PAPD5, a noncanonical poly(A) polymerase with an unusual RNA-binding motif [PAR-CLIP]
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE36987 | GEO | 2012/04/03
SECONDARY ACCESSION(S): PRJNA157515
REPOSITORIES: GEO
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Project description:PAPD5 is one of the seven members of non-canonical poly(A) polymerases in human cells. There are previous reports about polyadenylation dependent degradation of pre-ribosomal RNAs and uridylation dependent degradation of histone mRNAs in vivo. In this study, we observed polyadenylation but not polyuridylation activity of PAPD5 with in vitro assays. We aimed to get genome-wide targets of PAPD5 and used PAR-CLIP and deep sequencing for this purpose. Recombinant version of PAPD5 is expressed in HEK293 human cell lines and its genome wide targets are obtained with PAR-CLIP and deep sequencing as two replicate experiments. The short reads in the deep sequencing libraries of PAPD5 replicates and an unrelated protein to polymerization from a previous study, IGF2BP1, are aligned to the hg18 human genome assembly. The biological variance of the read counts in overlapping 100-nucleotide-long-windows is estimated between the PAPD5 replicates and further used in the differential expression estimations between the 100-nucleotide windows in PAPD5 replicates and IGF2BP1. The top differentially expressed windows in PAPD5 and IGF2BP1 are further annotated using gene and repeat tracks from UCSC.
2012-04-02 | E-GEOD-36987 | biostudies-arrayexpress
Project description:PAPD5 is one of the seven members of the family of noncanonical poly(A) polymerases in human cells. PAPD5 was shown to polyadenylate aberrant pre-ribosomal RNAs in vivo, similar to degradation-mediating polyadenylation by the noncanonical poly(A) polymerase Trf4p in yeast. PAPD5 has been reported to be also involved in the uridylation-dependent degradation of histone mRNAs. To test whether PAPD5 indeed catalyzes adenylation as well as uridylation of RNA substrates, we analyzed the in vitro properties of recombinant PAPD5 expressed in mammalian cells as well as in bacteria. Our results show that PAPD5 catalyzes the polyadenylation of different types of RNA substrates in vitro. Interestingly, PAPD5 is active without a protein cofactor, whereas its yeast homolog Trf4p is the catalytic subunit of a bipartite poly(A) polymerase in which a separate RNA-binding subunit is needed for activity. In contrast to the yeast protein, the C terminus of PAPD5 contains a stretch of basic amino acids that is involved in binding the RNA substrate.
| S-EPMC3162338 | biostudies-literature
Project description:Next-generation sequencing experiments have shown that microRNAs are expressed in many different isoforms (isomiRs), whose biological relevance is often unclear. We found that mature miR-21, the most widely researched microRNA because of its importance in human disease, is produced in two prevalent isomiR forms that differ by one nucleotide at their 3M-bM-^@M-^Y end, and moreover that the 3M-bM-^@M-^Y end of miR-21 is post transcriptionally adenylated by the noncanonical poly(A) polymerase PAPD5. PAPD5 knockdown caused an increase in the miR-21 expression level, suggesting that PAPD5-mediated adenylation of miR-21 leads to its degradation. Exoribonuclease knockdown experiments followed by small RNA sequencing suggested that PARN degrades miR-21 in the 3M-bM-^@M-^Y-to-5M-bM-^@M-^Y direction. In accordance with this model, microarray expression profiling demonstrated that PAPD5 knockdown results in a downregulation of miR-21 target mRNAs. We found that disruption of the miR-21 adenylation and degradation pathway is a general feature in tumors across a wide range of tissues, as evidenced by data from The Cancer Genome Atlas, as well as in the non-cancerous proliferative disease psoriasis. We conclude that PAPD5 and PARN mediate degradation of oncomiR miR-21 through a tailing and trimming process, and that this pathway is disrupted in cancer and other proliferative diseases. Gene expression levels were profiled by Illumina microarrays using RNA produced previously (PMID 20719920). Briefly, total RNA was extracted from THP1 cells in four replicates 72 hours after transfection of siRNA against PAPD5 or GLD2, or of calibrator negative control siRNA. Gene expression levels were profiled in two of the PAPD5 knockdown replicates, two of the GLD2 knockdown replicates, and two of the negative control replicates.
