Biophysical characterizations of the recognition of the AAUAAA polyadenylation signal

Hamilton, Keith; Sun, Yadong; Tong, Liang

doi:10.1101/503755

Cited by 5 publications

(6 citation statements)

References 24 publications

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“…Second, by searching for the most enriched motifs, we found that AAUAAA and U/GU rich motifs were the most significant motifs for WDR33 and CstF64, respectively (Supplemental Figure 5B), consistent with previous reports (16,49). Third, by focusing on the density of the reads closing to the canonical cleavage/polyadenylation (C/P) site, we observed peaks approximately 20 nt upstream of the C/P site for WDR33 and 10 nt downstream of the C/P site for CstF64 (Supplemental Figure 5B), which agree well with the current in vitro model for PAS recognition by CPSF/CstF (16,44,46,54,55). Two major observations were made from the iCLIP-seq analysis between the control and U1 AMO treated cells.…”

Section: U1 Amo Treatment Manipulates Cpsf/cstf Co-transcriptionallysupporting

confidence: 90%

U1 AMO (antisense morpholino oligo) disrupts U1 snRNP structure to promote intronic premature cleavage and polyadenylation (PCPA)

Feng

Lin

Deng

et al. 2023

Preprint

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Functional depletion of U1 snRNP with a 25 nt U1 AMO (antisense morpholino oligonucleotides) may lead to intronic premature cleavage and polyadenylation (PCPA) of thousands of genes, a phenomenon known as U1 snRNP telescripting; however, the underlying mechanism remains elusive. In this study, we demonstrated that U1 AMO could disrupt U1 snRNP structure both in vitro and in vivo, thereby affecting U1 snRNP/RNAP polymerase II (RNAPII) interaction. We further showed that U1 AMO treatment might promote RNAPII disassociation with pre-mRNA in an RNA pull-down assay. By performing ChIP-seq for phosphorylation of Ser2 (Ser2P) and Ser5 (Ser5P) of the C-terminal domain (CTD) of RNA polymerase II (RNAPII), we showed that transcription elongation was disturbed upon U1 AMO treatment, with a particular high Ser2P signal at intronic cryptic polyadenylation sites (PASs). In addition, we showed that core 3' processing factors CPSF/CstF are involved in the processing of intronic cryptic PAS. Their recruitment accumulated toward cryptic PASs upon U1 AMO treatment, as indicated by ChIP-seq and iCLIP-seq analysis. Furthermore, we showed that most of these PCPAed transcripts could be exported to cytoplasm and have the potential to be translated. Conclusively, our data provide more insight into U1 snRNP telescripting, and suggest a common theme that modulation of transcription elongation may be an important mode for the regulation of mRNA polyadenylation.

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Section: U1 Amo Treatment Manipulates Cpsf/cstf Co-transcriptionallysupporting

confidence: 90%

U1 AMO (antisense morpholino oligo) disrupts U1 snRNP structure to promote intronic premature cleavage and polyadenylation (PCPA)

Feng

Lin

Deng

et al. 2023

Preprint

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“…Our 3'RACE results suggest that the first PAS encountered (ATTAAA) is preferentially used both before and after UV treatment (Figure 2D), whereas the downstream PAS (AATAAA) was used with less efficiency (and only after UV treatment). Although ATTAAA is the first encountered, it also has 5-6 times lower affinity for cleavage and polyadenylation specificity factor (CPSF) than AATAAA (87). However, it has also been shown that short (<10 bp) distances between PAS and GU-rich sequences might inhibit processing (88), explaining the lower usage of the second PAS.…”

Section: Discussionmentioning

confidence: 99%

Long Non-Coding RNA Generated fromCDKN1AGene by Alternative Polyadenylation Regulates p21 Expression during DNA Damage Response

Murphy

Ramadei

Doymaz

et al. 2023

Preprint

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Alternative Polyadenylation (APA) is an emerging mechanism for dynamic changes in gene expression. Previously, we described widespread APA occurrence in introns during the DNA damage response (DDR). Here, we show that a DNA damage activated APA event occurs in the first intron of CDKN1A, inducing an alternate last exon (ALE)-containing lncRNA. We named this lncRNA SPUD (Selective Polyadenylation Upon Damage). SPUD localizes to polysomes in the cytoplasm and is detectable as multiple isoforms in available high throughput studies. SPUD has low abundance compared to the CDKN1A full-length isoform and is induced in cancer and normal cells under a variety of DNA damaging conditions in part through p53 transcriptional activation. RNA binding protein (RBP) HuR and the transcriptional repressor CTCF regulate SPUD levels. SPUD induction increases p21 protein, but not CDKN1A full-length levels, affecting p21 functions in cell-cycle, CDK2 expression, and cell viability. Like CDKN1A full-length isoform, SPUD can bind two competitive p21 translational regulators, the inhibitor calreticulin and the activator CUGBP1; SPUD can change their association with CDKN1A full-length in a DDR-dependent manner. Together, these results show a new regulatory mechanism by which a lncRNA controls p21 expression post-transcriptionally, highlighting lncRNA relevance in DDR progression and cell cycle.

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“…For example, the poly(A) polymerase enzyme is a constitutive subunit of the yeast but not the human complex (Chan et al, 2014;Kaufmann et al, 2004). Human CPSF has a nanomolar affinity for PAS-containing RNA (Hamilton et al, 2019), while the interaction of CPF with RNA is orders of magnitude weaker (Hill et al 2019). In addition, human CStF and CFIIm are separate complexes, whereas in yeast, they form a constitutive complex called CF IA (Gordon et al, 2011;Schäfer et al, 2018).…”

Section: Discussionmentioning

confidence: 99%

RBBP6 activates the pre-mRNA 3’-end processing machinery in humans

Boreikaitė

Elliott

Chin

et al. 2021

Preprint

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Abstract3’-end processing of most human mRNAs is carried out by the cleavage and polyadenylation specificity factor (CPSF; CPF in yeast). Endonucleolytic cleavage of the nascent pre-mRNA defines the 3’-end of the mature transcript, which is important for mRNA localization, translation and stability. Cleavage must therefore be tightly regulated. Here, we reconstitute specific and efficient 3’-endonuclease activity of human CPSF with purified proteins. This requires the sevensubunit CPSF as well as three additional protein factors: cleavage stimulatory factor (CStF), cleavage factor IIm (CFIIm) and, importantly, the multi-domain protein RBBP6. Unlike its yeast homologue Mpe1, which is a stable subunit of CPF, RBBP6 does not copurify with CPSF and is recruited in an RNA-dependent manner. Sequence and mutational analyses suggest that RBBP6 interacts with the WDR33 and CPSF73 subunits of CPSF. Thus, it is likely that the role of RBBP6 is conserved from yeast to human. Overall, our data are consistent with CPSF endonuclease activation and site-specific pre-mRNA cleavage being highly controlled to maintain fidelity in RNA processing.

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Biophysical characterizations of the recognition of the AAUAAA polyadenylation signal

Cited by 5 publications

References 24 publications

U1 AMO (antisense morpholino oligo) disrupts U1 snRNP structure to promote intronic premature cleavage and polyadenylation (PCPA)

U1 AMO (antisense morpholino oligo) disrupts U1 snRNP structure to promote intronic premature cleavage and polyadenylation (PCPA)

Long Non-Coding RNA Generated fromCDKN1AGene by Alternative Polyadenylation Regulates p21 Expression during DNA Damage Response

RBBP6 activates the pre-mRNA 3’-end processing machinery in humans

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