The ADAR1 editome reveals drivers of editing-specificity for ADAR1-isoforms

Adenosine deaminase acting on RNA (ADAR) (also known as ADAR1) promotes A-to-I conversion in double-stranded and highly structured RNAs. ADAR1 has two isoforms transcribed from different promoters: ADAR1p150, which is mainly cytoplasmic and interferon-inducible, and constitutively expressed ADAR1p110 that is primarily localized in the nucleus. Mutations in ADAR1 cause Aicardi – Goutières syndrome (AGS), a severe autoinflammatory disease in humans associated with aberrant IFN production. In mice, deletion of ADAR1 or selective knockout of the p150 isoform alone leads to embryonic lethality driven by overexpression of interferon-stimulated genes. This phenotype can be rescued by concurrent deletion of cytoplasmic dsRNA-sensor MDA5. These findings indicate that the interferon-inducible p150 isoform is indispensable and cannot be rescued by the ADAR1p110 isoform. Nevertheless, editing sites uniquely targeted by ADAR1p150 but also mechanisms of isoform-specificity remain elusive. Here we combine RIP-seq on human cells expressing ADAR1 isoforms and combine this with analysis of isoform-specific editing patterns in genetically modified mouse cells to extensively investigate ADAR1-isoform binding- and editing characteristics. Moreover, using mutated ADAR variants, we examine the effect of two unique features of ADAR1p150 on its target specificity: 1) cytoplasmic localization and 2) Z-DNA binding domain α. Our findings indicate that ZBDa contributes only minimally to p150 editing-specificity and that isoform-specific editing is directed mainly by the cytoplasmic localization of the editase.

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The Biological Impact of the Human Master Regulator p53 Can Be Altered by Mutations That Change the Spectrum and Expression of Its Target Genes

Molecular and Cellular Biology ◽

10.1128/mcb.26.6.2297-2308.2006 ◽

2006 ◽

Vol 26 (6) ◽

pp. 2297-2308 ◽

Cited By ~ 60

Author(s):

Daniel Menendez ◽

Alberto Inga ◽

Michael A. Resnick

Keyword(s):

Dna Binding ◽

Target Genes ◽

Tumor Development ◽

Human Tumor ◽

Human Cells ◽

Binding Domain ◽

Dna Binding Domain ◽

Missense Mutations ◽

The Relationship ◽

Biological Outcomes

ABSTRACT Human tumor suppressor p53 is a sequence-specific master regulatory transcription factor that targets response elements (REs) in many genes. p53 missense mutations in the DNA-binding domain are often cancer associated. As shown with systems based on the yeast Saccharomyces cerevisiae, p53 mutants can alter the spectra and intensities of transactivation from individual REs. We address directly in human cells the relationship between changes in the p53 master regulatory network and biological outcomes. Expression of integrated, tightly regulated DNA-binding domain p53 mutants resulted in many patterns of apoptosis and survival following UV or ionizing radiation, or spontaneously. These patterns reflected changes in the spectra and activities of target genes, as demonstrated for P21, MDM2, BAX, and MSH2. Thus, as originally proposed for “master genes of diversity,” p53 mutations in human cells can differentially influence target gene transactivation, resulting in a variety of biological consequences which, in turn, might be expected to influence tumor development and therapeutic efficacy.

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Spectroscopic Characterization of a DNA-Binding Domain, Zα, from the Editing Enzyme, dsRNA Adenosine Deaminase: Evidence for Left-Handed Z-DNA in the Zα−DNA Complex†

Biochemistry ◽

10.1021/bi9813126 ◽

1998 ◽

Vol 37 (38) ◽

pp. 13313-13321 ◽

Cited By ~ 45

Author(s):

Imre Berger ◽

William Winston ◽

Ramasamy Manoharan ◽

Thomas Schwartz ◽

Jens Alfken ◽

...

Keyword(s):

Dna Binding ◽

Adenosine Deaminase ◽

Spectroscopic Characterization ◽

Binding Domain ◽

Dna Binding Domain ◽

Left Handed ◽

Z Dna ◽

Editing Enzyme ◽

Dna Complex

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Centromeric transcription maintains centromeric cohesion in human cells

The Journal of Cell Biology ◽

10.1083/jcb.202008146 ◽

2021 ◽

Vol 220 (7) ◽

Author(s):

Yujue Chen ◽

Qian Zhang ◽

Zhen Teng ◽

Hong Liu

Keyword(s):

Dna Binding ◽

Gene Transcription ◽

Direct Consequence ◽

Human Cells ◽

Binding Domain ◽

Dna Binding Domain ◽

Major Function ◽

Krab Domain

Centromeric transcription has been shown to play an important role in centromere functions. However, lack of approaches to specifically manipulate centromeric transcription calls into question that the proposed functions are a direct consequence of centromeric transcription. By monitoring nascent RNAs, we found that several transcriptional inhibitors exhibited distinct, even opposing, efficacies on the suppression of ongoing gene and centromeric transcription in human cells, whereas under the same conditions, total centromeric RNAs were changed to a lesser extent. The inhibitor suppressing ongoing centromeric transcription weakened centromeric cohesion, whereas the inhibitor increasing ongoing centromeric transcription strengthened centromeric cohesion. Furthermore, expression of CENP-B DNA-binding domain or CENP-B knockdown moderately increased centromeric transcription without altering gene transcription; as a result, centromeric cohesion was accordingly strengthened. Targeting of the Kox1-KRAB domain with CENP-B DB to centromeres specifically decreased centromeric transcription and weakened centromeric cohesion. Thus, based on these findings, we propose that a major function of centromeric transcription is to maintain centromeric cohesion in human cells.

