Journal: Nucleic Acids Research

Article Title: Identification of LACTB2, a metallo-β-lactamase protein, as a human mitochondrial endoribonuclease

doi: 10.1093/nar/gkw050

Figure Lengend Snippet: LACTB2 is a mitochondrial, soluble and monomeric protein. ( A ) Mitochondria localization. HeLa cells were disrupted and the nuclear, cytoplasmic and mitochondria-containing fractions were separated and analyzed for the localization of LACTB2, using an immunoblotting assay. The nucleus-located protein, CPSF3L, was used as marker for nuclear proteins. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a cytosolic marker and cytochrome C (Cyt C) as a mitochondrial marker. ( B ) Immunoblot analysis of mitochondrial extract following Proteinase K accessibility assay. TX-100, Triton X-100. PK, Proteinase K. PNPase is a mitochondrial protein located mainly in the intermembrane space and a small amount in the matrix. HSP60 is a mitochondrial matrix protein. Tom20 is a mitochondrial outer membrane protein. ( C ) LACTB2 is a soluble protein. Immunoblotting analysis of mitochondrial extract following alkaline sodium carbonate (Na 2 CO 3 ) extraction of soluble and peripheral membrane proteins. T, total extract. S, supernatant. P, pellet. Subunit 5 of the ATP synthase (ATP5) was used as a marker for intrinsic membrane proteins and Cyt C as a marker of soluble proteins. (D) Immunoblot analysis of the recombinant LACTB2 (Rec.) and the native protein in isolated mitochondria (Mit.) showing the same size on SDS-PAGE. Therefore, the mitochondria targeting signal is not cleaved upon entering the mitochondria and remain an integral part of the mature protein. ( E ) LACTB2 is present as a monomer. Soluble proteins of bovine mitochondrial matrix were extracted from bovine liver as described in the methods section. This extract was fractionated on a size exclusion column superdex 200 and analyzed for LACTB2, using an immunoblotting assay. The following proteins were used as size markers: Tyroglobulin (669 kDa), polynucleotide phosphorylase (PNPase) (232 kDa), BSA (67 kDa), β-lactoglobulin (35 kDa) and ribonuclease A (13 kDa).

Article Snippet: The following antibodies were used: LACTB2 (Sigma HPA044391 or Thermo Scientific PA5–32043), CPSF3L (Sigma HPA029025), GAPDH (Santa Cruz Biotechnology FL-335), Tom 20 (Thermo Scientific PA5 39247), HSP60 (Thermo Scientific PA5–12484), PNPase (Thermo Scientific PA5–22396), CYT C (Santa Cruz Biotechnology H-104) and subunit V of the ATP-synthase ( ).

Techniques: Western Blot, Marker, Recombinant, Isolation, SDS Page

Journal: Nucleic Acids Research

Article Title: Identification of LACTB2, a metallo-β-lactamase protein, as a human mitochondrial endoribonuclease

