koko体育app
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摘要:
目的 通过大数据分析6月龄PS19小鼠体内异常选择性剪切（AS）事件是否在tauP301S诱导的神经退行性表型之前出现。 方法 采用axel从ENA数据库下载GSE182170数据集原始测序文件，通过STAR软件与ENSEMBL数据库参考基因组进行比对，rMATS和rmats2sashimiplot R包进行常见AS事件分析和结果可视化展示。RSEM软件进行基因转录本定量，Deseq2、edgeR、limma R包进行差异分析，采用clusterProfiler R包对差异基因进行GO富集分析。String和Cytoscape软件用来进行蛋白质间相互作用分析。采用ggcorrplt R包进行基因表达相关性分析。PCR结合琼脂糖电泳验证AS事件。 结果 通过rMATS鉴定出了8079个AS事件，并最终筛选出117个显著AS事件（ΔPSI>0.1，测序覆盖度>1）。外显子跳跃（SE）是最常见的AS事件（50.43%），其次是外显子3'端可变剪切（A3SS）和外显子互斥（MXE）。GO富集分析发现，突触组织基因发生异常SE事件，而剪切体基因主要发生异常A3SS事件。蛋白相互作用和相关性分析发现Snrpn剪切因子与最多数量的转录本表达显著相关。琼脂糖电泳证实PS19小鼠中Lrp8基因的异常AS事件。 结论 异常的剪切因子可能参与了tauP301S引起的异常AS改变。本研究扩展了tau蛋白病中tau蛋白循环和剪切因子的知识。 -
关键词:
- tau蛋白病 /
- P301S /
- koko体育app: 选择性剪切 /
- 外显子跳跃 /
- koko体育app: 突触
Abstract:Objective To explore through big data analysis whether aberrant alternative splicing (AS) events precede tauP301S-induced neurodegenerative phenotype in 6-month-old PS19 mice. Methods The original sequencing files of the GSE182170 dataset was downloaded from the European Nucleotide Archive (ENA) database with axel, aligned to the reference genome of the ENSEMBL database by using STAR software, and common AS event analysis and visualization were performed with rMATS and rmats2sashimiplot R packages. RSEM software was utilized for gene transcript quantification, Deseq2, edgeR, and limma R packages were used for differential expression analysis, and clusterProfiler R package was applied for GO enrichment analysis. String and Cytoscape were used for protein-protein interaction (PPI) analysis. Gene expression correlation analysis was performed with ggcorrplot R package. AS events were validated using PCR followed by agarose electrophoresis. Results A total of 8 079 AS events were identified with rMATS and 117 significant AS events (ΔPSI>0.1, sequencing coverage >1) were selected eventually. The most frequent type of AS event was skipped exon (SE) (50.43%), followed by alternative 3' splice site (A3SS) and mutually exclusive exons (MXE). GO enrichment analysis revealed that synapse organization genes were aberrantly spliced in SE events and spliceosome genes were spliced in A3SS events. PPI and correlation analyses showed that the splicing factor Snrpn was significantly associated with the largest number of transcripts. Agarose electrophoresis confirmed the aberrant AS event of the Lrp8 gene in PS19 mice. Conclusion Dysregulated splicing factors may contribute to tauP301S-induced aberrant AS changes. The study also increases the understanding of the cycling of tau protein and splicing factors in tauopathies. -
Key words:
- koko体育app: Tauopathy /
- koko体育app: P301S /
- koko体育app: Alternative splicing /
- Exon skipping /
- Synapse
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koko体育app
图 1 AS事件汇总
Figure 1. AS event summary
A3SS: alternative 3' splice site; A5SS: alternative 5' splice site; SE: skipped exon; RI: intron retention; MXE: mutually exclusive exons. A, Raw counts in each type of AS event. B, Number and proportion of each type of significant AS event. C, PCA analysis based on PSI values. PS19 and wild type groups were clearly distinguished. Each dot represents one sample. D, The percent of ΔPSI all significant AS events. E, Distribution of ΔPSI in all significant AS events. Each dot represents one AS event. ΔPSI=PS19－WT.
图 2 突触组织基因的异常SE事件
Figure 2. Aberrant SE even⛎ts of synapse organization genes
A, GO enrichment analysis of genes harbouring significant SE events. Each dot represents one AS event. B, Cnetplot illustrates genes in the enriched GO terms. Genes coloured in green represent decreased PSI level, while genes coloured in red represent increased PSI level. The sashimi plots are for Lrp8 (C) and Mecp2 (D).
