Alternative polyadenylation by sequential activation of distal and proximal PolyA sites

Tang, Peng; Yang, Yang; Li, Guangnan; Huang, Li; Wen, Miaomiao; Ruiying, Wen; Guo, Xiaolong; Zhang, Chen; Zuo, Xinxin; Luo, Daji; Xu, Yong; Fu, Xiang-Dong; Zhou, Yu

doi:10.1038/s41594-021-00709-z

Cited by 32 publications

(24 citation statements)

References 62 publications

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“…In general, each nascent mRNA molecule is cleaved and polyadenylated just once, so the poly(A) profile represents an ensemble of independent events. However, at some human genes, it has been suggested that longer isoforms can be retained in the nuclear matrix and be processed into shorter poly(A) isoforms ( Tang et al, 2022 ). In considering the link between elongation and polyadenylation, a key issue is the location of elongating Pol II, and hence, the length of accessible RNA at the time of cleavage and subsequent polyadenylation.…”

Section: Introductionmentioning

confidence: 99%

Nucleotide-level linkage of transcriptional elongation and polyadenylation

Geisberg

Moqtaderi

Fong³

et al. 2022

eLife

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Alternative polyadenylation yields many mRNA isoforms whose 3' termini occur disproportionately in clusters within 3' UTRs. Previously, we showed that profiles of poly(A) site usage are regulated by the rate of transcriptional elongation by RNA polymerase (Pol) II (Geisberg et., 2020). Pol II derivatives with slow elongation rates confer an upstream-shifted poly(A) profile, whereas fast Pol II strains confer a downstream-shifted poly(A) profile. Within yeast isoform clusters, these shifts occur steadily from one isoform to the next across nucleotide distances. In contrast, the shift between clusters from the last isoform of one cluster to the first isoform of the next - is much less pronounced, even over large distances. GC content in a region 13-30 nt downstream from isoform clusters correlates with their sensitivity to Pol II elongation rate. In human cells, the upstream shift caused by a slow Pol II mutant also occurs continuously at the nucleotide level within clusters, but not between them. Pol II occupancy increases just downstream of the most speed-sensitive poly(A) sites, suggesting a linkage between reduced elongation rate and cluster formation. These observations suggest that 1) Pol II elongation speed affects the nucleotide-level dwell time allowing polyadenylation to occur, 2) poly(A) site clusters are linked to the local elongation rate and hence do not arise simply by intrinsically imprecise cleavage and polyadenylation of the RNA substrate, 3) DNA sequence elements can affect Pol II elongation and poly(A) profiles, and 4) the cleavage/polyadenylation and Pol II elongation complexes are spatially, and perhaps physically, coupled so that polyadenylation occurs rapidly upon emergence of the nascent RNA from the Pol II elongation complex.

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Section: Introductionmentioning

confidence: 99%

Nucleotide-level linkage of transcriptional elongation and polyadenylation

Geisberg

Moqtaderi

Fong³

et al. 2022

eLife

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“…We used Iso-Seq to identify Polyadenylation sites (PASs) and RNA-seq to quantify APA changes. PASs within 50 nt were clustered into one PAS, and PASs with distance >50 nt were considered as different PASs, as defined previously ( Tang et al, 2022 ). If a PAS is within 50 nt of a known ncbiRefSeq PAS, it is considered as known, otherwise defined as novel ( Figure 5A ).…”

Section: Resultsmentioning

confidence: 99%

Integrative analysis of Iso-Seq and RNA-seq reveals dynamic changes of alternative promoter, alternative splicing and alternative polyadenylation during Angiotensin II-induced senescence in rat primary aortic endothelial cells

Wen

Chen

et al. 2023

Front. Genet.

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In eukaryotes, alternative promoter (AP), alternative splicing (AS), and alternative polyadenylation (APA) are three crucial regulatory mechanisms that modulate message RNA (mRNA) diversity. Although AP, AS and APA are involved in diverse biological processess, whether they have dynamic changes in Angiotensin II (Ang II) induced senescence in rat primary aortic endothelial cells (RAECs), an important cellular model for studying cardiovascular disease, remains unclear. Here we integrated both PacBio single-molecule long-read isoform sequencing (Iso-Seq) and Illumina short-read RNA sequencing (RNA-seq) to analyze the changes of AP, AS and APA in Ang II-induced senescent RAECs. Iso-Seq generated 36,278 isoforms from 10,145 gene loci and 65.81% of these isoforms are novel, which were further cross-validated by public data obtained by other techonologies such as CAGE, PolyA-Seq and 3′READS. APA contributed most to novel isoforms, followed by AS and AP. Further investigation showed that AP, AS and APA could all contribute to the regulation of isoform, but AS has more dynamic changes compared to AP and APA upon Ang II stimulation. Genes undergoing AP, AS and APA in Ang II-treated cells are enriched in various pathways related to aging or senescence, suggesting that these molecular changes are involved in functional alterations during Ang II-induced senescence. Together, the present study largely improved the annotation of rat genome and revealed gene expression changes at isoform level, extending the understanding of the complexity of gene regulation in Ang II-treated RAECs, and also provided novel clues for discovering the regulatory mechanism undelying Ang II caused vascular senescence and diseases.

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“…Very recently, a study from Yu Zhou and Xiang-dong Fu's lab revealed that the isoforms ended with stronger distal PAS could undergo additional round of posttranscriptional cleavage at a proximal PASs to generate the short 3′UTR isoform ( 57 ). If so, the perturbation of the distal PAS might result in more efficient use of proximal PASs with little effect on total mRNA abundance.…”

Section: Resultsmentioning

confidence: 99%

CRISPR-iPAS: a novel dCAS13-based method for alternative polyadenylation interference

Tian

Zhang

et al. 2022

Nucleic Acids Research

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Alternative polyadenylation (APA) plays an important role in gene regulation. With the recent application of novel sequencing technology in APA profiling, an ever-increasing number of APA genes/sites have been identified. However, the phenotypic relevance of most of these APA isoforms remains elusive, which is largely due to the lack of a convenient genetics tool for APA interference. To address this issue, herein, an efficient method is developed based on the CRISPR-dCas13 system, termed as CRISPR-iPAS. Out of eight different dCas13 proteins, Porphyromonas gulae (Pgu) dCas13b, is identified as the most effective one in blocking the usage of the polyadenylation site (PAS). With guide RNAs targeting at core regulatory elements, dPguCas13b enabled APA regulation of endogenous genes with different APA types, including tandem 3′UTR, alternative terminal exon, as well as intronic PAS. Finally, we demonstrated that the proposed APA perturbation tool could be used to investigate the functional relevance of APA isoforms.

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Alternative polyadenylation by sequential activation of distal and proximal PolyA sites

Cited by 32 publications

References 62 publications

Nucleotide-level linkage of transcriptional elongation and polyadenylation

Nucleotide-level linkage of transcriptional elongation and polyadenylation

Integrative analysis of Iso-Seq and RNA-seq reveals dynamic changes of alternative promoter, alternative splicing and alternative polyadenylation during Angiotensin II-induced senescence in rat primary aortic endothelial cells

CRISPR-iPAS: a novel dCAS13-based method for alternative polyadenylation interference

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