Journal: Molecular Cell

Article Title: Dinucleotide Degradation by REXO2 Maintains Promoter Specificity in Mammalian Mitochondria

doi: 10.1016/j.molcel.2019.09.010

Figure Lengend Snippet: Human REXO2 Is a Dinucleotidase (A) Activity of wild-type REXO2 upon RNA substrates of different lengths. Monophosphorylated radiolabeled substrate (10 fmol) was incubated with the indicated amount of REXO2 at 37°C for 30 min, separated by 18% urea-PAGE, and imaged by autoradiography. (B) Quantification of RNA degradation by REXO2 from (A). (C) Activity of wild-type REXO2 upon DNA substrates of different lengths. Monophosphorylated radiolabeled substrate (10 fmol) was incubated with the indicated amount of REXO2 at 37°C for 30 min, separated by 18% urea-PAGE, and imaged by autoradiography. (D) Quantification of DNA degradation by REXO2 from (C).

Article Snippet: Codon-optimized (Genscript) DNA constructs corresponding to amino acids 39-216 of human REXO2 was cloned in a pETM-11 vector (EMBL).

Techniques: Activity Assay, Incubation, Autoradiography

Journal: Molecular Cell

Article Title: Dinucleotide Degradation by REXO2 Maintains Promoter Specificity in Mammalian Mitochondria

doi: 10.1016/j.molcel.2019.09.010

Figure Lengend Snippet: Data Collection and Refinement Statistics

Article Snippet: Codon-optimized (Genscript) DNA constructs corresponding to amino acids 39-216 of human REXO2 was cloned in a pETM-11 vector (EMBL).

Techniques:

Journal: Molecular Cell

Article Title: Dinucleotide Degradation by REXO2 Maintains Promoter Specificity in Mammalian Mitochondria

doi: 10.1016/j.molcel.2019.09.010

Figure Lengend Snippet: Structural Basis of REXO2 Substrate Specificity (A) Front view of the overall structure of REXO2. Monomer A (gray) and monomer B (blue) are shown in cartoon representation with the DEDDh residues of monomer A shown as sticks. Stars indicate the active sites of monomer A and monomer B. Monomer B lacked clear density for regions encompassing helices α2–4 (residues 84–113) and a short loop corresponding to residues 189–192 and were therefore not built in the model. (B) Activity of truncated REXO2 variants (amino acids 39–216) upon RNA or DNA AA dinucleotide substrates. Monophosphorylated radiolabeled substrate (10 fmol) was incubated with the indicated amounts of REXO2 at 37°C for 30 min, separated by 18% urea-PAGE, and imaged by autoradiography. (C) Quantification of RNA degradation by REXO2 variants from (B). (D) Quantification of DNA degradation by REXO2 variants as in (B). (E) Surface representation of REXO2 with the RNA pApA dinucleotide (yellow) shown as sticks. The molecular surface is colored by the local electrostatic potential (blue, +5 kT; red, −5 kT). (F) Enlargement of REXO2 active site with bound RNA pApA dinucleotide as in (E). (G) The REXO2 active site. Residues interacting with the RNA pApA dinucleotide (orange) are shown as sticks and the polar bonds as yellow dashes. Zn 2+ and Mg 2+ are shown as pink and green spheres, respectively. Water molecules are shown as gray spheres.

Article Snippet: Codon-optimized (Genscript) DNA constructs corresponding to amino acids 39-216 of human REXO2 was cloned in a pETM-11 vector (EMBL).

Techniques: Activity Assay, Incubation, Autoradiography

Journal: Molecular Cell

Article Title: Dinucleotide Degradation by REXO2 Maintains Promoter Specificity in Mammalian Mitochondria

doi: 10.1016/j.molcel.2019.09.010

Figure Lengend Snippet: REXO2 Is an Obligate Homodimer (A) Top and front views of the surface of REXO2 with monomer A (blue) and monomer B (pink), where the interface interacting residues are highlighted in darker colors. K216 is highlighted in green, and R178, W179, and L175 are highlighted in yellow. (B) The R178-W179 binding pocket is shown in cartoon representation with R178, W179, and their interacting residues shown as sticks with the same color code as in (A). (C) Gel filtration profiles of the REXO2 dimer mutants. (D) Activity of REXO2 dimerization mutants upon RNA or DNA AA dinucleotides. Monophosphorylated radiolabeled substrate (10 fmol) was incubated with the indicated amounts of REXO2 protein at 37°C for 30 min, separated by 18% urea-PAGE, and imaged by autoradiography.

