1 (H3K9), KMT2 (H3K4), KMT3 (H3K36), KMT4 (H3K79), KMT5 (H4K20), and KMT6 (H3K27) methyltransferases . SET-dom Drosophila, Arabidopsis, and yeast. On the other hand, more and more recent studies give sufficient evidence of the fact that SET-domain-containing proteins also regulate many nonhistone substrates, including some transcriptional factors and tumor suppressors [ Bombyx mori. Here, we provide an overview of the common and unique features of silkworm SET-domain family members, which will provide more information and important reference to the whole SET-domain proteins.Members of this family have been studied extensively for their function in modifying histones by methylating them directly and thus changing the mode of chromatin to regulate the binding of cofactors in many research models, such as human, mouse,pressors –17. EpigThe silkworm strain, Dazao (p50), used in this study is maintained by the State Key Laboratory of Silkworm Genome Biology. The silkworm larvae were reared with fresh mulberry leaves under 25°C, with a 12 h/12 h photoperiod.http://silkworm.swu.edu.cn/silkdb/). Gene sequences of mammals, C. elegans, D. melanogaster, and some other insects were downloaded from GenBank (http://www.ncbi.nlm.nih.gov/).We combined the updated Silkworm 9x genomic sequencing database, silkworm EST database, CDS database, and silkworm predicted protein database  and Pfam (http://pfam.sanger.ac.uk/) to validate the SET domain in screening hits from silkworm database.Protein sequences of SET-domain family members from other species were used to query the silkworm database with μg of each RNA sample was used to synthesize the first strand of cDNA by M-MLV Reverse Transcriptase following the manufacturer's instructions .Different tissues of silkworm larvae were collected and stored in liquid nitrogen until use. Trizol reagent  was used to extract the total RNA of silkworm tissues. RNA concentration was calculated by spectrophotometer. RNA samples were digested by RNase-free DNase I  to get rid of genomic DNA contamination. 2 μL PCR reaction volume is as follows: initial denaturation at 94°C for 3 min, followed by 30 cycles of 30 s at 94°C, annealing at temperatures Tm  for 45 s and 1 min extension at 72°C, and extension at 72°C for 10 min. The PCR products were analyzed by 1% agarose gels.Primers were designed for most of the identified silkworm SET-domain family members to clone them from silkworm cDNA. Silkworm cytoplasmic gene Actin3  was used as an internal control. PCR amplification was performed using cDNA of deferent silkworm tissues to examine their expression profile. The 20 SET-domain amino acid sequences of silkworm SET-domain family candidates were aligned to each other and also with the representative SET domains of other species by the program ClustalX. The sequences of silkworm SET-domain-containing proteins were applied to construct phylogenetic trees by neighbor-joining algorithm (1000 bootstrap replicates) with the program MEGA4.0.We have identified the silkworm SET-domain family genes from silkworm database SilkDB for the first time. We have found 25 genes containing SET domain from silkworm genome by screening. Referring to the classification method of SET-domain family members in other species which is based on the SET-domain sequence and the feature of its flank motifs or domains, we were able to characterize 17 of them into six subfamilies of SUV39, SET1, SET2, SUV4-20, EZ, and SMYD see . SilkworAccording to the gene sequence from silkworm genome database, we designed primers to clone and verify whether these genes are true hits. We have cloned most members of the silkworm SET-domain family see . ThroughWe have selected the representative subfamily to make phylogenetic analysis; we found that the domain architecture of SUV4-20 subfamily and SETDB1 and Su(var)3-9 of SUV39 subfamily is highly conserved to other species. The three members of silkworm SET2 subfamily are clustered to their homolog of other species, respectively. Xenopus laevis have two SUV4-20 members, while in Drosophila and several other insects there is only one member. In silkworm, we identified only one SUV4-20 member like other insects. Phylogenetic analysis of SUV4-20 subfamily suggests that BmSuv4-20 clusters with Suv4-20 proteins of Drosophila, Anopheles gambiae, and other insects ( Rattus norvegicus are shorter than Suv4-20h1 (Suv4-20 homolog 1) and cluster together. BmSuv4-20 has the same domain structure as SUV4-20 proteins of other species that there is only one domain the SET located on the N-terminal of them , SETDB1 (ESET), and CLLL8 (SETDB2). The SET domains of this subfamily are located on the C-terminal of the proteins which are flanked by Pre-SET and Post-SET domain. The Pre-SET domain close to SET domain is a special character of this subfamily's proteins which assists SET domain in carrying out the enzyme activity . We iden Drosophila. HIF1 contains four conserved domains, SET, Post-SET, AWS, and WW. BmHIF1 identified from silkworm database have all domains but the WW domain, which may be because the sequence of BmHIF1 in silkworm is not complete, which is also confirmed by the relatively shorter sequence of BmHIF1 compared to Drosophila and other insects. ASH1 has four conserved domains except SET domain, AT hook, BROMO, BAH, and PHD. BmASH1 has all the domains except AT hook. Phylogenetic analysis of SET2 subfamily shows that members of silkworm SET2 subfamily cluster together with their homolog proteins in other species separately , and ASH1. NSD1, NSD2, and NSD3 share the conserved domains such as SET, AWS (Associated With SET), Post-SET, PWWP (a domain containing highly conserved Pro-Trp-Trp-Pro motif), PHD, and Ring finger. In silkworm, we just identified one homolog of the NSD proteins, BmNSD1, which has five of the above mentioned conserved domains except the Ring finger. However, so far, there is no homolog protein of NSD identified inparately .The expression profiles of silkworm SET-domain family members show that they have widely high expression level in gonad (testis and ovary) except BGIBMGA002076, which may be related to the gonad's function to propagate the genetic information .Through amplification of homolog gene of Su(var)3-9 in silkworm, we found that silkworm has two transcript isoforms. Using RACE technology to obtain the full-length cDNA sequence showed that the situation is different from other species; the two transcript isoforms have a different sequence of 846 bp at the 5′ end which belongs to the 5UTR of the longer spliceosome, and they encode a protein of 317 and 593 amino acids separately. Embryonic expression analysis of different period and different organs of three-day, fifth-instar larvae of silkworm displays that Su(var)3-9 is highly expressed during the 1–9 days of development of embryo, whereas it is relatively low expressed in the larvae organs 3-9 had been identified at the end of the last century, researchers confirmed the methyltransferase activity of Su(var)3-9 protein in mammals for the first time at the beginning of the 20th century. SET-domain proteins are widely identified from the lower eukaryote yeast to higher human. Human has more than 100 SET-domain-containing proteins. The great SET-domain family is linked to its great function in epigenetic regulation. SET-domain-containing proteins play important roles in the development process of human, Drosophila, and Arabidopsis. There are very few reports on the study of histone methylation in silkworm. Currently, the only one SET-domain protein reported is BmE(z) which is the main member of PcG (Polycomb group genes) proteins. The methylation of H3K27 by BmE(z) has been confirmed in silkworm by knocking down BmE(z) which leads to the reduction of H3K27me3 level [ Drosophila [SET-domain superfamily is a group of histone lysine methyltransferases which form different methylation marks  on histone by transferring the methyl from S-adenosyl-L-methionine (AdoMet) to lysine residues of histone proteins, with production of S-adenosyl-L-homocysteine (AdoHcy). The core enzymatic sites of the methyltransferases are located in the SET domain. Since the first SET-domain proteine3 level . Besidesosophila . Bombyx mori SUV39 subfamily members SETDB1 and su(VaR)3-9 domain composition are highly conserved among various species, especially SETDB1 unique for sequence insertion and splitting the set domain in the silkworm, Bombyx mori and other species are extremely conservative. In addition, G9a-like gene BGIBMGA007949 in the silkworm possessed the conservative composition and structure of G9a and group specificity. BGIBMGA001497, BGIBMGA003106, and BGIBMGA002246 in the silkworm were three respective homologous genes of SET2 subfamily. Interestingly, Su(VaR)3-9 in embryos from day 1 to day 9 and third day of the fifth larval stage were highly expressed, suggesting an important role possibly in the embryonic differentiation process.In our study, from the silkworm database, 24 genes of SET-domain family were identified and 17 out of them were included in SET1, SET2, SUV39, SUV4-20, EZ, and SMYD and six subfamilies. Except for three from SUV39 subfamily members, four from SET1 subfamily members, three from SET2 subfamily members, one from SUV4-20 subfamily members, one from EZ subfamily members, and five from SMYD subfamily members, the remaining five were independent members and two out of them were the homologous genes. Although the RIZ subfamily had not been identified in the silkworm, the certain members had already covered the common members of other species. Comparative analysis for identifying the SET-domain family members showed that they were highly conserved in each species, including the substrate binding site and catalytic site. Evolutionary analysis of the representative subfamily showed that the structure of the SUV4-20 family was highly consistent in the large number of species including the silkworm. Identified in the silkworm, Bombyx mori. We intend to study the common and unique features of silkworm SET-domain family members, which will provide more information and important reference to the whole SET-domain proteins.In summary, based on the silkworm genome database, we identified, cloned, and analyzed the SET-domains proteins in silkworm,


