Journal: microPublication Biology

Article Title: tRNA Arg binds in vitro TDP-43 RNA recognition motifs and ligand of Ate1 protein LIAT1

doi: 10.17912/micropub.biology.001224

Figure Lengend Snippet: ( A ) Schematic representation of human TDP-43: NTD- amino terminal domain, NLS- nuclear localization sequence, RRM – RNA recognition motif, LCR- low complexity region. ( B , C , D , E ) EMSA assays to asses in vitro interaction of fixed amount of human in vitro-transcribed tRNA Arg (left half of PAGE) or native yeast tRNA Phe (right half of PAGE) with increasing amounts of recombinant fragments of human TDP-43: ( B ) hTDP-43 NLS-RRM1-RRM2, ( C ) hTDP-43 RRM1-RRM2, ( D ) hTDP-43 NTD-ΔNLS-RRM1-RRM2, ( E ) quantitation of htRNA Arg binding to hTDP-43 fragments performed by gel image analysis. ( F ) Schematic representation of mouse LIAT1: “-“ - poly glutamate region, “+” - polylysine region, LIAT1 domain, R - LIAT1 repeat. ( G ) Size exclusion Superdex 75 HR10/30 chromatogram of mLIAT1 and SDS-PAGE gel of eluted mLIAT1. ( H ) Multiple sequence alignment of full-length vertebrate LIAT1 proteins shown in correspondence of polylysine region. ( I ) EMSA assay to asses in vitro interaction of fixed amount of htRNA Arg with full length recombinant mLIAT1; asterisks denote different migration of mLIAT1:htRNA Arg complexes.( J ) Ball-and-stick representation of cis peptide bond between mTDP-43 Lys224 and Pro225 and of interaction between Ile222 and Lys224 in crystal structure of mouse TDP-43 (mTDP-43) RRM2 (PDB 3D2W).

Article Snippet: Recombinant tags were removed by digestion with TEV protease, digestion reaction was applied on NiNTA resin and the flow through containing tagless protein was concentrated to ~ 200 μM and loaded on HiLoad Superdex 75 pg 10/30 column (Cytiva) in 150 mM KCl, 20 mM NaCl, 1 mM MgCl 2 , 5% glycerol, 50 mM Hepes-KOH pH 7.7, 2 mM 2- mercaptoethanol.

Techniques: In Vitro, Sequencing, Recombinant, Quantitation Assay, Binding Assay, SDS Page, Migration

Journal: microPublication Biology

Article Title: tRNA Arg binds in vitro TDP-43 RNA recognition motifs and ligand of Ate1 protein LIAT1

doi: 10.17912/micropub.biology.001224

Figure Lengend Snippet: ( A ) Schematic representation of human TDP-43: NTD- amino terminal domain, NLS- nuclear localization sequence, RRM – RNA recognition motif, LCR- low complexity region. ( B , C , D , E ) EMSA assays to asses in vitro interaction of fixed amount of human in vitro-transcribed tRNA Arg (left half of PAGE) or native yeast tRNA Phe (right half of PAGE) with increasing amounts of recombinant fragments of human TDP-43: ( B ) hTDP-43 NLS-RRM1-RRM2, ( C ) hTDP-43 RRM1-RRM2, ( D ) hTDP-43 NTD-ΔNLS-RRM1-RRM2, ( E ) quantitation of htRNA Arg binding to hTDP-43 fragments performed by gel image analysis. ( F ) Schematic representation of mouse LIAT1: “-“ - poly glutamate region, “+” - polylysine region, LIAT1 domain, R - LIAT1 repeat. ( G ) Size exclusion Superdex 75 HR10/30 chromatogram of mLIAT1 and SDS-PAGE gel of eluted mLIAT1. ( H ) Multiple sequence alignment of full-length vertebrate LIAT1 proteins shown in correspondence of polylysine region. ( I ) EMSA assay to asses in vitro interaction of fixed amount of htRNA Arg with full length recombinant mLIAT1; asterisks denote different migration of mLIAT1:htRNA Arg complexes.( J ) Ball-and-stick representation of cis peptide bond between mTDP-43 Lys224 and Pro225 and of interaction between Ile222 and Lys224 in crystal structure of mouse TDP-43 (mTDP-43) RRM2 (PDB 3D2W).

Article Snippet: Protein and RNA molecular size was assessed by size exclusion chromatography SEC applying 500 μL of protein sample (> 95% purity, in SEC buffer) on Superdex 75 pg 10/30 column (Cytiva) in SEC buffer and ran at 0.5 mL min -1 ; the calibration curve for the used column was obtained applying separately 500 μL of: myoglobin (150 kDa), polyhistidine-tagged T7 RNA polymerase (97 kDa), bovine serum albumin (66 kDa), ovalbumin (43 kDa), chimotripsinogen (25.9 kDa), and polyhistidine-tagged thioredoxin (15 kDa).

Techniques: In Vitro, Sequencing, Recombinant, Quantitation Assay, Binding Assay, SDS Page, Migration