Article Snippet: Codon-optimized (Genscript) DNA constructs corresponding to amino acids 39-216 of human REXO2 was cloned in a pETM-11 vector (EMBL).

Techniques: Binding Assay, Filtration, Activity Assay, Incubation, Autoradiography

Journal: Molecular Cell

Article Title: Dinucleotide Degradation by REXO2 Maintains Promoter Specificity in Mammalian Mitochondria

doi: 10.1016/j.molcel.2019.09.010

Figure Lengend Snippet: REXO2 Is Essential for Embryonic Development (A and B) Morphology of Rexo2 +/+ (A) and Rexo2 −/− (B) embryos at embryonic day 8.5. Scale bar, 500 μm. (C) Western blot of REXO2 levels in hearts from control (L/L) and tissue-specific Rexo2 knockout (L/L, cre) mice. VDAC is used as a loading control. (D) mtDNA copy number in control and Rexo2 knockout mice measured by qPCR using three TaqMan probe sets to different regions of the mitochondrial genome. mtDNA levels are normalized to the level of Actin and represent mean values from 3 independent experiments with total n = 15 mice for each group; error bars represent SEM. (E) Mitochondrial mRNA steady-state levels in control and Rexo2 knockout mice analyzed by northern blotting. Data are normalized to the level of 18S rRNA and presented as mean values from 3 independent experiments with total n = 10 mice for each group; error bars represent SEM. (F) mt-tRNA steady-state levels in control and Rexo2 knockout mice analyzed by northern blotting. Data are normalized to the level of 5.8S rRNA and presented as mean values from 3 independent experiments, with n = 15 mice for each group. (G) Level of the pApA RNA dinucleotide in heart tissue from control and Rexo2 knockout mice measured using LC-MS/MS. Data represent mean values from n = 3 mice for each group; error bars represent SEM, **p < 0.01. (H) Level of the pApA RNA dinucleotide in isolated mitochondria from heart tissue of control and Rexo2 knockout mice measured using LC-MS/MS. Data represent mean values from n = 3 mice for each group; error bars represent 1 SEM. n.p. indicates no peak.

Article Snippet: Codon-optimized (Genscript) DNA constructs corresponding to amino acids 39-216 of human REXO2 was cloned in a pETM-11 vector (EMBL).

Techniques: Western Blot, Control, Knock-Out, Northern Blot, Liquid Chromatography with Mass Spectroscopy, Isolation

Journal: Molecular Cell

Article Title: Dinucleotide Degradation by REXO2 Maintains Promoter Specificity in Mammalian Mitochondria

doi: 10.1016/j.molcel.2019.09.010

Figure Lengend Snippet: Loss of REXO2 Causes nanoRNA-Primed Non-canonical Mitochondrial Transcription Initiation (A) A complete map of changes in 5ʹ-end abundance (log2 fold change [knock out (KO)mean/control (Ctrl)mean]) from three control (L/L) and three Rexo2 knockout (L/L, cre) mice on heavy (top) and light (bottom) strands. Mean increases are shown in red and mean decreases in blue. A schematic of the mitochondrial genome is displayed in the center; rRNAs are displayed in green, mRNAs in blue, tRNAs in gray, and the non-coding region (NCR) in black. (B) Frequency of all possible dinucleotide combinations found at the 5ʹ ends of captured sequence tags in three control and Rexo2 knockout mice. (C) In vitro transcription reactions using supercoiled pBluescript II SK (+) DNA as a template. Reactions contain 20 μM ATP, CTP, and GTP; 1 μM UTP; 0.2 μCi 32 P α-UTP; and 20 μM of the indicated RNA or DNA oligonucleotide. (D) In vitro transcription reactions as in (C) but containing BamHI-linearized pBluescript II SK (+) DNA as a template. Reactions using a supercoiled template (lanes 12 and 13) are included as a positive control.