Repeated domains in proteins that have undergone duplication or loss, and sequence divergence, are especially informative about phylogenetic relationships. We have exploited divergent repeats of the highly structured, 50-amino acid WHEP domains that join the catalytic subunits of bifunctional glutamyl-prolyl tRNA synthetase (EPRS) as a sequence-informed repeat (SIR) to trace the origin and evolution of EPRS in holozoa. EPRS is the only fused tRNA synthetase, with two distinct aminoacylation activities, and a non-canonical translation regulatory function mediated by the WHEP domains in the linker. Investigating the duplications, deletions and divergence of WHEP domains, we traced the bifunctional EPRS to choanozoans and identified the fusion event leading to its origin at the divergence of ichthyosporea and emergence of filozoa nearly a billion years ago. Distribution of WHEP domains from a single species in two or more distinct clades suggested common descent, allowing the identification of linking organisms. The discrete assortment of choanoflagellate WHEP domains with choanozoan domains as well as with those in metazoans supported the phylogenetic position of choanoflagellates as the closest sister group to metazoans. Analysis of clustering and assortment of WHEP domains provided unexpected insights into phylogenetic relationships amongst holozoan taxa. Furthermore, observed gaps in the transition between WHEP domain groupings in distant taxa allowed the prediction of undiscovered or extinct evolutionary intermediates. Analysis based on SIR domains can provide a phylogenetic counterpart to palaentological approaches of discovering “missing links” in the tree of life. An increasing amount of whole genome sequence information from diverse organisms offers much promise for determining phylogenetic relationships between known taxa, and resolving the tree of life. However, recent analyses depending on linear sequence data from single gene or genome-scale datasets as indicators of historical relationships between taxa have proved problematic, frequently yielding incongruent trees and equivocal or conflicting phylogenetic hypotheses S. pombe and in protostomes and deuterostomes, in Gly(G)RS and Met(M)RS in protostomes and deuterostomes, but none have been reported in any non-synthetase proteins. An analysis based on intron positions has suggested that the domain was acquired by HRS and GRS early in eukaryote evolution and later spread to EPRS The utility of genome-level characters in inferring phylogenetic relationships is based on the concept that shared genomic changes are indicators of common descent Nematostella vectensis, indicating the evolution of WHEP domains and the non-canonical functionality of mRNA binding in the common cnidarian-bilaterian ancestor Recently, we demonstrated WHEP domains and GAIT element RNA-binding activity in the fused EPRS of the cnidarian Homo sapiens EPRS protein sequence as query. The sequence of EPRS WHEP domains in the elephant shark  was obtained from the elephant shark genome project database. In addition, the linker region that joins ERS and PRS domains of key branch point species, i.e., the primitive vertebrate lamprey (Petromyzon marinus) and the cephalochordate amphioxus (Branchiostoma floridae), were amplified using primers corresponding to flanking conserved regions, and sequenced. Identification as 50-amino acid WHEP domains was confirmed by Pustell dot matrix analysis against human WHEP domains Gallus gallus and Taenopygia guttata) repeats grouped closely with those of the lizard (Anolis carolinensis), supporting a common diapsid ancestor Ornithorhynchus anatinus) had four WHEP domains that grouped with the bird and reptile domains, with the closest mammalian WHEP domains being that of the early-branching marsupial, opossum (Mondelphus domestica). This suggested the relatedness of platypus to reptiles/birds, and supported the phylogenetic position of platypus as intermediate between reptiles/birds and mammals as indicated by the recently sequenced platypus genome Tetraodon nigroviridis, four in Gasterosteus aculeatus, and five in Danio rerio, resulting from lineage-specific duplications  WHEP 3 domains (WHEP 4 in birds and platypus) whereas WHEP 1 was an outgroup to vertebrate (tetrapod) WHEP 1 and 2 domains , the hemichordate acorn worm , and the cephalochordate amphioxus (Branchiostoma florida) showed lineage-specific amplification giving rise to 4 to 6 WHEP domains. Notably, amphioxus WHEP domains clustered with echinoderm and hemichordate domains, suggesting that cephalochordates are more closely related to echinoderms and hemichordates than to urochordates as is generally believed. This phylogenetic position for amphioxus is consistent with recent studies involving multigene datasets The EPRS WHEP domains of the echinoderm sea urchin  forming a strongly-supported separate cluster, whereas the domains from arthropods, annelids, and molluscs clustered together (C. elegans ERS (E6) and the single N-terminal WHEP domain in PRS (P1) formed the outgroup of nematode WHEP domains. This suggests these domains are closest to the EPRS WHEP domains of the last common ancestor of nematodes and arthropods as proposed Drosophila and WHEPs 5 and 6 from the wasp Nasonia vitripennis formed outgroups to all articulata WHEP domains, and showed the closest relationship with C. elegans ERS WHEP E6 and the PRS WHEP domain. The C-terminal WHEP domains of Drosophila and Nasonia EPRS and C. elegans ERS again constitutes a genomic signature of the last common ancestor of all protostomes, and suggests that the other WHEP domains in protostomes were derived by lineage-specific duplications from a single WHEP domain in the EPRS of the last common protostome ancestor.As the analysis of WHEP domain duplication and divergence provided insights into deuterostome phylogeny, a similar analysis was done using seventy-seven WHEP domain sequences from 18 protostomes that included 11 arthropod, two annelid, one mollusc, and four nematode species. Only together . This obB. malawi and L. loa grouped together, supporting a close relationship between these nematode species. Interestingly, Ascaris revealed seven EPRS WHEP domains, whereas the maximum number in EPRS of any other species found in this study is six. However, Ascaris EPRS WHEPs 5* and 6* were atypical and highly degenerate, suggesting genomic instability in that region of the gene. WHEP repeats exceeding six might lead to instability within the EPRS gene, and similarly might have led to fission of C. elegans EPRS, and formation of ERS with six WHEP domains and PRS with one. Similarly, tight grouping of WHEP domains indicated more recent lineage-specific duplications in the mosquito Anopheles gambiae, the wasp Nasonia vitripennis, the water flea Daphnia pulex, and the annelids Capitella capitata and Helobdella robusta. Except for the outgroup WHEP domains 5 and 6 from Nasonia vitripennis, WHEP repeats from the hymenopteran insects  grouped together. Ants exhibited a diversity of WHEP domain repeats; ant species with 2 , 3 (Solenopsis invicta), 4 (Acromyrmex echinatior), and 5 (Camponotus floridanus) WHEP domains formed a separate group with two bee species (Apis mellifera and Bombus terrestris) and showed lineage-specific domain amplification. This group excluded the wasp WHEP domains, suggesting an earlier divergence of ants and bees from wasps in the hymenopteran branch. However, one WHEP domain of the ponerine ant H. saltator grouped with the bee domains, whereas the other WHEP domain formed the outgroup to all hymenopteran WHEP domains. This suggests that H. saltator WHEP domains might be genomic signatures of the last common ancestor of wasps and bees/ants and supports the position of poneroid ants as basal in the ant phylogenetic tree as shown earlier Individual WHEP domains of Schistosoma mansoni, the body louse Pediculus humanus corporis, and the pea aphid Acrythosiphon pisum. Analysis of the pea aphid genome also showed the presence of a second EPRS variant with six WHEP domains. EPRS in the nematode Trichinella spiralis had four highly degenerate WHEP domains, recognizable as repeat domains by low-stringency dot plot analysis, but with little homology with other WHEP domains. Remarkably, the organisms with absent or degenerate EPRS WHEP domains, although belonging to divergent phyla, are all endoparasites or ectoparasites, suggesting a possible link between the absence of these domains and a parasitic life cycle. Because EPRS WHEP domains are involved in the innate immune response in humans, it is possible that these organisms, some of which lack multiple immune system genes Remarkably, we found linked EPRS lacking any WHEP domains in some protostome species, namely, the trematode platyhelminthes Nematostella vectensis as query sequence, showed fused EPRS with one to three WHEP domains in the coral Acropora millepora, sponges Oscarella carmela and Amphimedon queenslandica, placozoa Trichoplax adherens, choanoflagellates Monosiga brevicollis and Salpingoeca rosetta (Proterospongia), and filasterean Capsaspora owczarzaki. A maximum likelihood tree of WHEP repeat sequences