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Gene expression profiling in THP1 cells after PAPD5 or GLD2 knockdown

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE57939 | GEO | 2014/05/24

SECONDARY ACCESSION(S): PRJNA248456

REPOSITORIES: GEO

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Gene expression profiling in THP1 cells after PAPD5 or GLD2 knockdown

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

Homo sapiens strain:THP1, MCF7

Project description:PAPD5-mediated adenylation of miR-21

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Post-transcriptional manipulation of TERC reverses molecular hallmarks of telomere disease

Project description:The telomerase RNA component (TERC) is a critical determinant of cellular self renewal. Poly(A)-specific ribonuclease (PARN) is required for post-transcriptional maturation of TERC. PARN mutations lead to incomplete 3â² end processing and increased destruction of nascent TERC RNA transcripts, resulting in telomerase deficiency and telomere diseases. Here, we determined that overexpression of TERC increased telomere length in PARN-deficient cells and hypothesized that decreasing post-transcriptional 3â² oligo-adenylation of TERC would counteract the deleterious effects of PARN mutations. Inhibition of the noncanonical poly(A) polymerase PAP-associated domainâcontaining 5 (PAPD5) increased TERC levels in PARN-mutant patient cells. PAPD5 inhibition was also associated with increases in TERC stability, telomerase activity, and telomere elongation. Our results demonstrate that manipulating post-transcriptional regulatory pathways may be a potential strategy to reverse the molecular hallmarks of telomere disease. mRNA sequencing of induced pluripotent stem cells and 293 cell line.

2016-08-02 | E-GEOD-81507 | biostudies-arrayexpress

Mutant huntingtin induces neuronal apoptosis via derepressing the non-canonical poly(A) polymerase PAPD5

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.

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Mutant huntingtin induces neuronal apoptosis via derepressing the non-canonical poly(A) polymerase PAPD5 [ChIP-Seq]

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Maturation of human telomerase RNA component from an extended precursor

Project description:Here, we develop nascent RNAend-Seq, in which we isolate nascent RNA and sequence the 3' ends of RNA precursors. Using a pulse-chase experimental design, we follow extended precursors of the telomerase RNA component (hTR) and show that the mature telomerase RNA derives from these species with slow kinetics compared to other small non-coding RNAs. The human disease causing gene PARN further delays maturation of the hTR precursor in PARN- mutant cancer cell lines. Disruption of the poly(A)polymerase PAPD5 in PARN-mutant cells restores normal processing kinetics and levels of mature hTR. Unexpectedly, neither PARN nor PAPD5 is required for hTR processing. Instead, PAPD5 and PARN set the hTR maturation rate by controlling precursor adenylation.

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Post-transcriptional manipulation of TERC reverses molecular hallmarks of telomere disease

Project description:The telomerase RNA component (TERC) is a critical determinant of cellular self renewal. Poly(A)-specific ribonuclease (PARN) is required for post-transcriptional maturation of TERC. PARN mutations lead to incomplete 3′ end processing and increased destruction of nascent TERC RNA transcripts, resulting in telomerase deficiency and telomere diseases. Here, we determined that overexpression of TERC increased telomere length in PARN-deficient cells and hypothesized that decreasing post-transcriptional 3′ oligo-adenylation of TERC would counteract the deleterious effects of PARN mutations. Inhibition of the noncanonical poly(A) polymerase PAP-associated domain–containing 5 (PAPD5) increased TERC levels in PARN-mutant patient cells. PAPD5 inhibition was also associated with increases in TERC stability, telomerase activity, and telomere elongation. Our results demonstrate that manipulating post-transcriptional regulatory pathways may be a potential strategy to reverse the molecular hallmarks of telomere disease.

2016-08-02 | GSE81507 | GEO

Small-Molecule PAPD5 Inhibitors Restore Telomerase Activity in Patient Stem Cells

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.

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PAPD5, a noncanonical poly(A) polymerase with an unusual RNA-binding motif [PAR-CLIP]

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

PAPD5, a noncanonical poly(A) polymerase with an unusual RNA-binding motif [PAR-CLIP]

2012-04-03 | GSE36987 | GEO