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Genome-wide analysis of the effect of knockdown of the nuclear poly(A) binding proteins, PABPN1 and ZC3H14 on steady-state mRNA levels

ABSTRACT: The polyadenosine RNA binding proteins (Pabs) represent one class of RNA binding proteins that play critical roles in gene expression. This class includes the well-studied nuclear and cytoplasmic Pabs, PABPN1 and PABPC1, respectively, as well as the newly characterized nuclear Pab, zinc finger CCCH-type containing #14, or ZC3H14. ZC3H14 was recently linked to a form of intellectual disability, suggesting a critical role for ZC3H14 in neurons; however, the post-transcriptional function of ZC3H14 is unknown. In this study, we performed a microarray analysis of cells depleted of ZC3H14 or PABPN1 in MCF-7 breast cancer cells. These results revealed that PABPN1 significantly affected ~17% of expressed transcripts as compared to ZC3H14, which affected ~1% of expressed transcripts, suggesting that ZC3H14 has specific mRNA targets. The differentially expressed mRNAs identified in this analysis not only provide information about the classes and types of transcripts that are regulated by these proteins, but also represent a set of transcripts that could be directly bound by ZC3H14 and/or PABPN1. Total RNA isolated from MCF-7 cells treated for 48 hours with siRNA targeting PABPN1, ZC3H14 (all splice variants), or a control scramble siRNA.

ORGANISM(S): Homo sapiens

SUBMITTER: Callie Wigington

PROVIDER: E-GEOD-62067 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Genome-wide analysis of the effect of knockdown of the nuclear poly(A) binding proteins, PABPN1 and ZC3H14 on steady-state mRNA levels

Project description:The polyadenosine RNA binding proteins (Pabs) represent one class of RNA binding proteins that play critical roles in gene expression. This class includes the well-studied nuclear and cytoplasmic Pabs, PABPN1 and PABPC1, respectively, as well as the newly characterized nuclear Pab, zinc finger CCCH-type containing #14, or ZC3H14. ZC3H14 was recently linked to a form of intellectual disability, suggesting a critical role for ZC3H14 in neurons; however, the post-transcriptional function of ZC3H14 is unknown. In this study, we performed a microarray analysis of cells depleted of ZC3H14 or PABPN1 in MCF-7 breast cancer cells. These results revealed that PABPN1 significantly affected ~17% of expressed transcripts as compared to ZC3H14, which affected ~1% of expressed transcripts, suggesting that ZC3H14 has specific mRNA targets. The differentially expressed mRNAs identified in this analysis not only provide information about the classes and types of transcripts that are regulated by these proteins, but also represent a set of transcripts that could be directly bound by ZC3H14 and/or PABPN1.

2014-10-06 | GSE62067 | GEO

Regulation of alternative cleavage and polyadenylation by 3’ end processing and splicing factors

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

2015-04-01 | E-GEOD-62001 | biostudies-arrayexpress

Regulation of alternative cleavage and polyadenylation by 3’ end processing and splicing factors

2015-04-01 | GSE62001 | GEO

Phosphorylation of PABPN1 during mitosis protects mRNA from hyperadenylation and maintains transcriptome dynamics.

Project description:Polyadenylation controls mRNA biogenesis, nuclear export, translation, and decay. These processes are interdependent and coordinately regulated by poly(A)-binding proteins (PABPs), yet how PABPs are themselves regulated is not fully understood. Here, we show that human PABPN1, the nuclear PABP, is phosphorylated by mitotic kinases at four specific sites during mitosis, a time when nucleoplasm and cytoplasm mix. Phospho-inhibitory mutations decreased human cell proliferation. We therefore employed long-read sequencing to determine how PABPN1 phospho-site mutants affected poly(A) tails lengths of individual mRNAs, and TimeLapse-seq to monitor mRNA synthesis and decay. Phospho-inhibitory PABPN1 mutants lengthened poly(A) tails on both spliced and unspliced transcripts. In contrast, expression of phospho-mimetic PABPN1 resulted in shorter poly(A) tails and reduced transcriptome stability, indicating reduced polyadenylation activity of PABPN1 and targeting of long-tailed transcripts for decay. Thus, PABPN1 phosphorylation confers transcriptome instability that is associated with resetting the gene expression program as cells passage through cell cycle.

2024-06-26 | GSE247007 | GEO

Protein arginine methyltransferase 5 (PRMT5) is an actionable therapeutic target in CDK4/6 inhibitor-resistant ER+/RB-deficient breast cancer

Project description:PRMT5 Co-IP mass spectrometry: Lysates of MCF-7 RB1-knockout cells were pulled down with a PRMT5 antibody or IgG control. The pulldowns were subjected to mass spectrometry analysis. QEX2_981005, QEX2_981006, QEX2_981007: DMSO, IgG QEX2_981008, QEX2_981009, QEX2_981010: DMSO, PRMT5 antibody QEX2_981011, QEX2_981012, QEX2_981013, GSK, IgG QEX2_981014, QEX2_981015, QEX2_981016, GSK, PRMT5 antibody SDMA PTM analysis: MCF-7 RB1-knockout cells were transfected with a PRMT5 siRNA or a control siRNA. Lysates were collected three days after transfection and subjected to mass spectrometry analysis. LUM1_1000791, LUM1_1000792, LUM1_1000793: control SiRNA LUM1_1000794, LUM1_1000795, LUM1_1000796: PRMT5 siRNA [

2024-01-12 | PXD046996 | Pride

Phosphorylation of PABPN1 during mitosis protects mRNA from hyperadenylation and maintains transcriptome stability.