from various filozoan taxa including early branching metazoans, choanoflagellates, Capsaspora, and Sphaeroforma arctica, a sister group of filozoans, together with sequences from representative bilaterian taxa was constructed , which formed a sister group to WHEP 1 domain from Monosiga and WHEP 1 and WHEP 3 from the choanoflagellate Salpingoeca rosetta. Remarkably, the other Monosiga WHEP domains assorted between bilaterian and non-bilaterian metazoans; Monosiga WHEP 2 grouped with C. elegans ERS WHEP E6 and the PRS WHEP, forming an outgroup to all nematode WHEP domains, whereas Monosiga WHEP 3 formed an outgroup to all bilateran WHEP domains. This constitutes a genomic signature of a last common ancestor of choanoflagellates and metazoansand supports the phylogenetic position of choanoflagellates as the closest sister group to metazoans with the filastereans as an outgroup to choanoflagellates and metazoans Nematostella which expresses EPRS splice variants with both one and two WHEP domains Inferences regarding the sequence of events leading to the evolution of metazoans from unicellular ancestors require a robust phylogenetic framework of early branching metazoans and their immediate outgroups. However, phylogenetic signals at the base of the metazoan tree have been conflicting, with studies using different sets of DNA and amino acid sequences giving different and inconsistent tree topologies structed . The treCapsaspora has been found to be an outgroup to metazoans and choanoflagellates in several phylogenetic studies, although its exact phylogenetic position in relation to other sister groups of metazoans is uncertain Capsaspora EPRS had two nearly identical WHEP domains suggesting that the fused EPRS originated in a close ancestral organism with a single Capsaspora-like WHEP domain, which underwent a subsequent lineage-specific duplication. We hypothesized that the immediate outgroup of Capsaspora would contain separate ERS and PRS, with a single WHEP domain at the ERS C-terminus or PRS N-terminus, or possibly both, that would have preceded the fusion event. We investigated the genomic sequences of Sphaeroforma arctica, an icthyosporean suggested to be the sister group of filazoa, a clade consisting of filastarea, choanozoa, and metazoa S. arctica ERS harboured a C-terminus sequence recognized as a WHEP domain by dot plot comparison with other EPRS WHEP domains, and by multiple sequence alignment which revealed 46.4% pair-wise identity with Capsaspora EPRS WHEP domains  derived from the ancestral filasterea but contains the “b-like” and “c”-like” WHEP domains derived from choanoflagellates that assorted between the metazoan lineages. A search of newly sequenced metazoan organisms revealed that the deeply-diverging cnidarian Hydra magnipapillata satisfied these criteria, containing a “b-like” WHEP domain grouping with basal metazoans and two “c-like” domains grouping with Monosiga WHEP 2 and C. elegans WHEP domains (two other WHEP domains were highly degenerate). This suggests that EPRS WHEP domains in Hydra are genomic signatures of the predicted ancestor of basal and bilaterian metazoans. Similarly, collapse of bilaterian WHEP domains into a putative species tree reveals evolutionary relationships between the groups and predicts as-yet unknown linking branches . Total RNA from whole adult amphioxus and adult lamprey liver  was reverse-transcribed using oligo(dT) primers and the EPRS linker domain amplified using primers against the conserved enzymatic sequences, and sequenced. EPRS sequence for elephant shark was provided by Byrappa Venkatesh from the Elephant Shark Genome Project at the Institute of Molecular and Cell Biology, A-Star, Singapore (http://esharkgenome.imcb.a-star.edu.sg/). EPRS sequence of Nematostella vectensis was obtained from Stellabase, the Nematostella vectensis genome sequence database. Total RNA from adult Nematostella vectensis anemones  was reverse-transcribed, and EPRS linker domain amplified and sequenced as described Oscarella carmela EPRS sequence was provided by Bernard Degnan, University of Queensland and Amphimedon queenslandica EPRS sequence was obtained from its genome database (www.metazome.net/amphimedon). EPRS sequences for basal metazoans and filasporeans were obtained from the Joint Genome Institute (JGI) and Multicellularity Genome Project at the Broad Institute, Massachusetts Institute of Technology (http://www.broadinstitute.org/annotation/genome/multicellularity_project/MultiHome.html). EPRS sequence of choanoflagellates Monosiga brevicolis and Salpingoeca rosetta were provided by Nicole King, Berkeley University. EPRS linker domain was amplified from cDNA from Monosiga brevicolis (gift from Nicole King) using primers against flanking sequences.EPRS protein sequences for most taxa, unless otherwise stated, were extracted from the National Center for Biotechnology Information (NCBI) databases by BLAST search using protein query against translated nucleotide database (tBLASTn). Most vertebrate EPRS sequences were acquired from the Ensembl databases (www.geneious.com), and dot-plot analysis was performed to identify WHEP domain boundaries. The boundaries were manually applied to designate the 50-amino acid sequence of the WHEP domains. In the case of nucleotide sequence of EPRS linker domains, the sequence was translated in three reading frames and the translated sequences aligned with the human linker sequence to identify the WHEP domains.EPRS protein sequences were pairwise aligned with the human EPRS sequence using Geneious software (www.geneious.com). WAG model with gamma rate variation was utilized. Consensus trees were obtained from the sorted tree topologies after applying a burn-in fraction of 0.25. Maximum likelihood, neighbor-joining, and consensus Bayesian inference trees from each dataset were compared; branches predicted by two or three tree-building algorithms were indicated by * or **, respectively.Phylogenetic analyses were performed using MEGA 5 Figure S1Listing and alignment of all EPRS WHEP domains used in (TIF)Click here for additional data file.Figure S2S. arctica ERS WHEP domain with C. owczarzaki EPRS WHEP domains.Alignment of (TIF)Click here for additional data file.


A comparative model of the single C-terminal caspase-like domain in separase from C. elegans suggests similar binding modes of substrate peptides between these protein subfamilies, and enables differences in substrate specificity of separase proteins to be rationalised. We also modelled a newly identified putative death domain, located N-terminal to the caspase-like domain. The surface features of this domain identify potential sites of protein-protein interactions. Notably, we identified a novel conserved region with the consensus sequence WWxxRxxLD predicted to be exposed on the surface of the death domain, which we termed the WR motif. We envisage that findings from our study will guide structural and functional studies of this important protein family.Separases are large proteins that mediate sister chromatid disjunction in all eukaryotes. They belong to clan CD of cysteine peptidases and contain a well-conserved C-terminal catalytic protease domain similar to caspases and gingipains. However, unlike other well-characterized groups of clan CD peptidases, there are no high-resolution structures of separases and the details of their regulation and substrate recognition are poorly understood. Here we undertook an in-depth bioinformatical analysis of separases from different species with respect to their similarity in amino acid sequence and protein fold in comparison to caspases, MALT-1 proteins ( The separation of sister chromatids is a crucial step in cell division and is triggered by the activation of separase, a protease that cleaves the proteins that maintain the cohesion between sister chromatids. Knowledge of the molecular structure and activation mechanism of separase is limited by the difficulty of obtaining structural information on this large and flexible protein. Sequence conservation between separase homologues from diverse species is limited to the C-terminal region that contains the catalytically active protease domain. We conducted an in-depth bioinformatical analysis of separase and generated structural models of the two conserved domains that comprise the C-terminal region: a caspase-like domain and a putative death domain. This analysis provided insights into substrate recognition and identified potential sites of protein-protein interactions. Both the death domain and caspase-like domain are well-conserved in separases, which suggests an evolutionary pressure to keep these two domains together, perhaps to enable separase activity and/or provide stability. Insights into the molecular structures of separase gained in this study may provide a starting point for experimental structural studies on this protein and may aid therapeutic development against cancers where chromosomes are improperly segregated. Separase overexpression and aberrant nuclear localization are reported in a broad range of human tumours, and its overexpression in mouse models results in tumourigenesis , 2. A stEncephalitozoon and Drosophila species). However, in Drosophila, separase is made up of two genes, sse which is homologous to the C-terminal part of separases from other species and thr, which is homologous to the N-terminal part [Encephalitozoon species with apparently smaller separase proteins also encode for another protein homologous to the N-terminal region of separases.Throughout the cell cycle, separase forms a complex with its inhibitor securin which binds to the N-terminal part of separases while preventing access to the catalytic site –17. Thisnal part . The promucosa-associated lymphoidtissue lymphoma translocation protein 1) and gingipain, this catalytic dyad is brought into juxtaposition by association of two hydrophobic β-strands [Separases belong to clan CD of cysteine peptidases and are related to the clostripain, metacaspase, paracaspase, caspase and gingipain families –23. Each-strands –28. Viad-strands . In addi-strands , 29. The-strands , 19. Mor-strands .X. laevis and humans [Caspases recognize the acidic residue aspartate in P1 position, whereas gingipain-R and the paracaspase MALT-1 are specific for arginine in that position. Separases cleave their substrate Scc1 at two related sites, both with an arginine in the P1 position , 30, 31.d humans –35. Clead humans , a consed humans .Despite a plethora of information about separases’ involvement in the cell cycle, little is known about their structure and function at a molecular level. This is mainly due to an inherent instability of separases without their inhibitor securin, and the flexibility of the separase-securin complex. Most publications to date investigate separase in a cellular context with its inhibitor securin present. In order to gain insights into the structural make-up of separases, their domain organisation and their interaction with securin, we undertook an in-depth bioinformatical analysis of separases. We provide evidence for novel, highly conserved regions in separase that may be important for the overall three-dimensional arrangement of the domains. We also provide a homology model of a separase caspase-like catalytic domain and suggest how a substrate peptide might bind in its active site.H. sapiens, A. thaliana, S. cerevisiae and C. elegans using GLOBPLOT2 [C. elegans, one disordered stretch around residue 400 and another at the far C-terminus in the catalytic domain were predicted. Similar results were obtained from S. cerevisiae (disordered region around residue 950), A. thaliana (disordered regions around residues 410 and 1110) and H. sapiens , 2020 . This indicates that separases are globular proteins with a possible disordered region in the N-terminal region. This region might divide the N-terminal portion from the remainder of the protein and act as a ‘hinge region’, as has been observed in electron micrographs of the human separase-secuin complex [We first examined separases from LOBPLOT2 . GLOBPLO complex .C. elegans separase) as well as three β-strands (residues 720 to 750 in C. elegans separase). In some homologues these predicted β-strands were absent, only two strands predicted or interspersed with short helices. Finally, a well-conserved C-terminal region of approximately 240 amino acids was identified in all homologues that also contains the catalytically active residues histidine and cysteine  and human caspase 3 (PDB code 3EDQ) reveals that both structures are very similar , which  βIII in . Consoli βIII in  and JPre βIII in  revealedTo gain information about the degree of structural conservation between subfamilies of CD clan peptidases, we overlaid the crystal structures of human caspase 3 , gingipC. elegans separase we chose caspase 3 , in D. melanogaster TPEIIRC (putative) and DREIMRE in mouse (putative) [S. cerevisiae are SLEVGRR and SVEQGRR [C. elegans are LMEVERD200 or EVERDRD202  as tempf SIEVGR . Sullivautative) . The rec SVEQGRR . We aligERDRD202 . InteresThe final model shows a globular protein in which a central six-stranded β-sheet is flanked by seven helices in total, three on each face of the sheet and an additional helix (helix α2’) on the side opposite to the active site . The corSubstrate residues P1 to P5 are thought to be responsible for substrate specificity in caspase and separases , 44, 45.C. elegans, highlighted ~ in Gingipain-R and MALT-1 recognize the substrate P1-Arg through side-chain interactions with an aspartate residue  that is located in helix α1 . In contC.elegans sites uniquely have an acidic residue at P2. Our final model indicates that the acidic side chain of the P2 residue may interact with the basic side chains of Arg1120 (which is located in a well-conserved region preceding β6) and Arg1044 (which is located in a loop region between helix α3’ and β4), locking this residue in place  it is Asn267. Therefore, we enabled this residue to form a hydrogen bond to the main chain amide-NH of Lys1118 in our model. In our separase model, P4 may also interact with the side chain of Arg1116 , 780 to 812 (α2), 821 to 835 (α3), 842 to 849 (α4), 855 to 866 (α5) and 872 to 890 (α6) (Casp (329–566) yielded only oligomeric, poorly folded protein whereas the truncation variant hMALT1Casp-Ig3 (334–719) could be expressed in E. coli and was used to solve the hMALT1Casp-Ig3 apostructure [Secondary structure prediction on sequences immediately N-terminal of the caspase domain predicted six α-helices, and multiple sequence alignment of the respective regions in different separases reveals conservation of hydrophobic residues at certain positions . In separase from 890 (α6) . Interes890 (α6) . This dotructure . The cattructure . Moreovetructure , 50 and tructure . These dC. elegans separase and 1776 in human separase with the consensus sequence KAKWWKERxALDTRLGKLL , E3 SUMO-protein ligase ranbp2 (4GA2_A), TPR repeat of Lipoprotein NLPI (1XNF_A), a synthetic consensus TPR protein (2FO7_A) and a putative peptidylprolyl isomerase (3RKV_A). We could not detect sequence similarities to proteins containing HEAT or ARM repeats with confidence. However, prediction of HEAT or ARM repeats is challenging because of their great sequence variability [The N-terminal region of separase is thought to consist of helices, which may be assembled into ARM or HEAT repeats , 29 or t2  matched iability , and we et al. [et al. showed that securin efficiently hinders binding of a substrate to the separase active site but binding of securin to separase’s C-terminal region is weak [et al. identified a fragment of securin, residues 81 to 156 that physically interacted with separase via a DIE motif [S. cerevisiae and C. elegans and is located in an acidic region within securin. Recently, Han et al. identified His134 in human securin to be important for separase binding and initialisation of proteolytic activity [The C-terminal region of separase contains a catalytic domain belonging to the CD clan of proteases, including caspases, MALT-1 and gingipain-R. Specifically, we propose that the conserved C-terminal domain of separases is very similar to the joint smaller and larger subunits of caspases  and gingipain R, subdomain B, and we refer to it as a caspase-like domain. In contrast to Viadiu et al.  we couldet al. , MALT-1 et al.  and ginget al. . In theset al. –60. Vacuet al. , 62. Preet al. , 33. As et al.  and buddet al.  showed t is weak . They thIE motif . This moactivity . This reC. elegans separase with a proposed substrate peptide (sequence MEVER) is consistent with the predicted recognition sequences for separase from C. elegans (LMEVER/D200 or EVERDR/D202). In contrast to other known separase recognition sequences, these harbour an acidic residue in P2 position that is recognised by a pair of arginines. Comparison with known structures of caspases, MALT-1 and gingipain-R bound to peptide inhibitors revealed that while the P1 aspartate residue is locked into position via hydrogen bonding to arginines in caspases, in gingipain-R and MALT-1 this interaction is made by an acidic residue in a similar position. Notably, separases, which also recognize arginine in P1 position, all possess a glutamic or aspartic acid in place where gingipain-R harbours Asp163 in its S1 pocket. Our model suggests that additional interactions between the substrate peptide backbone and protease active site are similar and are therefore likely to be conserved. It is likely that dynamic changes in the substrate pocket also contribute to substrate recognition and specificity, and we believe that this will operate in a broadly similar way to the situation in caspases [Our model of the caspase-like domain of caspases . To enabDespite the clear importance of separase in the fundamental processes of chromosome segregation and licensing of centrosome duplication, there has only been a single published structural biology study of this enzyme . We enviAmino acid sequences of separases from different organisms were taken from NCBI. Great care was taken to include as many classes of animals and plants as possible, avoiding a bias of the multiple sequence alignment towards mammals. Separate alignments were carried out for the C-terminal caspase-like domain, the central proposed death domain and the N-terminal domain. Initial alignments were performed in ClustalW  using deCaenorhabditis elegans were generated using Modeller 9.12 [www.modelarchive.org) as access code ma-ajsa8 and the model for the death domain as access code ma-awsce.The theoretical models of the caspase-like domain and the proposed death domain from separase of ler 9.12 , 69. Comler 9.12 . Energy ler 9.12  using stSurface electrostatics and positions of hydrogen bonds were determined using PyMOL . All figures of structures were prepared using PyMOL.S1 FileAligned sequences of caspase domains from caspases, gingipain R and separases from different species.(TXT)Click here for additional data file.S2 FileAligned sequences of Scc1 proteins from different species. Putative and confirmed cleavage sites are indicated as well as predictions of secondary structure elements.(TXT)Click here for additional data file.S3 FileAligned sequences of separases from different species and sequences of known death domains (PDB code indicated). Predictions of secondary structure elements are indicated.(TXT)Click here for additional data file.