doi: 10.1093/nar/gkw050

Figure Lengend Snippet: LACTB2 is a member of the metallo-β-lactamase (MBL) superfamily. ( A ) Domain analysis comparison of LACTB2, ELAC1, ELAC2, CPSF-73 of human, as well as bacterial, archaeal and plant proteins that are members of β-CASP MBL subfamilies. The core domains of the following proteins were aligned: human metallo-β-lactamase protein like 2 (LACTB2) (Q53H82), human cleavage and polyadenylation specificity factor 73 (CPSF-73) (Q9UKF6), human nuclear tRNAseZ (ELAC1), human mitochondrial RNaseZ (ELAC2) (Q9BQ52), bacteria Bacillus Subtilis RNase J1 ( B. subtilis J1) (Q45493), archaea Methanocaldoccous jannaschii RNase J2 and J3 ( M. jannaschii J2 and M. jannaschii J3) (MJ1236, MJ0861), and plant Arabidopsis thaliana RNase J that is located in the chloroplast ( A. Thaliana J) (Q84W56). The conserved motifs of the MBL, β-CASP and RRM (I–IV; A–C) are indicated in green, blue and purple, respectively, along with the diagnostic amino acid residues. The MBL and β-CASP proteins were mapped based on their structural alignment ( , ). Motif V of LACTB2, ELAC1 and ELAC2 shares a linear sequence alignment with Motif C of the β-CASP proteins, which is represented as C/V. Mitochondria and chloroplast transit peptides are colored in pink and gray, respectively. The archaeal N- terminal region corresponds to the KH RNA-binding domain and the plant C-terminal region includes a putative GT1 DNA-binding domain . The number of amino acids of each protein is indicated to the right. ( B ) Alignment of the amino acid sequence of Motif II of LACTB2 and several MBL ribonucleases. CLUSTAL W and JalView were used for the alignment. The numbers above the amino acid sequence indicate the position of amino acids in human LACTB2. Signature MBL motif II, HXHXDH, is underlined with a thick black line and His (H) residues are highlighted in cyan and Asp (D) residues in purple. The intense cyan and purple colors represent the highest degree of conservation among the eight proteins while pale blue and green colors signify a lower degree of conservation. Uncolored residues represent no conservation within the annotated threshold. ( C ) Structure-based alignment of the MBL domains of LACTB2, CPSF73 and the DNA exonucleases SNM1A and SNM1B (all from humans). Secondary structural elements (blue arrows: β strands; red cylinders: α-helices) are shown above the alignment for LACT2B and below for CPSF73. The Roman numerals I–IV indicate the conserved MBL motifs, and the specific residues are indicated highlighted in yellow; less conserved sequences are highlighted in cyan. The alignment ends where the structures diverge (β-CASP region in CPSF73, SNM1A and SNM1B, and the unrelated C-terminal region of LACTB2).

Article Snippet: The following antibodies were used: LACTB2 (Sigma HPA044391 or Thermo Scientific PA5–32043), CPSF3L (Sigma HPA029025), GAPDH (Santa Cruz Biotechnology FL-335), Tom 20 (Thermo Scientific PA5 39247), HSP60 (Thermo Scientific PA5–12484), PNPase (Thermo Scientific PA5–22396), CYT C (Santa Cruz Biotechnology H-104) and subunit V of the ATP-synthase ( ).

Techniques: Diagnostic Assay, Sequencing, RNA Binding Assay, Binding Assay

Journal: Nucleic Acids Research

Article Title: Identification of LACTB2, a metallo-β-lactamase protein, as a human mitochondrial endoribonuclease

doi: 10.1093/nar/gkw050

Figure Lengend Snippet: Crystal structure of LACTB2 and comparisons with β-CASP ribonucleases. ( A ) Comparison of the overall fold of LACTB2 and RNase J1 (PDB ID: 3T3N). The chains are colored in blue to red from the N- to the C-terminal and the Zn ions are shown as cyan spheres. The RNase J structure also includes a bound RNA oligonucleotide, shown in stick representation. The common MBL fold is shown on the top in both proteins, while the CASP domain is present only on RNaseJ. The chain is colored as a rainbow spectrum from the N (blue) to C (red) terminus. ( B ) Superposition of the MBL domains of LACTB2 (aa 1–211 orange) and CPSF-73 (PDB ID: 2I7T, residues 9–207 and 395–459; cyan). ( C ) Closeup of the active sites of LACTB2 (orange) and CPSF-73 (cyan). The numbers of the homologous residues from LACTB2 (shown in the figure) and CPSF73 are, respectively, Motif II: His 77/71, His 79/73, Asp 81/75 and His 82/76. Motif III: His 145/158. Motif IV: Asp 164/179, and motif C/V: His 199/418. Motif I (Asp 46/39), which helps to orient His 82/76 but does not coordinate the zinc ions, is not shown. Motif A (Thr 230 in CPSF73), which helps to orient His 418, is conserved in SNM1A/B, but absent in LACTB2; His 199 of LACTB2 is instead supported by an interaction with the main-chain carbonyl of Gly 30. The Zn 2+ ions are presented as spheres; coordination bonds as dashed orange lines, and selected hydrogen bonds are depicted as dotted lines. The His 396 residue belonging to motif B of the β-CASP domain of CPSF-73 that is lacking in LACTB2, is also indicated.

Article Snippet: The following antibodies were used: LACTB2 (Sigma HPA044391 or Thermo Scientific PA5–32043), CPSF3L (Sigma HPA029025), GAPDH (Santa Cruz Biotechnology FL-335), Tom 20 (Thermo Scientific PA5 39247), HSP60 (Thermo Scientific PA5–12484), PNPase (Thermo Scientific PA5–22396), CYT C (Santa Cruz Biotechnology H-104) and subunit V of the ATP-synthase ( ).