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A Z-DNA binding domain present in the human editing enzyme, double-stranded RNA adenosine deaminase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.94.16.8421 ◽

1997 ◽

Vol 94 (16) ◽

pp. 8421-8426 ◽

Cited By ~ 206

Author(s):

A. Herbert ◽

J. Alfken ◽

Y.-G. Kim ◽

I. S. Mian ◽

K. Nishikura ◽

...

Keyword(s):

Dna Binding ◽

Adenosine Deaminase ◽

Binding Domain ◽

Dna Binding Domain ◽

Double Stranded Rna ◽

Z Dna ◽

Editing Enzyme

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Phosphorylation of a Conserved Serine in the Deoxyribonucleic Acid Binding Domain of Nuclear Receptors Alters Intracellular Localization

Molecular Endocrinology ◽

10.1210/me.2006-0300 ◽

2007 ◽

Vol 21 (6) ◽

pp. 1297-1311 ◽

Cited By ~ 57

Author(s):

Kai Sun ◽

Vedrana Montana ◽

Karthikeyani Chellappa ◽

Yann Brelivet ◽

Dino Moras ◽

...

Keyword(s):

Dna Binding ◽

Nuclear Receptors ◽

Nuclear Localization ◽

Thyroid Hormone Receptor ◽

Zinc Fingers ◽

Binding Domain ◽

Retinoid X Receptor ◽

Common Mechanism ◽

Dna Binding Domain ◽

Cytoplasmic Localization

Abstract Nuclear receptors (NRs) are a superfamily of transcription factors whose genomic functions are known to be activated by lipophilic ligands, but little is known about how to deactivate them or how to turn on their nongenomic functions. One obvious mechanism is to alter the nuclear localization of the receptors. Here, we show that protein kinase C (PKC) phosphorylates a highly conserved serine (Ser) between the two zinc fingers of the DNA binding domain of orphan receptor hepatocyte nuclear factor 4α (HNF4α). This Ser (S78) is adjacent to several positively charged residues (Arg or Lys), which we show here are involved in nuclear localization of HNF4α and are conserved in nearly all other NRs, along with the Ser/threonine (Thr). A phosphomimetic mutant of HNF4α (S78D) reduced DNA binding, transactivation ability, and protein stability. It also impaired nuclear localization, an effect that was greatly enhanced in the MODY1 mutant Q268X. Treatment of the hepatocellular carcinoma cell line HepG2 with PKC activator phorbol 12-myristate 13-acetate also resulted in increased cytoplasmic localization of HNF4α as well as decreased endogenous HNF4α protein levels in a proteasome-dependent fashion. We also show that PKC phosphorylates the DNA binding domain of other NRs (retinoic acid receptor α, retinoid X receptor α, and thyroid hormone receptor β) and that phosphomimetic mutants of the same Ser/Thr result in cytoplasmic localization of retinoid X receptor α and peroxisome proliferator-activated receptor α. Thus, phosphorylation of this conserved Ser between the two zinc fingers may be a common mechanism for regulating the function of NRs.

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Cytosine base editing systems with minimized off-target effect and molecular size

10.21203/rs.3.rs-414230/v1 ◽

2021 ◽

Author(s):

Ang Li ◽

Hitoshi Mitsunobu ◽

Shin Yoshioka ◽

Suzuki Takahisa ◽

Akihiko Kondo ◽

...

Keyword(s):

Dna Binding ◽

Molecular Size ◽

Human Cells ◽

Binding Domain ◽

Enzyme Function ◽

Dna Binding Domain ◽

Aav Vector ◽

Base Editing ◽

Rational Engineering ◽

Target Effect

Abstract Structure-based rational engineering of the cytosine base editing system Target-AID was performed to minimize its off-target effect and molecular size. By intensive and careful truncation, DNA-binding domain of its deaminase PmCDA1 was eliminated and additional mutations were introduced to restore enzyme function. The resulting tCDA1EQ was effective in N-terminal fusion (AID-2S) or inlaid architecture (AID-3S) with Cas9, showing minimized gRNA-independent off-targets, as assessed in yeast and human cells. Combining with the smaller Cas9 ortholog system, the smallest cytosine base editing system was created that is within the size limit of AAV vector.

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