Having been reported from only seven current insect orders, the exact phylogenetic distribution of dsx within the largest Arthropod sub-phylum, the Hexapoda, is unknown. To understand the evolution of this integral gene relative to other arthropods, we tested for the presence of dsx within public EST and genome sequencing projects representative of all 32 hexapod orders. We find the dsx gene to be ubiquitous, with putative orthologs recovered from 30 orders. Additionally, we recovered both alternatively spliced and putative paralogous dsx transcripts from several orders of hexapods, including basal lineages, indicating the likely presence of these characteristics in the hexapod common ancestor. Of note, other arthropods such as chelicerates and crustaceans express two dsx genes, both of which are shown to lack alternative splicing. Furthermore, we discovered a large degree of length heterogeneity in the common region of dsx coding sequences within and among orders, possibly resulting from lineage-specific selective pressures inherent to each taxon. Our work serves as a valuable resource for understanding the evolution of sex determination in insects.The  Doublesex/Mab-3 Related Transcription factor (DMRT) family of zinc-finger proteins145doublesex, the gene that acts as the terminal “double-switch” in the insect sex determination cascade.Somatic sexual differentiation is fundamentally conserved among metazoans. The development of distinct male and female phenotypes integral to sexual reproduction is directed by the Drosophila melanogaster7910sexlethal (sxl) and transformer (tra). Functional SXLF protein  then splices tra pre-mRNA to encode functional TRAF protein, which in turn splices dsx pre-mRNA in the female specific manner. Male-specific SXLM and TRAM are truncated and do not appear functional, resulting in male-specific dsx mRNA. As sxl is limited to Drosophila and tra appears lost in several insect lineages1213F and male-specific DSXM transcription factors remain elusive, however recent work has shown that D. melanogaster DSX binds thousands of targets from multiple tissues in both sexesdsx is also known to influence the development of both behavioral and morphological secondary sex characteristics1617Best elucidated in the model fly doublesexDSX proteins retain conserved domains essential for transcription factor activity and oligomerization: an atypical zinc-finger DNA-binding DM domain (herein OD1) conserved throughout the DMRT gene family that exhibits a characteristic C2H2C4 Cys-His configuration1920Dsx transcripts have been recovered in the crustacean Daphnia magnaMetaseiulus occidentalisdsx pre-mRNA shows no sign of alternative splicing and directs sexual fate via sex-biased expression in males. The extent of conservation of this critical gene throughout hexapods however remains unknown, as thus far dsx homologs have been recovered from genome and/or transcriptome data of only seven insect orders  from 30 orders, 22 of which were represented by at least one EST encoding both an OD1 and downstream OD2 domain. Included in this latter group were species of primitive entognathous hexapods (i.e. Protura) and the derived pterygote insects, confirming the presence of doublesex in the common ancestor of the HexapodaTo understand the breadth and functional conservation of these genes throughout insects (as well as other Metazoa) we used public transcriptome datasets, including recent EST data generated for all 32 currently recognized hexapod ordersdoublesex EST contigs encoding both an OD1 and OD2 domain (designated herein as “high-confidence” dsx EST contigs) from 22 hexapod orders (de novo contigs from short read RNAseq libraries (see Methods) for Sipyloidea sipylus (Phasmatodea) and Lepismachilis y-signata (Archaeognatha) that contained high-confidence ESTs.We recovered d orders  online. doublesex ESTs recovered maintained a conserved Lysine amino acid residue at position 14 of the DM/OD1 domain alignment, most frequently with a Thr-Pro-Pro-Asn-like motif at positions 1–4 , we find type-B singletons alone to be poor evidence for the presence of insect dsx and may represent a convergent zinc-finger domain from an alternative DMRT-superfamily gene . Of note, the conserved lysine at position 21 responsible for maintaining a salt bridge with the target DNA moleculeWith the exception of four orders, all high-confidence doublesex from the termite (Isoptera) data, as only a single type-A OD1 motif was recovered with no evidence for OD2. Two BLAST hits to a type-B-like OD1 motif (contigs Znev_05388 and Znev_16235) were recorded from the Zootermopsis nevadensis predicted proteomecoupling of ubiquitin to ER degradation) DMA domain in the C-terminus  characteristic of the DMRT family but not of doublesex. No BLAST hits to OD2 were reported from Z. nevadensis. A profile HMM search using the model we created (see Methods) failed to identity an OD2 motif. As a final check, we searched the Nasutitermes takasagoensis raw sequencing reads generated by Hayashi et al.DRR013047 again using TBLASTN and profile HMM. This returned a single read  transcriptome data, thus we scanned their constitutive raw short-read RNAseq libraries  from the other insects. Loss of the dimerization (OD2) domain would almost certainly impact the DNA-binding affinity of DSXSimilarly, we did not recover an OD2 domain from assembled Embiopteran , we chose to query the assembled genome of the German cockroach 01136787  online. Metallyticus splendidus, Mantis religiosa, Corydalus cornutus, Xenophysella greensladeae), however further protein biochemistry will be required to qualify the exact effects on DNA-binding affinity.Several taxa were found to possess modified zinc finger structures responsible for binding target DNA and modulating transcription. The canonical C2H2C4 Cys-His motifdoublesex homologs are the water flea Daphnia magna (Arthropoda: Crustacea: Branchiopoda: Cladocera: Daphniidae)Metaseiulus occidentalis (Arthropoda: Chelicerata: Acari: Phytoseiidae)dsx and lack sex-specific alternative splicing in favor of sex-biased expression in males. The possibility thus exists that the hexapod common ancestor may have encoded two or more dsx genes.The nearest hexapod outgroup taxa with reported dsx EST (encoding both OD1 and OD2 domains) and either an additional divergent OD2-encoding singleton or a second high-confidence EST  from eight hexapod orders: Protura, Zygentoma, Ephemeroptera, Zoraptera, Phasmatodea, Mantodea, Hymenoptera and Diptera  than did the singleton, and produced higher domain e-values in homology searches against the profile HMM. Expression of the diverged transcript also appears to be much higher than that of the canonical form; the zygentoman libraries SRR921654, SRR921648 and SRR921568  contained 148, 114 and 164 respective reads that mapped to the OD2 domain of the singleton EST, while 2, 2 and 5 reads from the same libraries mapped to the OD2 domain on the high-confidence EST (not shown). Interestingly, high-confidence ESTs identified within the Zygentoman data exhibit a greater degree of amino acid similarity to the high-confidence ESTs identified within the Ephemeroptera than to their conspecific singleton reads encoding an OD2 domain , Coleoptera (Meloe violaceus) and Diptera (Belgica antarctica). Further taxon sampling will be required to determine whether these represent individual cases of gene duplication, or retention of an ancestral state. Additionally, multiple members of the Coleoptera  were found to encode a second OD2 domain downstream of the first  splicing within the hexapods (currently reported only from within the holometabolous insects), we mapped raw sequencing reads (retrieved from the NCBI SRA and corresponding to the transcriptome assemblies used in this paper) from basal orders  to the 3′ end of the respective OD2 domain identified for each. We chose this location as it has been shown to harbor the sex-specific exon in the Diptera343542Lepismachilis y-signata) and Collembola (Anurida maritima) that support an alternative dsx isoform that diverges immediately downstream of the Glutamic acid residue encoded in the 3′ OD2 mRNA  will be required to confirm these alternate isoforms, and to qualify their sex-specific expression.Insect male and female-specific sexual development has been shown in multiple species to proceed via sex-specific splicing of OD2 mRNA . This sidoublesex homologs. The length (in nucleotides) between the OD1 and OD2 domains varied widely both within and between orders . Whether this pattern is maintained throughout the Orussidae is of future interest, as a second member of the “Symphyta” (Tenthredo koehleri) had a much shorter inter-domain distance. The phasmid Aretaon asperrimus had the smallest distance bridging the two domains at only 84bp. Signs of positive selection have been reported within the common region of DSX among closely related members of the Anastrepha fraterculus species groupPapiliodsx are diverse and differ even among closely related