2014-05-24 | E-GEOD-57939 | biostudies-arrayexpress
Project description:Next-generation sequencing experiments have shown that microRNAs are expressed in many different isoforms (isomiRs), whose biological relevance is often unclear. We found that mature miR-21, the most widely researched microRNA because of its importance in human disease, is produced in two prevalent isomiR forms that differ by one nucleotide at their 3’ end, and moreover that the 3’ end of miR-21 is post transcriptionally adenylated by the noncanonical poly(A) polymerase PAPD5. PAPD5 knockdown caused an increase in the miR-21 expression level, suggesting that PAPD5-mediated adenylation of miR-21 leads to its degradation. Exoribonuclease knockdown experiments followed by small RNA sequencing suggested that PARN degrades miR-21 in the 3’-to-5’ direction. In accordance with this model, microarray expression profiling demonstrated that PAPD5 knockdown results in a downregulation of miR-21 target mRNAs. We found that disruption of the miR-21 adenylation and degradation pathway is a general feature in tumors across a wide range of tissues, as evidenced by data from The Cancer Genome Atlas, as well as in the non-cancerous proliferative disease psoriasis. We conclude that PAPD5 and PARN mediate degradation of oncomiR miR-21 through a tailing and trimming process, and that this pathway is disrupted in cancer and other proliferative diseases.
2014-05-24 | GSE57939 | GEO
Project description:MicroRNAs (miRNAs) are small non-coding RNAs that play crucial roles in post-transcriptional gene regulation. Poly(A) RNA polymerase D5 (PAPD5) catalyzes the addition of adenosine to the 3′ end of miRNAs. In this study, we demonstrated that the Yin Yang 1 protein, a transcriptional repressor of PAPD5, was recruited to both RNA foci and protein aggregates, resulting in an upregulation of PAPD5 expression in Huntington’s disease (HD). Additionally, we identified a subset of PAPD5-regulated miRNAs with increased adenylation and reduced expression in our disease model. We focused on miR-7-5p and found that its reduction caused the activation of the TAB2-mediated TAK1–MKK4–JNK pro-apoptotic pathway. This pathway was also activated in induced pluripotent stem cell-derived striatal neurons and post-mortem striatal tissues isolated from HD patients. In addition, we discovered that a small molecule PAPD5 inhibitor, BCH001, could mitigate cell death and neurodegeneration in our disease models. This study highlights the importance of PAPD5-mediated miRNA dysfunction in HD pathogenesis and suggests a potential therapeutic direction for the disease.
2024-08-01 | GSE272903 | GEO
Project description:MicroRNAs (miRNAs) are small non-coding RNAs that play crucial roles in post-transcriptional gene regulation. Poly(A) RNA polymerase D5 (PAPD5) catalyzes the addition of adenosine to the 3′ end of miRNAs. In this study, we demonstrated that the Yin Yang 1 protein, a transcriptional repressor of PAPD5, was recruited to both RNA foci and protein aggregates, resulting in an upregulation of PAPD5 expression in Huntington’s disease (HD). Additionally, we identified a subset of PAPD5-regulated miRNAs with increased adenylation and reduced expression in our disease model. We focused on miR-7-5p and found that its reduction caused the activation of the TAB2-mediated TAK1–MKK4–JNK pro-apoptotic pathway. This pathway was also activated in induced pluripotent stem cell-derived striatal neurons and post-mortem striatal tissues isolated from HD patients. In addition, we discovered that a small molecule PAPD5 inhibitor, BCH001, could mitigate cell death and neurodegeneration in our disease models. This study highlights the importance of PAPD5-mediated miRNA dysfunction in HD pathogenesis and suggests a potential therapeutic direction for the disease.
