Analysis of sequences downstream of the polyadenylation signal and their influence on gene expression
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE215681 | GEO | 2022/10/18
REPOSITORIES: GEO
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Project description:Differences in gene regulation contribute to phenotypic differences between humans and other primates. While co-transcriptional gene regulatory mechanisms such as alternative polyadenylation (APA) can help explain how variation in gene regulation manifests, such mechanisms remain understudied. We measured polyadenylation site (PAS) usage in a panel of 6 human and 6 chimpanzee lymphoblastoid cell lines (LCLs). While APA is largely conserved between humans and chimpanzees, genes with divergent APA patterns are enriched among differentially expressed and differentially translated genes. Differential usage of 3’ UTR and intronic PAS are both significantly correlated with differential mRNA expression effect sizes but in opposite directions. For many genes, there is one PAS dominant, meaning it is used much more often than others. The dominant PAS for these gene is overwhelmingly shared between species, however, differences in dominant PAS are enriched for genes with expression differences. Finally, through post-translational mechanisms, we believe APA contributes to genes differentially expressed at the protein level but not in mRNA. As this is the first primate comparative study of APA, our study establishes APA as a key mechanism underlying the genetic regulation of gene and protein expression levels in primates.
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Project description:We employ a massively parallel reporter assay (MPRA) to measure the ex vivo activities of hundreds of K562 and HepG2 enhancers with known transcription factor motif instances. For seven selected motifs that correspond to known or predicted activators and repressors in the two cell types, we make directed modifications of the bases corresponding to these motifs and observe the changes in enhancer activity. Reporter mRNA-seq from HepG2 and K562 cells transfected with a ~55,000-plex MPRA plasmid pool containing 5,418 mutated human enhancer sequences, each linked to 10 distinct 10-nt tags. The reporter mRNA tags facilitate quantitation of their abundances. The same tags were also sequenced from the transfected MPRA plasmid pool to facilitate normalization to plasmid copy numbers.
2013-03-15 | E-GEOD-33367 | biostudies-arrayexpress
Project description:Homo sapiens and Macaca fascicularis neural progenitor cell lines were transduced with a lentiviral MPRA (Massively Parallel Reporter Assay) library. MPRA barcode sequencing and RNA-seq was performed on the extracted RNA. MPRA data was used to compare activity of regulatory sequences across 75 mammalian species with a focus on primates and correlate these activities with the Phenotype of gyrencephaly.
2021-05-12 | E-MTAB-9952 | biostudies-arrayexpress
Project description:Homo sapiens and Macaca fascicularis neural progenitor cell lines were transduced with a lentiviral MPRA (Massively Parallel Reporter Assay) library. MPRA barcode sequencing and RNA-seq was performed on the extracted RNA. RNA-seq data was used to confirm transcription factor expression.
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Project description:Alternative polyadenylation (APA) of mRNAs has emerged as an important mechanism for post-transcriptional gene regulation in higher eukaryotes. Although microarrays have recently been used to characterize APA globally, they have a number of serious limitations that prevents comprehensive and highly quantitative analysis. To better characterize APA and its regulation, we have developed a deep sequencing-based method called Poly(A) Site Sequencing (PAS-Seq) for quantitatively profiling RNA polyadenylation at the transcriptome level. PAS-Seq not only accurately and comprehensively identifies poly(A) junctions in mRNAs and noncoding RNAs, but also provides quantitative information on the relative abundance of polyadenylated RNAs. We first analyzed HeLa cell transcriptome using PAS-Seq to demonstrate that PAS-Seq not only accurately and comprehensively identifies poly(A) junctions, but also provide quantitative information on the relativel abundance of APA isoforms. Next we analyzed the mouse embryonic stem cells, neural stem/progenitor cells, and neurons by PAS-Seq to characterize the dynamic changes of mRNA polyadenylation during stem cell differentiation.
