Biology subjects

Dai, J.

Publications and source records attributed to Dai, J..

6 recordsLinked to original sources

bioRxiv2018

Integrative analysis of Zika virus genome RNA structure reveals critical determinants of viral infectivity

Since its outbreak in 2007, Zika virus (ZIKV) has become a global health threat that causes severe neurological conditions. Here we perform a comparative in vivo structural analysis of the RNA genomes of two ZIKV strains to decipher the regulation of their infection at the RNA level. Our analysis identified both known and novel functional RNA structural elements. We discovered a functional long-range intramolecular interaction specific for the Asian epidemic strains, which contributes to their infectivity. Our findings illuminate the structural basis of ZIKV regulation and provide a rich resource for the discovery of RNA structural elements that are important for ZIKV infection.

molecular biology↗

bioRxiv2018

Dissecting PCNA function with a systematically designed mutation library in yeast

Proliferating cell nuclear antigen (PCNA), encoded by POL30 in Saccharomyces cerevisiae, is a key component of DNA metabolism. Here a library consisting of 308 PCNA mutants was designed and synthesized to probe the contribution of each residue to its biological function. Five regions were identified with elevated sensitivity to DNA damaging reagents using high-throughput phenotype screening. Using a series of genetic and biochemical analyses, we demonstrated that one particular mutant, K168A, which displayed severe DNA damage sensitivity, abolished the DNA damage tolerance (DDT) pathway by disrupting interactions between PCNA and Rad5p. Subsequent domain analysis showed that the PCNA/Rad5p interaction is prerequisite for the function of Rad5p in DDT. Our study not only provides a resource in the form of a library of versatile mutants to study PCNA functions, but also reveals a key regulatory function of Rad5p, which highlights the importance of the PCNA-Rad5p interaction.\n\nAuthor summaryPCNA is regarded as the maestro of DNA replication fork because of the astonishing ability to interact with lots of partner proteins that participate in various DNA metabolism processes. However, it has remained elusive as to how does PCNA orchestrate these functions in harmony. Here, we constructed a systematic mutation library of PCNA, which covers every amino acid to map the functional sites of it. This carefully designed synthetic mutant pool could be generally useful and serve as a flexible resource, such as dissecting the functional mechanism of PCNA by genetic relationship analysis with key proteins through Synthetic genetic array. We further dissected the intrinsic mechanism for damage sensitivity of PCNAK168A, the most severe DNA damage sensitive mutant in our alanine scanning mutation library, this helps us to get better understanding of how PCNA participates in DNA damage tolerance (DDT) pathways. Our findings indicate that K168 site is vital for the interaction between DDT related partner proteins and PCNA, and also highlight the importance of the PCNA-Rad5p interaction.

genetics↗

bioRxiv2018

Analysis of the role of Nidogen/entactin in basement membrane assembly and morphogenesis in Drosophila

Basement membranes (BMs) are thin sheet-like specialized extracellular matrices found at the basal surface of epithelia and endothelial tissues. They have been conserved across evolution and are required for proper tissue growth, organization, differentiation and maintenance. The major constituents of BMs are two independent networks of Laminin and Type IV Collagen interlinked by the proteoglycan Perlecan and the glycoprotein Nidogen/entactin (Ndg). The ability of Ndg to bind in vitro Collagen IV and Laminin, both with key functions during embryogenesis, anticipated an essential role for Ndg on morphogenesis linking the Laminin and Collagen IV networks. This was supported by results from in vitro and cultured embryonic tissues experiments. However, the fact that elimination of Ndg in C. elegans and mice did not affect survival, strongly questioned this proposed linking role. Here, we have isolated mutations in the only Ndg gene present in Drosophila. We find that while, similar to C.elegans and mice, Ndg is not essential for overall organogenesis or viability, it is required for appropriate fertility. We also find, alike in mice, tissue-specific requirements of Ndg for proper assembly and maintenance of certain BMs, namely those of the adipose tissue and flight muscles. In addition, we have performed a thorough functional analysis of the different Ndg domains in vivo. Our results support an essential requirement of the G3 domain for Ndg function and unravel a new key role for the Rod domain in regulating Ndg incorporation into BMs. Furthermore, uncoupling of the Laminin and Collagen IV networks is clearly observed in the larval adipose tissue in the absence of Ndg, indeed supporting a linking role. In light of our findings, we propose that BM assembly and/or maintenance is tissue-specific, which could explain the diverse requirements of a ubiquitous conserved BM component like Nidogen.\n\nAuthor SummaryBasement membranes (BMs) are thin layers of specialized extracellular matrices present in every tissue of the human body. Its main constituents are two networks of Laminin and Type IV Collagen linked by Nidogen (Ndg) and proteoglycans. They form an organized scaffold that regulates organ morphogenesis and function. Mutations affecting BM components are associated with organ dysfunction and several congenital diseases. Thus, a better comprehension of BM assembly and maintenance will not only help to learn more about organogenesis but also to a better understanding and, hopefully, treatment of these diseases. Here, we have used Drosophila to analyse the role of Ndg in BM formation in vivo. Elimination of Ndg in worms and mice does not affect survival, strongly questioning its proposed linking role, derived from in vitro experiments. Here, we show that in the fly Ndg is dispensable for BM assembly and preservation in many tissues, but absolutely required in others. Furthermore, our functional study of the different Ndg domains challenges the significance of some interactions between BM components derived from in vitro experiments, while confirming others, and reveals a new key requirement for the Rod domain in Ndg function and incorporation into BMs.