图 3 剪切体基因异常A3SS事件
Figure 3. Aberrant A3SS events of spli🅷ceosome genes
A, GO enrichment analysis of genes harbouring significant A3SS events. Each dot represents one AS event. B, Cnetplot illustrates genes in the enriched GO terms. Genes coloured in green indicate decreased PSI level, while genes coloured in red represent increased PSI level. The sashimi plots are for Celf1 (C) and Celf2 (D).
图 4 差异表达的转录本
Figure 4. Differentially expressed transcripts
A, Sample correlation analysis based on transcript expression. B, Heatmap of dysregulated transcripts between PS19 and wild type groups. C, Venn plot of dysregulated transcripts among Deseq2, edgeR, and limma R packages. The left panel showed up-regulated transcripts and the right panel showed down-regulated transcripts. D, GO enrichment analysis of biological processes using host genes of differentially expressed transcripts. The GO terms were mainly enriched in synapse organization. E, GO enrichment analysis of cellular components using host genes of differentially expressed transcripts. The GO terms were mainly enriched in synapse membrane.
图 5 转录本表达与AS事件的相关性
Figure 5. Correlation between transcript expressi𒉰on and AS events
A, Volcano plot of dysregulated transcripts. Genes carried differentially expressed transcripts with opposite trends were labelled. B, Genomic information of Ankrd16. Red arrow indicates the protein coding transcript and the green arrow indicates the intron retained transcript. C, The sashimi plots for Ankrd16 show a decreased inclusion level of intron retained transcript, ENSMUST00000125024, in the PS19 group. D, Relꦛative expr♔ession of different transcripts. ENSMUST00000125024 represents the intron retained transcript and ENSMUST00000056108 represents the protein coding transcript.
图 6 上游剪切因子分析
Figure 6. Analysis of upstream splicing factors
A, Volcano plot of dysregulated SFs. 10 SFs were upregulated and 5 SFs were downregulated. B, PPI networks of the 15 dysregulated SFs. C, The screened cluster of SFs using the MCODE application in Cytoscape. D, Correlation between the dysregulated SFs and transcripts involved in AS events.
图 7 UNC0642逆转PS19小鼠异常转录基因与AS事件关联分析
Figure 7. Intersection between dysregulat♕eജd PS19 mice genes reversed by UNC0642 and AS events
The expression of 98 downregulated and 173 upregulated genes in PS19 mice had been reversed by UNC0642 treatment. The list of these genes was intersected with genes in significant AS events and the result showed that only Ksr1 was found in AS events.
图 8 PS19小鼠异常AS事件验证
Figure 8. Validation of AS events of PS19 mice
RNA from the prefrontal cortex was extracted and Lrp8 was amplified in the range 🐠of exon 18 to exon 20 to detect exon 19 skipping. The PCR products were observed by electrophoresis with 1% agarose.
表 1 AS事件汇总
Table 1. Summary of AS events
Event type Total events junction counts Average coverage>1 FDR<0.05, ΔPSI>0.1 Ratio in WT Ratio in PS19 A3SS 1215 629 29 16.1% 32.8% A5SS 642 300 11 10.7% 8.2% SE 4476 2853 59 53.6% 47.5% RI 1375 325 5 7.1% 1.6% MXE 371 237 13 12.5% 9.8% Total 8079 4344 117 100% 100% 
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表 2 前10个显著AS事件
Table 2. The top 10 significant AS events
ID P FDR ΔPSI Gene Type 7954 4.17E-11 1.87E-07 −0.945 Rims1 SE 10181 1.13E-10 2.53E-07 −0.891 Fermt2 SE 5088 3.83E-10 5.71E-07 1 Ep400 SE 1464 1.54E-09 2.11E-06 −0.834 Ankrd16 RI 4546 1.98E-08 3.86E-06 0.786 Smarca4 A3SS 4694 5.62E-09 5.03E-06 −0.762 Ube2j1 SE 6202 4.57E-08 2.05E-05 −0.758 Pex2 SE 19029 1.31E-07 5.35E-05 0.585 Qrich1 SE 564 3.22E-07 0.000109 −0.678 Spire1 SE 7952 3.18E-07 0.000109 −0.798 Rims1 SE