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Project description:Alternative cleavage and polyadenylation (APA) generates diverse mRNA isoforms. We developed 3' region extraction and deep sequencing (3'READS) to address mispriming issues that commonly plague poly(A) site (pA) identification, and we used the method to comprehensively map pAs in the mouse genome. Thorough annotation of gene 3' ends revealed over 5,000 previously overlooked pAs (~8% of total) flanked by A-rich sequences, underscoring the necessity of using an accurate tool for pA mapping. About 79% of mRNA genes and 66% of long noncoding RNA genes undergo APA, but these two gene types have distinct usage patterns for pAs in introns and upstream exons. Quantitative analysis of APA isoforms by 3'READS indicated that promoter-distal pAs, regardless of intron or exon locations, become more abundant during embryonic development and cell differentiation and that upregulated isoforms have stronger pAs, suggesting global modulation of the 3' endM-bM-^@M-^Sprocessing activity in development and differentiation. 3'READS to map pAs in mouse genome
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Project description:Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage and polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3’UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1, and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly influencing C/P events in 5’ introns and U2 impacting those in efficiently spliced introns. Furthermore, PABPN1 regulates expression of transcripts with pAs near the transcription start site, a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results indicate that the abundance of different C/P factors and splicing factors plays diverse roles in APA, and is relevant to APA regulation in biological conditions. knockdown experiments of 23 C/P factors, 3 splicing factors and U1D in mouse C2C12 myoblast cells
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Project description:Transcription of the mammalian genome is pervasive, but productive transcription outside of protein-coding genes is limited by unknown mechanisms. In particular, although RNA polymerase II (RNAPII) initiates divergently from most active gene promoters, productive elongation occurs primarily in the sense-coding direction. Here we show in mouse embryonic stem cells that asymmetric sequence determinants flanking gene transcription start sites control promoter directionality by regulating promoter-proximal cleavage and polyadenylation. We find that upstream antisense RNAs are cleaved and polyadenylated at poly(A) sites (PASs) shortly after initiation. De novo motif analysis shows PAS signals and U1 small nuclear ribonucleoprotein (snRNP) recognition sites to be the most depleted and enriched sequences, respectively, in the sense direction relative to the upstream antisense direction. These U1 snRNP sites and PAS sites are progressively gained and lost, respectively, at the 5' end of coding genes during vertebrate evolution. Functional disruption of U1 snRNP activity results in a dramatic increase in promoter-proximal cleavage events in the sense direction with slight increases in the antisense direction. These data suggest that a U1-PAS axis characterized by low U1 snRNP recognition and a high density of PASs in the upstream antisense region reinforces promoter directionality by promoting early termination in upstream antisense regions, whereas proximal sense PAS signals are suppressed by U1 snRNP. We propose that the U1-PAS axis limits pervasive transcription throughout the genome. 3' end sequencing of poly (A) + RNAs in mouse ES cells with and without U1 snRNP inhibition using antisense morpholino oligonucleotides (AMO). Each with two biological replicates.
2013-06-28 | E-GEOD-46433 | biostudies-arrayexpress
Project description:Genome-wide association studies (GWAS) have successfully identified 145 genomic regions that contribute to schizophrenia risk, but linkage disequilibrium (LD) makes it challenging to discern causal variants. Computational finemapping prioritized thousands of credible variants, ~98% of which lie within poorly characterized non-coding regions. To functionally validate their regulatory effects, we performed a massively parallel reporter assay (MPRA) on 5,173 finemapped schizophrenia GWAS variants in primary human neural progenitors (HNPs). We identified 440 variants with allelic regulatory effects (MPRA-positive variants), with 72% of GWAS loci containing at least one MPRA-positive variant. Transcription factor binding had modest predictive power for predicting the allelic activity of MPRA-positive variants, while GWAS association, finemap posterior probability, enhancer overlap, and evolutionary conservation failed to predict MPRA-positive variants. Furthermore, 64% of MPRA-positive variants did not exhibit eQTL signature, suggesting that MPRA could identify yet unexplored variants with regulatory potentials. MPRA-positive variants differed from eQTLs, as they were more frequently located in distal neuronal enhancers. Therefore, we leveraged neuronal 3D chromatin architecture to identify 273 genes that physically interact with MPRA-positive variants. These genes annotated by chromatin interactome displayed higher mutational constraints and regulatory complexity than genes annotated by eQTLs, recapitulating a recent finding that eQTL- and GWAS-detected variants map to genes with different properties. Finally, we propose a model in which allelic activity of multiple variants within a GWAS locus can be aggregated to predict gene expression by taking chromatin contact frequency and accessibility into account. In conclusion, we demonstrate that MPRA can effectively identify functional regulatory variants and delineate previously unknown regulatory principles of schizophrenia.
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