taxa3435We observed significant length heterogeneity between n orders . The avedoublesex to be present throughout the Hexapoda, and likely present in the last common ancestor of the subphylum. The recovery of EST contigs that encode both the DNA-binding (DBD/OD1) and dimerization domains (OD2) from members of the primitive Entognatha to the derived orders of holometabolous insects attests to the conservation of this integral double-switch gene in the sex-determination cascade. Of note, we were unable to recover evidence for the OD2 domain in the embiopteran and isopteran datasets. Despite the use of alternative short-read libraries in addition to transcriptome sequence archives, the domain (if present) has diverged beyond our ability to isolate it via profile hidden Markov model and/or BLAST in these orders.Our results show dsx likely existed as a paralogous gene in the hexapod common ancestor, as at least two copies are transcribed in the basal Entognatha (among other orders), and also present in sequenced crustacean and chelicerate outgroups. Additionally, the phenomenon of sex-specific alternative splicing, currently undescribed outside of the insects, appears to occur in the most basal orders and is thus likely to have been present in the proto-hexapod. The advent of sex-specific alternative splicing coupled with mutation and eventual loss of dsx gene copy in the higher insect orders are perhaps concomitant, with neo-functionalization of the alternate isoform replacing the lost copy. Tandem duplications of the OD2 domain in the Coleoptera reported here, perhaps once functional, appear to have been inactivated via accrual of mutations. The extreme variation in coding sequence length and conservation observed among and within orders may provide evidence of a punctuated molecular response to sexual selection pressures influenced by the phenotypes that dsx modulates. The transcripts identified here serve as initial candidates for studying pan-insect evolution, and for development of transgenic sexing techniques for insect pestsWe present evidence that Doublesex dimerization domain [OD2]). Contigs with both domains present were extracted and translated in the proper reading frame as reported by BLAST. We then extracted and manually aligned the two protein domain motifs with their respective orthologs from other orders. These data compose the ‘high-confidence’ dataset, as the presence of both domains on a single EST is strong evidence for doublesex homology. We entered the remaining EST contigs encoding a singular OD1 or OD2 domain into the alignment in the same manner. To search for remote (or diverged) homologs, an alignment of OD2 domain sequences (OD1 maintained strong sequence similarity) from the high-confidence dataset was used to construct a profile hidden Markov model (HMM) using HMMER v3.1b1We queried the NCBI Transcriptome Shotgun Assembly (TSA), Whole-Genome Shotgun (WGS), and non-redundant (nr) databases via tBLASTn/BLASTp  with the Pfam seed alignments for pfam00751 (DM DNA binding domain [OD1]) and pfam08828 (dsx EST. The Lepismachilis y-signata (Archaeognatha) Illumina-generated RNAseq data deposited in NCBI SRA accessions ERR424579, ERR392013, ERR392014, and ERR392008 were downloaded and assembled using the CLC Genomics Workbench de-novo assembler . A BLAST database was created from the resultant contigs and queried with the OD1 and OD2 consensus sequences. Both domains were recovered on assembled contigs 383881 and 336923, respectively  of Misof et al.We used several data sources outside of the NCBI TSA and nr databases as secondary sources of evidence for orders in which we failed to recover a high-confidence ectively  online. Zootermopsis nevadensishttp://termitegenome.org and queried via BLASTP with the OD1/2 protein sequences reported here for the roaches  in an attempt to isolate a high-confidence genomic contig encoding both OD1 and OD2 domains. We queried the assembled genome scaffolds via TBLASTN with roach OD1/2 sequences identified within the NCBI TSA, and with the OD2 profile HMM.The predicted proteome of the dampwood termite dsx transcripts within the basal hexapods, we retrieved the sequencing reads from the NCBI SRA corresponding to species for which we had recovered an OD2 domain: Protura (SRR921562 [Acerentomon sp. AD-2013]), Collembola , Diplura (SRR921624 [Occasjapyx japonicas]), Archaeognatha  and Zygentoma . These reads were six-frame translated and pattern matched to the 3′ end of the OD2 domain to discern divergent splice forms.To search for alternative splicing of How to cite this article: Price, D. C. et al. The ubiquity and ancestry of insect doublesex. Sci. Rep. 5, 13068; doi: 10.1038/srep13068 (2015).


We found a significantly increased number of total boutons and satellite boutons in conjunction with compromised endocytosis in the neuromuscular junctions (NMJs) of ube3a mutants compared to the wild type. Genetic and biochemical analysis showed upregulation of bone morphogenetic protein (BMP) signaling in the nervous system of ube3a mutants. An immunochemical study revealed a specific increase in the protein level of Thickveins (Tkv), a type I BMP receptor, but not other BMP receptors Wishful thinking (Wit) and Saxophone (Sax), in ube3a mutants. Ube3a was associated with and specifically ubiquitinated lysine 227 within the cytoplasmic tail of Tkv, and promoted its proteasomal degradation in Schneider 2 cells. Negative regulation of Tkv by Ube3a was conserved in mammalian cells. These results reveal a critical role for Ube3a in regulating NMJ synapse development by repressing BMP signaling. This study sheds new light onto the neuronal functions of UBE3A and provides novel perspectives for understanding the pathogenesis of UBE3A-associated disorders.Altered expression of the E3 ubiquitin ligase UBE3A, which is involved in protein degradation through the proteasome-mediated pathway, is associated with neurodevelopmental and behavioral defects observed in Angelman syndrome (AS) and autism. However, little is known about the neuronal function of UBE3A and the pathogenesis of UBE3A-associated disorders. To understand the  Drosophila melanogaster as a model system in conjunction with an array of biochemical and physiological assays, we showed that mutants of ube3a had excess synaptic boutons and endocytic defects at the neuromuscular junction terminals due to an elevated level of bone morphogenetic protein (BMP) signaling. Specifically, Ube3a directly binds and ubiquitinates the BMP receptor Thickveins for proteasomal degradation; a function that is conserved in mammalian cells. Negative regulation of BMP signaling by UBE3A suggests a previously unknown molecular mechanism that underlies the pathogenesis of UBE3A-associated AS and autism.Angelman syndrome (AS), characterized by severe mental retardation, developmental delay, ataxia, seizures, speech impairment, and happy disposition, is caused by mutation of E3 ubiquitin ligase UBE3A; a critical enzyme involved in proteasome-mediated protein degradation. Increasing evidence demonstrates that overexpression or hyperactivation of UBE3A is associated with autism. Thus, both loss and gain of UBE3A functions result in neurodevelopmental and cognitive defects. However, the neuronal functions of UBE3A and the mechanism by which altered expression of UBE3A leads to developmental and cognitive defects are poorly understood. Using  UBE3A results in autism [Angelman syndrome (AS) is a neurodevelopmental disorder characterized by severe mental retardation, developmental delay, ataxia, seizures, speech impairment, and happy disposition , 2. It in autism –7. A poin autism . Thus, bUbe3a mutant mice, a valid model of AS, show impairment in hippocampal long-term potentiation (LTP) [Ube3a mice [Ube3a mutant mice are attributed to decreased Ube3a-mediated proteasomal degradation of RhoA guanine exchange factor (GEF) Ephexin5 and synaptic protein Arc [UBE3A is involved in ubiquitin–proteasome-mediated protein degradation. Although several substrates of UBE3A have been identified, few have been implicated in neural development and function . Ube3a mon (LTP) , 11 and on (LTP) , 13, togon (LTP)  and alteon (LTP) . Moreoveon (LTP) –18. Whiltein Arc , 20. Howtein Arc , 22. Thuube3a, the Drosophila homolog of human UBE3A [ube3a loss-of-function mutants, overexpression of wild-type ube3a also leads to motor abnormalities, circadian rhythm defects, and decreased dendritic branching [Drosophila ube3a regulates synapse development. No direct targets of Ube3a-involved proteasomal degradation have been definitely identified in Drosophila, despite a series of proteomic studies [Null mutants of an UBE3A , show dean UBE3A , 25, as an UBE3A . Similarranching –26. So f studies , 27, 28.Drosophila Ube3a regulates synapse development and function at neuromuscular junctions (NMJs) by ubiquitinating and promoting proteasome-mediated degradation of the type I BMP receptor Tkv, and that the negative regulation of Tkv by Ube3a is conserved in