Lysine acetylation in proteins is a dynamic and reversible PTM and plays an important role in diverse cellular processes. In this study, using lysine-acetylation (Kac) peptide enrichment coupled with nano HPLC/MS/MS, we initially identified the acetylome in the silkworms. Overall, a total of 342 acetylated proteins with 667 Kac sites were identified in silkworm. Sequence motifs analysis around Kac sites revealed an enrichment of Y, F, and H in the +1 position, and F was also enriched in the +2 and -2 positions, indicating the presences of preferred amino acids around Kac sites in the silkworm. Functional analysis showed the acetylated proteins were primarily involved in some specific biological processes. Furthermore, lots of nutrient-storage proteins, such as apolipophorin, vitellogenin, storage proteins, and 30 K proteins, were highly acetylated, indicating lysine acetylation may represent a common regulatory mechanism of nutrient utilization in the silkworm. Interestingly, Ser2 proteins, the coating proteins of larval silk, were found to contain many Kac sites, suggesting lysine acetylation may be involved in the regulation of larval silk synthesis. This study is the first to identify the acetylome in a lepidoptera insect, and expands greatly the catalog of lysine acetylation substrates and sites in insects.
BackgroundA transposable element (TE) is a DNA fragment that can change its position within a genome. Transposable elements play important roles in maintaining the stability and diversity of organisms by transposition. Recent studies have shown that approximately half of the genes in Bombyx mori are TEs.ResultsWe systematically identified and analyzed the BmAGO2-associated TEs, which exceed 100 in the B. mori genome. Additionally, we also mapped the small RNAs associated with BmAGO2 in B.mori. The transposon Bm1645 is the most abundant TE associated with BmAGO2, and Bm1645-derived small RNAs represent a small RNA pool. We determined the expression patterns of several Bm1645-derived small RNAs by northern blotting, and the results showed there was differential expression of multiple small RNAs in normal and BmNPV-infected BmN cells and silkworms from various developmental stages. We confirmed that four TE-siRNAs could bind to BmAGO2 using EMSA and also validated the recognition sites of these four TE-siRNAs in Bm1645 by dual-luciferase reporter assays. Furthermore, qRT-PCR analysis revealed the overexpression of the four TE-siRNAs could downregulate the expression of Bm1645 in BmN cells, and the transcription of Bm1645 was upregulated by the downregulation of BmAGO2.ConclusionsOur results suggest Bm1645 functions as a source of small RNAs pool and this pool can produce many BmAGO2-associated small RNAs that regulate TE’s expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3598-5) contains supplementary material, which is available to authorized users.
Background: Microsatellite instability (MSI) is an important indicator of larger genome instability and has been linked to many genetic diseases. MMR gene mutations usually lead to the loss of MMR proteins, resulting in high instability of tumor DNA microsatellites (MSI-H), and leading to a high tumor mutation burden (TMB-H). In current clinical practice, MSI detection is performed by experimental methods such as MSI-PCR and MMR-IHC. However, the PCR-based detection procedure is laborious and time-consuming, the accuracy of the results depends on the naked eye judgment of the analysts. With the development of next-generation sequencing (NGS) technology, a large number of MSI detection software based on high-throughput sequencing data have been developed. In this study, we compared the two methods provide a computational method that quantifies MSI status based on genome sequencing data. Methods: 2523 tested samples, including 80 MSI-H samples and 2443 MSS samples, detected by MSI-PCR and NGS-Seq simultaneously, were enrolled in this study. In the MSI-PCR method, MSI status was classified as Microsatellite stable (MSS) and MSI-high (MSI-H, over 2 markers unstable). For the NGS-Seq method, the status of 54 microsatellite site was evaluated by the MSI sensor (https://github.com/ding-lab/msisensor) and the proportion of unstable microsatellite site (RatioUMS) was calculated, and MSI status was classified as MSS (RatioUMS<35), MSS-To Be Determined (MSI-TBD, 35≤RatioUMS≤55), and MSI-H (RatioUMS>55). For MSI-TBD, either TMB is greater than 30 (TMB>30) or TMB is greater than 10 and had a non-silent mutation in an MMR gene (TMB>10&MMR+), MSI-H is considered, otherwise, MSS is considered. Results: In our test, NGS results were inconsistent with MSI-PCR method in 8 samples, among which 5 samples were categorized incorrectly by MSI-PCR after reverification. According to the above results, the consistency of two methods was 99.9%. We also tested the classifier using 114 microsatellite sites in 425 samples and 309 microsatellite sites in 2098 samples. Compared with MSI-PCR, the consistency was 100% and 99.6%, respectively. For the 25 MSI-TBD samples, adding two parameters, MMR gene mutation and TMB, greatly improved the accuracy of the judgment. Conclusion: The results show NGS method are highly consistent with the gold standard MSI-PCR, and the judgmental errors caused in MSI-PCR by human factors can be avoided. The different number of microsatellite sites used in the NGS method has little effect on the final results. NGS approach offers the advantages to simultaneous detection of predictive markers, including MSI, TMB and clinically relevant genomic alterations, will save valuable material, time and costs, which can provide more comprehensive and reasonable recommendations for medication and treatment. Citation Format: Hongling Yuan, Danhua Wang, Honglin Zhu. Tumor microsatellite instability detection method using paired tumor-normal sequence data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2080.
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