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表 3 异常AS事件中涉及的异常表达转录本
Table 3. Dysregulated transcripts involved in aberrant AS even🅷t💯s
Gene Transcript logFC P Change Czib ENSMUST00000030347 1.659523 0.025186 Up Ctdspl2 ENSMUST00000036647 1.762767 0.012691 Up Ppp2r2d ENSMUST00000041097 0.999524 0.028762 UP Ep400 ENSMUST00000041558 1.140912 0.011476 Up Setd5 ENSMUST00000042889 −0.66614 0.033083 Down Armcx1 ENSMUST00000051256 0.998508 0.007322 Up Ablim2 ENSMUST00000054598 −1.36966 0.008548 Down Ankrd16 ENSMUST00000056108 1.277506 0.009568 Up Celf1 ENSMUST00000068747 0.603695 0.012954 Up St6galnac6 ENSMUST00000072111 −0.77398 0.003676 Down Plec ENSMUST00000073418 −1.03616 0.011021 Down Fbxw11 ENSMUST00000076383 0.861714 0.015902 Up St6galnac6 ENSMUST00000095044 1.606891 0.008143 Up Rims1 ENSMUST00000097808 −1.32182 0.005498 Down Celf2 ENSMUST00000100429 0.734255 0.029349 Up Usp48 ENSMUST00000105837 −0.78614 0.032728 Down Lrp8 ENSMUST00000106733 −1.0628 0.03465 Down Shank1 ENSMUST00000107935 −0.62579 0.010369 Down Zmiz2 ENSMUST00000109787 1.700795 0.015008 Up Celf1 ENSMUST00000111455 −2.20973 0.044164 Down Ep400 ENSMUST00000112436 −1.52965 0.000674 Down Armcx ENSMUST00000113197 −2.1344 0.033295 Down St6galnac6 ENSMUST00000113290 −1.69228 0.023443 Down Adgrl3 ENSMUST00000117407 −0.72072 0.047625 Down Ankrd16 ENSMUST00000125024 −2.8623 0.013749 Down Adgrl3 ENSMUST00000132375 −1.67991 0.023895 Down Eef1d ENSMUST00000134222 −1.26873 0.02715 Down Tmem126a ENSMUST00000136652 1.757229 0.00223 Up Ncln ENSMUST00000136721 1.847606 0.006957 Up Ctdspl2 ENSMUST00000138920 1.534733 0.03553 Up Afdn ENSMUST00000139666 −0.58987 0.025895 Down Nbr1 ENSMUST00000146452 1.34011 0.02221 Up Nbr1 ENSMUST00000148805 −1.51325 0.034956 Down Ecpas ENSMUST00000149301 0.653901 0.035459 Up Ecpas ENSMUST00000149513 −2.4474 0.039962 DOWN Zcchc17 ENSMUST00000149755 0.928164 0.03315 Up Ankrd16 ENSMUST00000150320 −0.83481 0.035656 Down Mib2 ENSMUST00000151843 −0.61662 0.036781 Down Tbce ENSMUST00000159966 −1.6102 0.005239 Down Ttll11 ENSMUST00000160906 1.295583 0.006904 Up Rbm26 ENSMUST00000163499 −1.27127 0.019638 Down Rbm26 ENSMUST00000163545 0.850966 0.01284 Up Nt5c2 ENSMUST00000168536 2.836236 0.018041 Up Dnajc13 ENSMUST00000185503 1.366736 0.038832 Up Pcdh9 ENSMUST00000192221 0.82386 0.012156 Up Pcdh9 ENSMUST00000195376 2.469259 0.000393 Up Fnbp1l ENSMUST00000197259 1.71466 0.01799 Up Mtf2 ENSMUST00000198662 −1.70095 0.015405 Down Immt ENSMUST00000206625 −1.13042 0.045971 Down Picalm ENSMUST00000207225 1.17105 0.013091 Up Btrc ENSMUST00000225662 −0.63403 0.040695 Down Lrp8 ENSMUST00000238570 1.068084 0.037582 Up
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Table 4. Dysregulated splicing factors
Gene Transcript logFC P Change Prpf3 ENSMUST00000015892 0.963048 0.01293 Up Hnrnpa1 ENSMUST00000036004 1.301391 0.008566 Up Gemin8 ENSMUST00000056410 2.91523 0.00033 Up Snrpn ENSMUST00000098402 1.47411 0.017551 Up Aqr ENSMUST00000102543 1.764123 0.002358 Up Pabpn1 ENSMUST00000116476 −0.76628 0.010123 Down Son ENSMUST00000140312 0.783944 0.038661 Up Hnrnph3 ENSMUST00000140743 1.921598 0.017536 Up U2af1 ENSMUST00000166526 1.040334 0.012575 Up Tnpo3 ENSMUST00000170350 0.964117 0.003083 Up Rbm4b ENSMUST00000183332 −1.01892 0.015464 Down Sf3b1 ENSMUST00000188859 −1.56249 0.010113 Down Rps13 ENSMUST00000205548 −2.44238 0.001799 Down Cd2bp2 ENSMUST00000206026 −0.90113 0.001208 Down Lsm4 ENSMUST00000210987 1.621149 0.013662 Up
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