mammalian cells. These results offer novel insight into the intricate regulation of BMP signaling in the nervous system, and suggest a potential intervention strategy for UBE3A-associated disorders by manipulating BMP signaling via genetic and pharmacological means.Bone morphogenetic protein (BMP) pathway plays a crucial role in neural development throughout evolution –31. In tube3a mutants through P-element-mediated excisions of EP3214, an insertion in the first exon. Multiple imprecise excisions  were obtained (ube3a549 and ube3a689) induced by the chemical mutagen ethyl methanesulfonate (EMS) were identified via a TILLING service (see ube3a549/Df(3L)ED4470 (which removes the ube3a locus completely) and ube3a689/Df(3L)ED4470 mutants and in homozygous ube3a35 mutants, whereas a truncated protein of 110 kDa was detected in ube3a8 mutants ube3a mut mutants . Immunos mutants  Figs. Boratories , 26, werUbe3a mutant mice [Drosophila ube3a may play an important role at synapses. To test this possibility, we examined the NMJs of wandering third instar larvae by staining them with anti-cysteine string protein (CSP), a synaptic vesicle marker, and anti-horseradish peroxidase (HRP), which labels the presynaptic neuronal membrane. In ube3a35 null mutants, the total bouton number for muscle 4 NMJs in abdominal segments A2 and A3 was 33.4 ± 1.2, which was significantly higher than 26.8 ± 1.1 in wild-type (WT) larvae (P < 0.001) ED4470 and ube3a549/Df(3L)ED4470 also showed more boutons than the wild type. The number of satellite boutons budding from a parental bouton in ube3a35 mutants was also significantly higher than in wild-type larvae; these satellites were more frequent at the distal ends of NMJ terminals  . Similarerminals . To conf mutants . However did not , althougube3a exerts its effect on the pre- or postsynaptic side, we knocked down Ube3a expression by RNA interference (RNAi) in a tissue-specific manner. Targeted knockdown of ube3a in presynaptic neurons by elav-Gal4-driven Thu3266 RNAi resulted in a modest but significant increase in the number of total boutons (20.15%) and a dramatic increase in satellite boutons (371.7%) compared to wild-type larvae  within a 200 nm radius of the active zone of transmitter release was normal in ube3a mutants compared to wild type  and miniature EJPs (mEJPs) in ube3a35 mutants did not differ from those in the controls (ube3a mutants was similar to that in wild-type larvae (ube3a35 mutant (B) are shown in ube3a mutants. We then examined the ability of ube3a mutants to maintain neurotransmitter release during intense stimulation. The EJP amplitudes in ube3a mutants under high-frequency stimulation of 10 Hz in the presence of 0.5 mM extracellular Ca2+ for 10 min declined much faster and remained at 64% of the initial response , which then translocates to the nucleus to activate target gene transcription. Elevated pMad levels are thus indicative of active BMP signaling , 40, 41. mutants . These r by half .ube3a mutants to determine whether any are Ube3a substrates. Western blotting of larval brain extracts showed that pMad level was significantly increased by 203%, while Mad level was increased by 71% in ube3a mutants, consistent with the staining results (ube3a mutants compared to wild type (P < 0.001), while the level of the endogenous Sax and Wit, the other type I and type II BMP receptors, respectively, was unaltered  against GFP, Tkv level decreased markedly from 1.5 h to 9 h upon drug treatment , which was blocked by the proteasome inhibitor MG132  through P-element-mediated excisions of EP3214. Transgenic lines expressing genomic ube3a+ and ube3aC941A  were from J. Fischer. UAS-Tkv-GFP was from T. B. Kornberg. UAS-DTS5 was a gift from J. Belote via K. Broadie [ube3a549 and ube3a689 were obtained from a TILLING  service (http://tilling.fhcrc.org). For tissue-specific rescue experiments, the pan-neural elav-Gal4 (from Bloomington) and muscle-specific C57-Gal4 (from V. Budnik) lines were used. RNAi lines P{GD450}v45876 and Thu3266 were obtained from the Drosophila RNAi CenterVienna  (VDRC) and Tsinghua University , respectively. A yellow fluorescent protein (YFP) genetrap line labeling the endogenous Tkv with YFP was obtained from the Drosophila Genome Research Center . EP(3)3214, Df(3L)ED4470, tkv8, tkvK16713, mad12, and witB11 were obtained from the Bloomington Stock Center.All  Broadie . The EMSube3a from a home-made cDNA pool derived from whole adults, and cloned into the vector pUAST. An UAS-ube3a insertion in the X chromosome was used in this study.For overexpression studies, UAS-ube3a was obtained by amplifying the full-length cDNA of Drosophila Ube3a with an N-terminal 6×His tag was produced in E. coli. Mouse monoclonal antibodies produced by clone 8F7 were shown to be specific for Ube3a by western blotting and immunostaining.To generate specific antibodies against Ube3a, a fusion protein containing amino acids 568–973 of Immunohistochemical analysis of larval NMJ synapses was performed as previously described . The folFor statistical analysis of NMJ morphology, the number of total boutons and satellite boutons of muscle 4 NMJ in abdominal segments A2 and A3 was quantified as previously described .2+-free HL3.1 saline and recorded in HL3.1 saline containing 0.5 mM CaCl2. Intracellular microelectrodes with a resistance greater than 5 MΩ filled with 3 M KCl were used for the assay. Excitatory junction potential (EJPs) and miniature EJPs (mEJPs) were recorded from muscle 6 in the abdominal segments A2 or A3 for 120 seconds. EJPs were elicited by low frequency (0.3 Hz) stimulation. We analyzed recordings from muscles cells with physiological resting potentials ≤ -60 mV and input resistances > 5 MΩ. For examining synaptic transmission under tetanic stimulation, synapses were stimulated at 10 Hz for 10 minutes and recorded in a modified HL3.1 saline with 0.5 mM extracellular Ca2+. For the FM1-43 uptake assay, we followed a previously published protocol [Electrophysiological recordings were performed at 20°C essentially following a conventional procedure with minor modifications , 34. Forprotocol .ube3a and gfp, DNA fragments were synthesized by PCR using primers containing a T7 promoter sequence : ube3a sense: 5’-TAA TAC GAC TCA CTA TAG GGA TTG CCG GAA ACC ACT GAT A-3’ and antisense: 5’-TAA TAC GAC TCA CTA TAG GGC TCC GTT CTC AAA TGG TGT G-3’, and gfp sense: 5’-TAA TAC GAC TCA CTA TAG GGA TGG TGA GCA AGG GCG AGG A-3’ and antisense: 5’-TAA TAC GAC TCA CTA TAG GGC TTG TAC AGC TCG. The dsRNAs were transcribed from both strands of the PCR-amplified DNA fragments using a Megascript T7 kit , and purified using an RNeasy kit (Qiagen).S2 cells were cultured in Schneider’s medium  and transfected using Cellfectin II reagent (Invitrogen). For the production of double-strand RNA (dsRNA) targeting ube3a or gfp (control) at an average transfection efficiency of about 46%, and treated 48 h later with 40 μg/ml cycloheximide (Sigma) for 0, 1.5, 3, 6, and 9 h. To determine whether the degradation of Tkv was proteasome-dependent, S2 cells were co-transfected with flag-Ube3a or flag-GFP plasmid together with myc-Tkv. After 48 h, cells were treated with 20 μM MG132 (Sigma) or mock treated for 6 h before being harvested for western blotting.To examine Tkv protein turnover, S2 cells were transfected with dsRNAs against 2. For efficient RNAi knockdown of human UBE3A, UBE3A siRNA1 (5’-GGG UCU ACA CCA GAU UGC UTT-3’) [UBE3A and ALK3  clones were provided, respectively, by Dr. Peter M Howley and William C. Hahn at Harvard Medical School . UBE3A and ALK3 coding sequences were subcloned to generate pCMV-Tag2B-HA-UBE3A and pCDNA3-myc-ALK3, respectively.Human HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Invitrogen) at 37°C in a humidified incubator with 5% CO UTT-3’) , siRNA2  UTT-3’) , and a sIP assays of larval brains and ventral ganglia were carried out according to a previous report . ImmunopTo analyze protein levels in different genotypes, larval brains and ventral ganglia were homogenized in ice-cold lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% (v/v) NP-40, 0.1% (w/v) SDS, 1% (v/v) proteinase inhibitor). Western blots were probed with primary antibodies anti-Ube3a, anti-pMad , anti-Wit, anti-GFP, and anti-α-tubulin, followed by HRP-conjugated secondary antibody .in vivo ubiquitination assay, S2 cells were transfected with plasmids using Cellfectin II reagent (Invitrogen). The ubiquitination assay was performed according to the protocols described previously [For eviously . In brieelav-Gal4; tkv-GFP and elav-Gal4; tkv-GFP; ube3a35) were dissected and incubated with 50 μM MG132 (Sigma) in Schneider’s medium for 4 h to inhibit proteasome-mediated degradation before proceeding with IP. The brains were then homogenized in lysis buffer and the lysates were immunoprecipitated with anti-GFP. The ubiquitinated Tkv was examined by anti-ubiquitin antibody. For in vitro ubiquitination assay, the myc-tagged Tkv C-terminus (myc-TkvC) was synthesized by an in vitro