Techniques:

Journal: Nucleic Acids Research

Article Title: Identification of LACTB2, a metallo-β-lactamase protein, as a human mitochondrial endoribonuclease

doi: 10.1093/nar/gkw050

Figure Lengend Snippet: Recombinant LACTB2 displays endoribonuclease activity. ( A ) LACTB2 is an endoribonuclease. Recombinant and purified LACTB2 was incubated with 5΄ end [ 32 P], or 3΄ end [ 32 P]Cp, radiolabeled 30 nt-long RNA and unlabeled yeast tRNA. The reaction conditions included incubation at 37°C for 15, 30 and 60 min, followed by the addition of formamide dye and analysis by denaturing gel and autoradiography. Lane (L): nucleotide ladder created by alkaline hydrolysis of the substrate RNA. Lane (1): [ 32 P]—AMP marker of 5΄ [ 32 P]-labeled RNA, created by digestion with RNase One. Lane (−): RNA incubation for 60 min, with no addition of protein. Forms of the same shape presented to the right of the autoradiogram indicate the matching cleavage products of the complementary 5΄ and 3΄ RNAs and can be identified at the sequences presented below the figure, in panel C . ( B ) LACTB2 does not display exoribonucleolytic activity. LACTB2 was incubated with 40 nt body-labeled [ 32 P]-UTP RNA substrate, with the nucleotide sequence shown in panel C. Lane (L): nucleotide ladder described in panel A. Lane (1): [ 32 P]-UMP marker, prepared by the digestion of body-labeled RNA, using Methanocaldoccous jannaschii RNase J3, at 60°C for 1 h . Lane : [ 32 P]-UDP marker, obtained by digesting body-labeled RNA with Escherichia coli PNPase. Other lanes are labeled as described for panel A. (C) RNA sequence of the substrates. The cleavage sites corresponding to the shape shown in panel A are indicated. The uridines labeled in the body-labeled RNA used in the experiment described in panel B are colored gray.

Article Snippet: The following antibodies were used: LACTB2 (Sigma HPA044391 or Thermo Scientific PA5–32043), CPSF3L (Sigma HPA029025), GAPDH (Santa Cruz Biotechnology FL-335), Tom 20 (Thermo Scientific PA5 39247), HSP60 (Thermo Scientific PA5–12484), PNPase (Thermo Scientific PA5–22396), CYT C (Santa Cruz Biotechnology H-104) and subunit V of the ATP-synthase ( ).

Techniques: Recombinant, Activity Assay, Purification, Incubation, Autoradiography, Marker, Labeling, Sequencing

Journal: Nucleic Acids Research

Article Title: Identification of LACTB2, a metallo-β-lactamase protein, as a human mitochondrial endoribonuclease

doi: 10.1093/nar/gkw050

Figure Lengend Snippet: Recombinant LACTB2 prefers U, but not G or poly(A). ( A ) LACTB2 was incubated for 15, 30 and 60 min, at 37°C with poly(GU) 12 , poly(U) 20 or poly(A) 20 , in the presence of yeast tRNA. The RNA substrates were labeled with [ 32 P] at the 5΄ end. Lane (L): Nucleotide ladder prepared by alkaline hydrolysis of poly(U) 20 . Lane (−): RNA substrate incubated with no protein for 60 min. Lanes under the triangle—incubation for 15, 30 and 60 min. Lanes labeled ΔmotifII: the RNAs were incubated for 60 min with the Δmotif II mutated protein in which the six amino acids of motif II were deleted (see Figure ). Following incubation, the RNA was purified and analyzed by denaturing PAGE and autoradiography. ( B ) Poly(GU) 12 was digested in the presence of yeast tRNA with LACTB2, RNase A (cleaves following U) and RNase T1 (cleaves following G). Lane (−) is the same as in panel A. ( C ) LACTB2 cleavage products have 3΄-OH termini and therefore are sensitive to digestion by the exoribonuclease PNPase. 5΄ [ 32 P] (GU) 12 RNA was digested with the enzymes as indicated on the top. When using two enzymes, the RNA was purified by phenol extraction and EtOH precipitation between the incubations. The full length 24 nt RNA contains a 3΄-OH and therefore is sensitive to PNPase. The cleavage products of RNase A have 3΄-phosphate while those of Nuclease P1 contain 3΄-OH. Some leakage of the digestion products of PNPase to the lane of no protein (−) happened when the gel was loaded with the samples. Nucl. P1: Nuclease P1.