2024-08-01 | GSE273082 | GEO
Project description:Ferroptosis is a unique iron-dependent form of non-apoptotic cell death characterized by devastating lipid peroxidation. Whilst growing evidence suggests that ferroptosis is a type of autophagy-dependent cell death, the underlying molecular mechanisms regulating ferroptosis are largely unknown. In this study, through an unbiased RNA-sequencing screening, we demonstrate the activation of a multi-faceted tumor-suppressor protein Par-4/PAWR during ferroptosis. Functional studies reveal that genetic depletion of Par-4 effectively blocks ferroptosis, whereas Par-4 overexpression sensitizes cells to undergo ferroptosis. More importantly, we have determined that Par-4-triggered ferroptosis is mechanistically driven by the autophagic machinery. Upregulation of Par-4 promotes activation of ferritinophagy (autophagic degradation of ferritin) via the nuclear receptor co-activator 4 (NCOA4), resulting in excessive release of free labile iron and, hence, enhanced lipid peroxidation and ferroptosis. Inhibition of Par-4 dramatically suppresses the NCOA4-mediated ferritinophagy signaling axis. Our results also establish that Par-4 activation positively correlates with reactive oxygen species (ROS) production, which is critical for ferritinophagy-mediated ferroptosis. Furthermore, Par-4 knockdown effectively blocked ferroptosis-mediated tumor suppression in the mouse xenograft models. Collectively, these findings reveal that Par-4 has a crucial role in ferroptosis, which could be further exploited for cancer therapy.
2024-05-24 | GSE267823 | GEO
Project description:Genetic lesions that reduce telomerase cause a range of incurable diseases including dyskeratosis congenita (DC) and pulmonary fibrosis (PF), and restoring telomere length in these patients would be curative. Ectopic expression of telomerase reverse transcriptase (TERT) risks cellular immortalization, and how to target telomerase in stem cells throughout the body remains unclear. Here we describe a successful screen for small molecules that augment TERC, the non-coding telomerase RNA component, and thereby specifically elongate telomeres in stem cells. PAPD5 is a non-canonical polymerase that oligo-adenylates and destabilizes TERC. Using a high-throughput screen we identify BCH001, a specific PAPD5 inhibitor that decreases TERC 3′- oligo(A) tailing and increases telomerase activity and telomere length by thousands of nucleotides in DC patient induced pluripotent stem cells (iPSCs). BCH001 does not result in immortalization or telomere elongation in somatic cells which lack TERT, establishing a favorable safety profile. When human hematopoietic stem and progenitor cells (HSPCs) engineered by CRISPR-Cas9 to carry PARN mutations that cause DC and PF are xenotransplanted into immunodeficient mice, oral treatment with PAPD5 inhibitors rescues TERC 3′-end maturation and telomere length. Our data demonstrate telomere restoration in human stem cells in vivo using small molecules.
2020-04-21 | GSE144986 | GEO
Project description:PAR-CLIP sequencing experiments for SOX2 in HEK293T cells.
2020-01-30 | GSE115273 | GEO
Project description:In mammals, homologous chromosomes rarely pair outside meiosis. One exception is the X chromosome, which transiently pairs during X-chromosome inactivation (XCI). How two chromosomes find each other in 3D space is not known. Here, we reveal a required interaction between the X-inactivation center (Xic) and the telomere in mouse embryonic stem (ES) cells. The subtelomeric, pseudoautosomal regions (PARs) of the two sex chromosomes (X and Y) also undergo pairing in both female and male cells. PARs transcribe a class of telomeric RNA, dubbed PAR-TERRA, which accounts for a vast majority of all TERRA transcripts. PAR-TERRA binds throughout the genome, including to the PAR and Xic. During X-chromosome pairing, PAR-TERRA anchors the Xic to the PAR, creating a ‘tetrad’ of pairwise homologous interactions (Xic–Xic, PAR–PAR, and Xic–PAR). Xic pairing occurs within the tetrad. Depleting PAR-TERRA abrogates pairing and blocks initiation of XCI, whereas autosomal PAR-TERRA induces ectopic pairing. We propose a ‘constrained diffusion model’ in which PAR-TERRA creates an interaction hub to guide Xic homology searching during XCI.
2017-07-10 | GSE69887 | GEO