translation kit containing rabbit reticulocyte lysates (Promega). Ube3a was fused to GST, then expressed and purified from E. coli. E1, E2, HA-Ub , and the potential substrate myc-TkvC, together with the GST control, the E3 ligase Parkin control, or GST-Ube3a, were incubated at 30°C for 1.5 h in 40 μl ubiquitination reaction buffer (50 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol, 50 mM NaCl, 5 mM MgCl2, 2 mM ATP). Reactions were terminated by adding SDS-PAGE sample buffer and analyzed by western blotting.To detect the levels of ubiquitinated Tkv in vivo, 200 larval brains of each genotype . Power SYBR Green PCR Master Mix (Applied Biosystems) was used for quantitative real-time PCR. Actin mRNA level was amplified as an internal control using published primers , whereast tests were used for statistical comparisons between two groups. Multiple group means were compared by one-way ANOVA. Asterisks above a column indicate comparisons between a specific genotype and wild type, whereas asterisks above a bracket denote comparisons between two specific genotypes. Data are presented as mean ± standard error of the mean (SEM). P-values < 0.05 were considered statistically significant.Student’s S1 Figube3a35 ventral ganglion. (C) Ube3a is expressed in muscles and axons with specific enrichment at presynaptic NMJ terminals compared with background expression of Ube3a in mutants. Larval preparations were double-stained with anti-Ube3a (green) and anti-HRP (magenta). Ube3a is cytoplasmic and expressed in the adult ovary (D) and adult testis (E).(A) Ube3a protein is cytoplasmic and is highly expressed in the ventral ganglion of a third instar larva. The ventral ganglion was double-labeled with a monoclonal antibody 8F7 against Ube3a (green) and propidium iodide (magenta) to visualize nuclei. (B) No specific Ube3a staining was detected in (TIF)Click here for additional data file.S2 Figen-Gal4/+; RNAi /+; A) or an upregulated level  of Ube3a in the posterior compartment by en-Gal4. Tkv protein level remains unchanged upon altered expressions of Ube3a. Scale bar = 40 μm. (C) The ventral ganglion of a OK6-Gal4/UAS-CD8-GFP larva was double-labeled with GFP (bright enough without staining) and anti-Ube3a. Ube3a expresses in GFP-positive motoneurons as well as GFP negative interneurons. (D) No specific Ube3a staining was detected in ube3a35 ventral ganglion. Scale bar = 40 μm. The specificity of anti-Ube3a was demonstrated by staining wing disc expressing a reduced level ((TIF)Click here for additional data file.S3 FigOK6-Gal4-driven Thu3266 RNAi against ube3a in motoneurons led to more total boutons and satellite boutons compared with the control. Scale bar = 10 μm. (B) Quantification of total boutons and satellite boutons in control  and OK6-Gal4/+; RNAi/+ animals. n ≥ 16 NMJs; ***P < 0.001 by t test; error bars represent SEM. (C) The NMJ growth appeared normal in ube3a15B homozygotes, but there were more total boutons and more satellite boutons in ube3a15B/ube3a35 and ube3a15B/Def mutants compared with ube3a15B. (D) Quantification of the total boutons and satellite boutons in the three genotypes. n ≥ 16 NMJs; ***P < 0.001 by one-way ANOVA test; error bars represent SEM.(A) Muscle 4 NMJ synapses were double-stained with anti-HRP (green) and anti-CSP (magenta). (TIF)Click here for additional data file.S4 Figube3a35 mutants (B). Compared with wild type, ube3a35 mutants exhibited significantly more cisternae with diameters >60 nm (arrowheads in B). Arrows in A and B indicate active zone. Scale bar, 500 nm.  High magnification view of representative active zones from wild type (C) and ube3a35 mutants (D). Dashed line defines a 200 nm radius around the active zone for quantitative analysis of SVs. Scale bar, 100 nm. (E) High magnification view of a presynaptic bouton from ube3a35 mutants. Arrowheads indicate cisternae. Scale bar, 100 nm.  Quantification of the number of SVs within a 200 nm radius of the active zone (F) and the mean number of cisternae per cross-sectioned presynaptic bouton (G). n ≥ 38 boutons from more than 4 larvae analyzed, t test, mean ± s.e.m., ***p < 0.001. Electron micrographs of synaptic boutons from wild type (A) and (TIF)Click here for additional data file.S5 Figube3a35 mutant (B) NMJs. Scale bars for EJP and mEJP are annotated. Representative traces of EJP and mEJP of wild type (A) and (TIF)Click here for additional data file.S6 Figube3a35 mutants were rescued by a heterozygous mutation of tkv8. Scale bars, 5 μm and 10 μm in A and B, respectively. (C) Quantification of the fluorescence intensities of pMad at NMJ and nuclei of different genotypes including wild type, ube3a35, and tkv8/+; ube3a35. ***p < 0.001 by one-way ANOVA; error bars indicate SEM. (D) NMJ4 synapses in abdominal segment A3 loaded with FM1-43 in wild type, ube3a35, and tkv8/+; ube3a35. Scale bar, 5 μm. (E) Quantification of FM1-43 intensities in NMJ boutons following high K+-stimulated endocytosis. n ≥ 14 NMJs; ***p < 0.001 by one-way ANOVA; error bars indicate SEM. (F) Percentage of average EJP amplitudes under tetanic stimulation for 10 min in wild type, ube3a35, and tkv8/+; ube3a35. n ≥ 10 animals. Representative traces are shown on the right. Increased levels of pMad at NMJ synapses (A) and motoneuron nuclei (B) of (TIF)Click here for additional data file.S7 Fig(A) The ventral ganglion of a wild-type larva was double-labeled with anti-GFP and anti-HRP. No GFP signals were detected in the ventral ganglion. (B) Tkv-YFP under the control of the endogenous promoter expresses in the soma of various neurons including motoneurons along the midline of a ventral ganglion. Scale bar = 40 μm.  Tkv-YFP is functional as the genetrap line shows NMJ growth (D) comparable to wild-type control (C).(TIF)Click here for additional data file.S8 Fig(A) Schematic representation of full-length and various truncated Ube3a used for co-IP assays. (B) The N-terminal regions of Ube3a mediate the interaction with myc-Tkv. S2 cell lysates were transfected with different combinations of constructs and immunoprecipitated with anti-flag antibody, followed by western blotting using anti-flag and anti-myc. IgG heavy chain is indicated by an asterisk. α-tubulin was used as a loading control. (C) Schematic diagram of full-length Tkv and its various truncations. Different functional domains are indicated. (D) Co-IP assays showed that the C-terminus of Tkv (TkvC), lacking the extracellular and transmembrane (TM) regions, was able to bind Ube3a. More precisely, the dual-specificity serine-threonine/tyrosine protein kinase catalytic (STYKc) domain exhibited strong binding to Ube3a; consistently, deletion of this domain greatly reduced the interaction.(TIF)Click here for additional data file.S9 Fign ≥ 16; **P < 0.01, ***p < 0.001 by one-way ANOVA; error bars indicate SEM. (C) Expressing DTS5, a dominant temperature-sensitive mutation of the β6 subunit of the 26s proteasome , by elav-Gal4  also led to an increase of pMad at NMJ synapses and the motoneuron nuclei. (D) Statistical analysis of fluorescence intensities of pMad at NMJs and motoneuron nuclei of elav/+ control and elav-Gal4-driven DTS5 larvae. n ≥ 18; **P < 0.01, ***p < 0.001 by one-way ANOVA; error bars represent SEM.(A) The level of pMad increased at NMJ synapses and motoneuron nuclei of dissected wild-type larvae treated with 50 μM MG132 inhibitor in Schneider’s medium for 4 h. (B) Quantification of fluorescence intensities of pMad at NMJ and nuclei of non-treated and MG1320-treated larvae. (TIF)Click here for additional data file.S10 Fig(A) Sequence alignment of Tkv and human ALK3 spanning the ubiquitination site K227. (B) Wild-type and mutant ALK3 showed a similar level of proteins in the presence of UBE3A. Tubulin was probed as a loading control.(TIF)Click here for additional data file.S11 Figube3a35 mutants  labeled with anti-Dynamin (A and B), anti-Eps15 (C and D), and anti-Endophilin A (E and F).Confocal images of NMJ4 from wild type  and (TIF)Click here for additional data file.S12 Figelav-Gal4 but not in muscles by C57-Gal4 leads to overgrown NMJ terminals.  Neuronal overexpression of Ube3a does not drive down BMP signaling as pMad staining appears normal at both NMJs (B) and motoneuron nuclei (C).(A) Overexpression of Ube3a in neurons by (TIF)Click here for additional data file.S13 FigS6KL or ube3a had no effect on the number of satellite boutons, while the number of satellite boutons increased significantly in S6KL140 and ube3a35 trans-heterozygotes and double mutants. (C) Quantification of satellite bouton number in different genotypes. n ≥ 16 NMJs analyzed, one-way ANOVA, mean ± s.e.m., ***p < 0.001.(A) Different plasmid constructs were singly or co-transfected to S2 cells. Transfected cells were harvested 48 hr later. Cell lysates were used in a two-step immunoprecipitation step by anti-myc and anti-HA sequentially. (B) Confocal images of NMJ4 labeled with anti-HRP. Loss of one copy of (TIF)Click here for additional data file.S1 Text(DOC)Click here for additional data file.