Article Snippet: The following antibodies were used: LACTB2 (Sigma HPA044391 or Thermo Scientific PA5–32043), CPSF3L (Sigma HPA029025), GAPDH (Santa Cruz Biotechnology FL-335), Tom 20 (Thermo Scientific PA5 39247), HSP60 (Thermo Scientific PA5–12484), PNPase (Thermo Scientific PA5–22396), CYT C (Santa Cruz Biotechnology H-104) and subunit V of the ATP-synthase ( ).

Techniques: Recombinant, Incubation, Labeling, Purification, Autoradiography

Journal: Nucleic Acids Research

Article Title: Identification of LACTB2, a metallo-β-lactamase protein, as a human mitochondrial endoribonuclease

doi: 10.1093/nar/gkw050

Figure Lengend Snippet: Mutation of certain amino acids in the vicinity of the catalytic site, as well as at other locations, significantly inhibits the ribonucleolytic activity. ( A ) The locations of nine LACTB2 mutations introduced in this work are indicated in the structural image of the protein, created using Chimera. The mutated amino acids that significantly inhibited the endoribonucleolytic cleavage activity are colored in blue, and those not impairing the activity are colored in orange. The ΔMotif II mutation is a deletion of the entire motif II (amino acids HWHRDH). The other mutations, located in the vicinity of the cleavage catalytic site, were D81A and D164A. Additional mutated amino acids were: R110A, N116A, E118A, H216A::R217T, R220T and the double mutant H259A::N260A. The two zinc ions are presented as purple spheres and hydrogen bonds are presented as with purple dashed lines. ( B ) Non-mutated (WT) and LACTB2 mutants ΔMotif II, D81A, D164A, R110A, N116A, E118A, H216A::R217T, R220T, H259A::N260A were incubated with 5΄ [ 32 P] −110 nt RNA corresponding to the mitochondrial COX1 transcript (left panel) or 30 nt RNA described in Figure at 37°C, followed by the addition of formamide dye and analysis by denaturing gel and autoradiography. Deca RNA Marker was fractionated in the first lane to the right and the nucleotide ladder of the 30 nt RNA substrate is shown in the next lane. Lane (−): RNA incubation for 60 min with no added protein. Lanes under the triangle: incubation for 15, 30 and 60 min. ( C ) Kinetic graphs displaying the quantification of the disappearance of the full-length substrate RNA, when incubated with LACTB2 WT or the mutated proteins. The amount of RNA present in the lane marked (−) was taken as 100%. WT is colored in black, RNA cleavage in mutants displaying similar activity to WT is colored in orange. The activity of mutated proteins in which a significant inhibition was observed is colored in blue.

Article Snippet: The following antibodies were used: LACTB2 (Sigma HPA044391 or Thermo Scientific PA5–32043), CPSF3L (Sigma HPA029025), GAPDH (Santa Cruz Biotechnology FL-335), Tom 20 (Thermo Scientific PA5 39247), HSP60 (Thermo Scientific PA5–12484), PNPase (Thermo Scientific PA5–22396), CYT C (Santa Cruz Biotechnology H-104) and subunit V of the ATP-synthase ( ).

Techniques: Mutagenesis, Activity Assay, Incubation, Autoradiography, Marker, Inhibition

