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Paf1C plays novel subunit-specific roles in alternative cleavage and polyadenylation

ABSTRACT: The PAF complex (Paf1C) has been shown to regulate chromatin modifications, gene transcription, and PolII elongation. Here, we provide the first genome-wide analysis of chromatin occupancy by the entire PAF complex in mammalian cells. We show that Paf1C is recruited not only to promoters and gene bodies, but also to regions downstream of cleavage/polyadenylation (pA) sites at 3’ ends, a profile that sharply contrasted with the yeast complex. Remarkably, our studies identified novel, subunit-specific links between Paf1C and regulation of alternative cleavage and polyadenylation (APA) and upstream antisense transcription. Moreover, we found that depletion of Paf1C subunits also resulted in the accumulation of RNA polymerase II (PolII) over gene bodies, which coincided with APA. Depletion of specific Paf1C subunits leads to global loss of histone H2B ubiquitylation, but surprisingly, there is little impact of Paf1C depletion on other histone modifications, including the tri-methylation of histone H3 on lysines 4 and 36 (H3K4me3 and H3K36me3), previously associated with this complex. Our results provide surprising differences with yeast, while unifying observations that link Paf1C with PolII elongation and RNA processing, and suggest that Paf1C could play a role in protecting transcripts from premature cleavage by preventing PolII accumulation at TSS-proximal pA sites.

ORGANISM(S): Mus musculus

PROVIDER: GSE72574 | GEO | 2016/01/15

SECONDARY ACCESSION(S): PRJNA294381

REPOSITORIES: GEO

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Paf1C plays novel subunit-specific roles in alternative cleavage and polyadenylation

Project description:The PAF complex (Paf1C) has been shown to regulate chromatin modifications, gene transcription, and PolII elongation. Here, we provide the first genome-wide analysis of chromatin occupancy by the entire PAF complex in mammalian cells. We show that Paf1C is recruited not only to promoters and gene bodies, but also to regions downstream of cleavage/polyadenylation (pA) sites at 3â ends, a profile that sharply contrasted with the yeast complex. Remarkably, our studies identified novel, subunit-specific links between Paf1C and regulation of alternative cleavage and polyadenylation (APA) and upstream antisense transcription. Moreover, we found that depletion of Paf1C subunits also resulted in the accumulation of RNA polymerase II (PolII) over gene bodies, which coincided with APA. Depletion of specific Paf1C subunits leads to global loss of histone H2B ubiquitylation, but surprisingly, there is little impact of Paf1C depletion on other histone modifications, including the tri-methylation of histone H3 on lysines 4 and 36 (H3K4me3 and H3K36me3), previously associated with this complex. Our results provide surprising differences with yeast, while unifying observations that link Paf1C with PolII elongation and RNA processing, and suggest that Paf1C could play a role in protecting transcripts from premature cleavage by preventing PolII accumulation at TSS-proximal pA sites. ChIP-seq, RNA-seq and 3'READS of Paf1C factors in mouse C2C12 myoblast cells

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Analysis of alternative cleavage and polyadenylation by 3' region extraction and deep sequencing

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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DNA Methylation Regulates Alternative Polyadenylation via CTCF and the Cohesin Complex

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Project description:The development of the central nervous system (CNS) is a complex process that must be exquisitely controlled at multiple levels to ensure the production of appropriate types and quantity of neurons. RNA alternative polyadenylation (APA) contributes to transcriptome diversity and gene regulation and has recently been shown to be widespread in the CNS. However, previous studies have been primarily focused on the tissue specificity of APA and developmental APA change of whole model organisms, a systematic survey of APA usage is lacking during CNS development. Here we conducted global analysis of APA during mouse retinal development, and identified stage-specific polyadenylation (pA) sites that are enriched for genes critical for retinal development and visual perception. Moreover, we demonstrated 3’UTR lengthening and increased usage of intronic pA sites over development that would result in gaining many different RBP (RNA binding protein) and miRNA target sites. Furthermore, we showed that a considerable number of polyadenylated lncRNAs are co-expressed with protein-coding genes involved in retinal development and functions. Together, our data indicate that APA is highly and dynamically regulated during retinal development and maturation, suggesting that APA may serve as a crucial mechanism of gene regulation underlying the delicate process of CNS development.

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