ABSTRACT
OBJECTIVE: To perform a genome-wide alternative polyadenylation (APA) profiling in both mouse female germline stem cells (FGSCs) and embryonic stem cells (ESCs) and explore the role of germline-specific APA in the biological behaviors of FGSCs. METHODS: We used a high-throughput sequencing-based method 3T-Seq to profile the genome-wide 3' termini of the transcripts and delineate all the APA sites in mouse FGSCs and ESCs. The genes with altered APA sites in FGSCs compared with ESCs were analyzed with DAVID Gene Ontology tool for their biological roles. RESULTS: We identified a total of 50243 APA sites in 16973 genes. In FGSCs, 1148 genes were shown to have alterations in 3'UTR length, among which 795 ( 66%) genes had shortened and 353 (34%) had lengthened 3'UTR. Some of the genes with shortened 3'UTR were involved in germ cell development. CONCLUSIONS: Our genome-wide APA profiling analysis reveals a cell type-specific APA alternation in FGSCs, and APA-mediated 3'UTR alteration contributes to germline-related biological process. This study provides a framework for understanding the post-transcriptional regulation mechanisms in FGSCs.
Subject(s)
Embryonic Germ Cells/metabolism , Embryonic Stem Cells/metabolism , Polyadenylation , 3' Untranslated Regions , Animals , Cell Differentiation , Female , Gene Expression Regulation , Genome , MiceABSTRACT
Alternative polyadenylation (APA) is an important post-transcriptional modification implicated in many diseases, including cancer. Although extensively characterized, the functional consequence of APA modulation on tumorigenesis remains elusive. Here, we developed a deep sequencing-based approach that specifically profiles 3' termini of polyadenylated RNAs (herein termed 3T-seq) and analyzed APA events in two gastric cancer cell lines and one non-transformed counterpart. Overall, we identified >28 000 poly(A) sites, 70% of which are potentially novel. Further, we observed widespread APA-mediated 3' UTR shortening of 513 genes (false discovery rate < 0.05) across gastric cancer genome. We characterized one of these genes, NET1, in detail and found that the shortening of NET1 3' UTR significantly enhances transcriptional activity. Moreover, the NET1 isoform with short 3' UTR promotes cellular migration and invasion in vitro. Collectively, our work provides an effective approach for genome-wide APA site profiling and reveals a link between APA modulation and gastric cancer metastasis.
Subject(s)
Neoplasms/genetics , Neoplasms/pathology , Polyadenylation/genetics , RNA, Messenger/genetics , 3' Untranslated Regions , Cell Line , Cell Movement/genetics , Gene Expression Profiling , High-Throughput Nucleotide Sequencing , Humans , Neoplasm Metastasis , Stomach Neoplasms/genetics , Transcription, GeneticABSTRACT
The identification of structural and functional elements encoded in a genome is a challenging task. Although the transcriptome of budding yeast has been extensively analyzed, the boundaries and untranslated regions of yeast genes remain elusive. To address this least-explored field of yeast genomics, we performed a transcript profiling analysis through paired-end ditag (PET) approach coupled with deep sequencing. With 562,133 PET sequences we accurately defined the boundaries and untranslated regions of 3,409 ORFs, suggesting many yeast genes have multiple transcription start sites (TSSs). We also identified 85 previously uncharacterized transcripts either in intergenic regions or from the opposite strand of reported genomic features. Furthermore, our data revealed the extensive 3' end heterogeneity of yeast genes and identified a novel putative motif for polyadenylation. Our results indicate the yeast transcriptome is more complex than expected. This study would serve as an invaluable resource for elucidating the regulation and evolution of yeast genes.