Journal: Nucleic Acids Research

Article Title: Identification of LACTB2, a metallo-β-lactamase protein, as a human mitochondrial endoribonuclease

doi: 10.1093/nar/gkw050

Figure Lengend Snippet: Recombinant LACTB2 cleaves ssRNA but not dsRNA or ssDNA. ( A ) LACTB2 does not cleave ssDNA. LACTB2 was incubated with 5΄ [ 32 P] 30 nt-long RNA or ssDNA of the same sequence for 15, 30 and 60 min at 37°C. Lane (−): RNA or ssDNA incubated for 60 min, with no added protein. Lanes under the triangle: incubation for 15, 30 and 60 min. ( B ) A stem-loop structured RNA is cleaved by LACTB2 at the loop. LACTB2 was incubated with 5΄ [ 32 P] 30 nt stem-loop-shaped RNA, as presented below the figure. The reaction was incubated at 25°C, a temperature in which the stem is formed, for 15, 30 and 60 min in the presence of yeast tRNA. Lane (L): nucleotide ladder of the 30 nt RNA. Lane (−): incubation for 60 min, with no added protein. Lanes under the triangle: incubation for 15, 30 and 60 min. The sequence and predicted stem-loop structure formed at 25°C is shown at the bottom. ( C ) LACTB2 cleaves the substrate at a temperature in which the stem-loop structure is not formed. LACTB2 was incubated at 25°C in the presence of yeast tRNA with a linear 16 nt RNA corresponding to the nucleotide sequence of the stem of the molecule used in (B), in order to show the presence of cleavage sites in this sequence. In addition, the same reaction presented in panel B, was repeated at 37°C, where the stem-loop structure is not predicted to be formed. The sequence and structure of the RNA at 37°C are shown below the autoradiogram. Black arrows indicate the cleavage sites.

Article Snippet: The following antibodies were used: LACTB2 (Sigma HPA044391 or Thermo Scientific PA5–32043), CPSF3L (Sigma HPA029025), GAPDH (Santa Cruz Biotechnology FL-335), Tom 20 (Thermo Scientific PA5 39247), HSP60 (Thermo Scientific PA5–12484), PNPase (Thermo Scientific PA5–22396), CYT C (Santa Cruz Biotechnology H-104) and subunit V of the ATP-synthase ( ).

Techniques: Recombinant, Incubation, Sequencing

Journal: Nucleic Acids Research

Article Title: Identification of LACTB2, a metallo-β-lactamase protein, as a human mitochondrial endoribonuclease

doi: 10.1093/nar/gkw050

Figure Lengend Snippet: Downregulation of LACTB2 expression resulted in modest accumulation of mitochondrial transcripts. ( A ) Quantitation of the amount of LACTB2 in the siRNA treated cells. siRNA duplex targeted for LACTB2 or scrambled siRNAs duplexes, as a negative control, were transfected into HEK-293 cells. Cells were collected 48 h post-transfection and the amount of LACTB2 was determined using immunoblotting assay. Proteins from equal number of cells were loaded in the lanes labeled 100%. Proteins from 50, 25 and 10% of the control and 150 and 200% of the siRNA LACTB2 treated cells were analyzed for the amount of LACTB2, in order to determine the degree of down-expression. ( B ) Medium acidification due to treatment with siRNA duplex, 48 h post-transfection. Ctrl—cells treated with scrambled siRNAs duplexes as a negative control. RNAi—cells treated with siRNA duplex targeted to LACTB2. ( C ) The amount of mitochondrial transcripts in the cells treated with the siRNA duplex targeted to LACTB2 (turquoise bars), or with scrambled siRNAs duplexes as a negative control (brown bars), was analyzed by qRT-PCR. The GAPDH transcript was used as the reference gene for normalization of the expression. The error bars display the standard errors of at least three independent experiments. ( D ) Northern blot analysis of HEK-293 cells that were not transfected (WT), transfected with scrambled siRNAs as control (Ctrl) or transfected with LACTB2 siRNA (RNAi). Hybridizations were performed with probes specific to the mitochondrial mRNAs, rRNAs and, stained cytosolic 28S and 18S rRNAs as loading control.

Article Snippet: The following antibodies were used: LACTB2 (Sigma HPA044391 or Thermo Scientific PA5–32043), CPSF3L (Sigma HPA029025), GAPDH (Santa Cruz Biotechnology FL-335), Tom 20 (Thermo Scientific PA5 39247), HSP60 (Thermo Scientific PA5–12484), PNPase (Thermo Scientific PA5–22396), CYT C (Santa Cruz Biotechnology H-104) and subunit V of the ATP-synthase ( ).

Techniques: Expressing, Quantitation Assay, Negative Control, Transfection, Western Blot, Labeling, Quantitative RT-PCR, Northern Blot, Staining