Project description:Polyadenylation controls mRNA biogenesis, nuclear export, translation, and decay. These processes are interdependent and coordinately regulated by several poly(A)-binding proteins (PABPs). How PABPs are functionally regulated to control RNA fate is not fully understood. Here, we show that human PABPN1, the nuclear PABP, is phosphorylated by mitotic kinases at four specific sites during mitosis when nucleoplasm and cytoplasm mix. We employed long-read sequencing to detect altered activities of PABPN1 mutants on poly(A) tails lengths of individual mRNAs and TimeLapse-seq to monitor mRNA turnover rates. Phospho-inhibitory PABPN1 mutants lengthened poly(A) tails on both spliced and unspliced transcripts, increased mRNA half-lives and decreased synthesis, and blocked cell proliferation. Although phospho-mimetic PABPN1 mutants still bind RNA, poly(A) tails were shorter in vivo. Thus, PABPN1 phosphorylation reduces the polyadenylation activity of PABPN1 and increases mRNA instability. We conclude that PABPN1 regulation balances mRNA synthesis and decay during cell cycle to achieve transcriptome homeostasis.

2024-10-17 | PXD047110 | Pride

Protein arginine methyltransferase 5 (PRMT5) is an actionable therapeutic target in CDK4/6 inhibitor-resistant ER+/RB-deficient breast cancer

2023-06-30 | MSV000092321 | MassIVE

Canonical poly(A) polymerase activity promotes the decay of a wide variety of mammalian nuclear RNAs

Project description:The human nuclear poly(A)-binding protein PABPN1 has been implicated in the decay of nuclear noncoding RNAs (ncRNAs). In addition, PABPN1 stimulates hyperadenylation by poly(A) polymerase, and this activity is thought to be required for decay. Here, we inactivated hyperadenylation by two distinct mechanisms and examined changes in gene expression in HEK293 cells by RNAseq. We observed the upregulation of various ncRNAs, including snoRNA host genes, primary miRNA transcripts, and upstream antisense RNAs, confirming that hyperadenylation is broadly required for the degradation of PABPN1-targets. In addition, we found that mRNAs with retained introns are susceptible to PABPN1 and PAPα/γ-mediated decay (PPD). Transcripts are targeted for degradation due to inefficient export, which is a consequence of reduced intron number or incomplete splicing. We conclude that PPD is an important mammalian nuclear RNA decay pathway for the removal of poorly spliced and nuclear-retained transcripts.

2015-10-03 | GSE73678 | GEO

A complex with poly(A) binding protein cytoplasmic 1 and EWS facilitates oncogenic ETS transcription factor function in prostate cells

Project description:The ETS transcription factor ERG is aberrantly expressed in approximately 50% of prostate tumors due to chromosomal rearrangements such as TMPRSS2/ERG. The ability of ERG to drive oncogenesis in prostate epithelial cells requires interaction with distinct co-activators, such as the RNA-binding protein EWS. Here, we find that ERG has both direct and indirect interactions with EWS, and the indirect interaction is mediated by the poly-A RNA-binding protein PABPC1. PABPC1 directly bound both ERG and EWS. ERG expression in prostate cells promoted PABPC1 localization to the nucleus and recruited PABPC1 to ERG/EWS binding sites in the genome. Knockdown of PABPC1 in prostate cells abrogated ERG-mediated phenotypes and decreased the ability of ERG to activate transcription. These findings define a complex including ERG and the RNA-binding proteins EWS and PABPC1 that represents a novel therapeutic target for ERG-positive prostate cancer and identify a novel nuclear role for PABPC1.

2023-11-06 | GSE226472 | GEO

Effects of HIST1H2ac knockdown on the DNA methylation landscape of breast cancer cell line MCF-7

Project description:There are 64 replication-dependent histone genes with different isotypes associated with each class of the five histone types, H1, H2a, H2b, H3 and H4 in the human genome. In a previous report, we have showed that HIST1H2ac serves as a master regulator of ERalpha-dependent gene activation via ERalpha recruitment, and mediates chromatin looping of regulatory elements of estrogen receptor-targeted genes. Here, we performed whole genome bisulfite sequencing of MCF-7 upon suppressing H2ac expression through siRNA transfection using two different siRNAs to determine the knockdown effect on DNA methylation. Our analysis showed the DNA methylation of ~99% of the genome were mostly unchanged comparing to normal MCF-7 (methylation levels difference within 0.2).

2020-12-31 | E-MTAB-3320 | biostudies-arrayexpress

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