Article Snippet: Codon-optimized (Genscript) DNA constructs corresponding to amino acids 39-216 of human REXO2 was cloned in a pETM-11 vector (EMBL).

Techniques: Knock-Out, Control, Sequencing, In Vitro, Positive Control

Journal: Molecular Cell

Article Title: Dinucleotide Degradation by REXO2 Maintains Promoter Specificity in Mammalian Mitochondria

doi: 10.1016/j.molcel.2019.09.010

Figure Lengend Snippet: Loss of REXO2 Causes Transcription Dysregulation at Mitochondrial Promoters (A and B) Genome browser view (log scaled) of the abundance from three control (L/L) and three Rexo2 knockout (L/L, cre) mice (mean normalized count) and relative changes (log2 fold change[KOmean/WTmean]) in RNA-seq and PARE libraries showing the abundance of 5ʹ ends mapped to the HSP (A) and LSP (B). The direction of transcription from the LSP and HSP is indicated, and the cleavage site between tRNA-Phe (TF) and 12S rRNA is indicated by an upward arrow. (C) Schematic (top) shows the experimental setup of in vitro transcription reactions and complementarity between RNA oligonucleotides and the human LSP promoter sequence. Reactions (bottom) contain linearized or supercoiled LSP-containing plasmid template; 20 μM ATP, CTP, and GTP; 1 μM UTP; 0.2 μCi 32 P α-UTP; and 20 μM of the indicated RNA oligonucleotide (red). Oligonucleotide sequences are designed to anneal to the promoter sequence at the transcription start site as indicated. “RO” indicates the runoff transcription product, and “PT” indicates transcription products prematurely terminated at CSB2. (D) In vitro transcription reactions as in (C) but containing linearized or supercoiled human HSP-containing plasmid as a template. A base in the AAAA RNA oligonucleotide (red) that is not complementary to the HSP sequence is shown in yellow in the schematic. (E) In vitro transcription reactions as in (C) but using poly(A) RNA (red) or DNA (black) oligonucleotides. (F) In vitro transcription reactions as in (D) but using poly(A) RNA (red) or DNA (black) oligonucleotides.

Article Snippet: Codon-optimized (Genscript) DNA constructs corresponding to amino acids 39-216 of human REXO2 was cloned in a pETM-11 vector (EMBL).

Techniques: Control, Knock-Out, RNA Sequencing, In Vitro, Sequencing, Plasmid Preparation

Journal: Molecular Cell

Article Title: Dinucleotide Degradation by REXO2 Maintains Promoter Specificity in Mammalian Mitochondria

doi: 10.1016/j.molcel.2019.09.010

Figure Lengend Snippet: Loss of REXO2 Causes an RNA Processing Defect (A and B) A complete map of mtRNA abundance and mean (log2 fold change [KOmean/Ctrlmean]) determined by RNA-seq coverage from control (n = 3) and Rexo2 knockout (n = 3) mice on the heavy (upper) and light (lower) strand with the mtDNA genome positioned in the centre. Increases (red) and decreases (blue) are depicted for the coding regions (A) and rRNA regions (B). (C) Changes in the levels of canonical mtRNA junctions were determined from the PARE datasets.

Article Snippet: Codon-optimized (Genscript) DNA constructs corresponding to amino acids 39-216 of human REXO2 was cloned in a pETM-11 vector (EMBL).

Techniques: RNA Sequencing, Control, Knock-Out

Journal: Molecular Cell

Article Title: Dinucleotide Degradation by REXO2 Maintains Promoter Specificity in Mammalian Mitochondria

doi: 10.1016/j.molcel.2019.09.010

Figure Lengend Snippet:

Article Snippet: Codon-optimized (Genscript) DNA constructs corresponding to amino acids 39-216 of human REXO2 was cloned in a pETM-11 vector (EMBL).

Techniques: Virus, Recombinant, Filtration, Western Blot, High Molecular Weight, Reverse Transcription, SYBR Green Assay, Labeling, Northern Blot, Mass Spectrometry, Plasmid Preparation, Software