developmental biology↗

bioRxiv2018

Understanding the limit of open search in the identification of peptides with post-translational modifications -- A simulation-based study

MotivationAnalyzing tandem mass spectrometry data to recognize peptides in a sample is the fundamental task in computational proteomics. Traditional peptide identification algorithms perform well when identifying unmodified peptides. However, when peptides have post-translational modifications (PTMs), these methods cannot provide satisfactory results. Recently, Chick et al., 2015 and Yu et al., 2016 proposed the spectrum-based and tag-based open search methods, respectively, to identify peptides with PTMs. While the performance of these two methods is promising, the identification results vary greatly with respect to the quality of tandem mass spectra and the number of PTMs in peptides. This motivates us to systematically study the relationship between the performance of open search methods and quality parameters of tandem mass spectrum data, as well as the number of PTMs in peptides.\n\nResultsThrough large-scale simulations, we obtain the performance trend when simulated tandem mass spectra are of different quality. We propose an analytical model to describe the relationship between the probability of obtaining correct identifications and the spectrum quality as well as the number of PTMs. Based on the analytical model, we can quantitatively describe the necessary condition to effectively apply open search methods.\n\nAvailabilitySource codes of the simulation are available at http://bioinformatics.ust.hk/PST.html.\n\nContactboningli@ust.hk or eeyu@ust.hk\n\nSupplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2017

Xolik: finding cross-linked peptides with maximum paired scores in linear time

MotivationCross-linking technique coupled with mass spectrometry (MS) is widely used in the analysis of protein structures and protein-protein interactions. In order to identify cross-linked peptides from MS data, we need to consider all pairwise combinations of peptides, which is computationally prohibitive when the sequence database is large. To alleviate this problem, some heuristic screening strategies are used to reduce the number of peptide pairs during the identification. However, heuristic screening criteria may ignore true findings.\n\nResultsWe directly tackle the combination challenge without using any screening strategies. With the additive scoring function and the data structure of double-ended queue, the proposed algorithm reduces the quadratic time complexity of exhaustive searching down to the linear time complexity. We implement the algorithm in a tool named Xolik, and the running time of Xolik is validated using databases with different number of proteins. Experiments using synthetic and empirical datasets show that Xolik outperforms existing tools in terms of running time and statistical power.\n\nAvailabilitySource code and binaries of Xolik are freely available at http://bioinformatics.ust.hk/Xolik.html.\n\nContacteeyu@ust.hk\n\nSupplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2017

Intracellular BH3 profiling reveals shifts in anti-apoptotic dependency in B-cell maturation and activation

Apoptosis is critical to B-cell maturation, but studies of apoptotic regulation in primary human B cells is lacking. Previously, we found that infecting human B cells with Epstein-Barr virus induces two different survival strategies (Price et al., 2017). Here, we sought to better understand the mechanisms of apoptotic regulation in normal and activated B cells. Using intracellular BH3 profiling (iBH3), we defined the Bcl2-dependency of B-cell subsets from human peripheral blood and tonsillar lymphoid tissue as well as mitogen-activated B cells. We found that naive and memory B cells were BCL-2 dependent, while germinal center B cells were MCL-1 dependent and plasma cells were BCL-XL dependent. Proliferating B cells activated by CpG or CD40L/IL-4 became more dependent upon MCL-1 and BCL-XL. As B-cell lymphomas often rely on survival mechanisms derived from normal and activated B cells, these findings offer new insight into potential therapeutic strategies for lymphomas.

immunology↗