Journal: Nucleic Acids Research

Article Title: Reconstitution of the human tRNA splicing endonuclease complex: insight into the regulation of pre-tRNA cleavage

doi: 10.1093/nar/gkaa438

Figure Lengend Snippet: The heterotetrameric human TSEN complex retains nuclease activity in the absence of CLP1. (A) Expression of the human TSEN complex in E. coli was possible through use of a polycistronic vector in which each TSEN protein is under independent ribosome binding sites. (B) Separation of the TSEN complex via gel filtration produces three distinct TSEN peaks. (C) A protein gel reveals the peaks contain the heterotetramer, a trimer (TSEN2, TSEN34 and TSEN15), and pool of soluble TSEN15. (D) Cleavage activity assays were performed on fractions from gel filtration separation of the TSEN complex. Cleavage reactions were separated on denaturing TBE-Urea gels, which were stained with SYBR Gold to visualize total RNA. (E) SEC-MAL analysis of the peak centered around 10.5 mL reveals the TSEN complex is a single heterotetramer with an average mass of 191.9 kDa.

Article Snippet: tRNA cleavage reactions were carried out in RNA Cleavage Buffer (50 mM KCl, 50 mM Tris pH 7.5, 5 mM MgCl 2 , 1 mM DTT and 1 unit/ml RNAsin (Promega)) at room temperature for 30 min (unless otherwise noted).

Techniques: Activity Assay, Expressing, Plasmid Preparation, Binding Assay, Filtration, Staining

Journal: Nucleic Acids Research

Article Title: Reconstitution of the human tRNA splicing endonuclease complex: insight into the regulation of pre-tRNA cleavage

doi: 10.1093/nar/gkaa438

Figure Lengend Snippet: Cleavage of tRNA substrates by the human TSEN complex. The heterotetrameric TSEN complex is efficient at cleaving (A) tRNA-R1 full-length (1.7 μM) and (B) anticodon stem loop (0.5 μM) tRNA substrates. Representative time courses (0, 1, 2.5, 5, 10, 20, 40 min) are shown for the cleavage of each tRNA substrates by 1.5 μM TSEN complex (both wildtype and endonuclease dead (AAA2) complexes). RNA cleavage products were separated on TBE–Urea denaturing gels and visualized using a typhoon. The average of three replicates is graphed, standard deviation is shown for each data point.

Article Snippet: tRNA cleavage reactions were carried out in RNA Cleavage Buffer (50 mM KCl, 50 mM Tris pH 7.5, 5 mM MgCl 2 , 1 mM DTT and 1 unit/ml RNAsin (Promega)) at room temperature for 30 min (unless otherwise noted).

Techniques: Standard Deviation

Journal: Nucleic Acids Research

Article Title: Reconstitution of the human tRNA splicing endonuclease complex: insight into the regulation of pre-tRNA cleavage

doi: 10.1093/nar/gkaa438

Figure Lengend Snippet: Depletion of the Drosophila melanogaster CLP1 orthologue, cbc, in S2 cells does not prevent the generation of tricRNAs or tRNA maturation. S2 cells were treated with dsRNA to knock down cbc in vivo. Cells were then transfected with a D. melanogaster tRNA-Tyr reporter containing Broccoli in the intron (23). Total RNA was isolated from the cells. (A) RT-PCR was used to measure successful cbc knockdown by dsRNAs, with 5S rRNA and unamplified RNA as controls. (B) A cartoon depicting the final products formed from the in vivo cleavage of endogenous Drosophila tRNA CR31905 and the transfected reporter tRNA-Broccoli-intron tRNA. Only tric31905 is visualized by the tric31905 probe whereas introns, intron containing tRNAs, and the 3′ exon are detected for the dual reporter. (C) A northern blot probing tric31905 was performed to measure levels of endogenous circular intron production. We also carried out a northern blot to measure levels of pre-tRNA and mature-tRNA from the same sample. Levels of the snRNA U1 in bottom panel is the loading control for both northern blots. (D) Levels of tric31905 (n = 3), tricBroc (n = 4), mature tRNA (n = 4), and pre-tRNA (n = 4) levels were quantified and normalized to the U1 or 5S controls on their respective gels/blots. The average and standard error for biological replicates is shown. A student's t-test was used to calculate significance: *P < 0.05 and **P < 0.005. (E) RNA was run on a TBE-Urea gel and visualized using DFHBI-1T. The gel was then re-stained with ethidium bromide, and the 5S rRNA is shown as a loading control.

Article Snippet: tRNA cleavage reactions were carried out in RNA Cleavage Buffer (50 mM KCl, 50 mM Tris pH 7.5, 5 mM MgCl 2 , 1 mM DTT and 1 unit/ml RNAsin (Promega)) at room temperature for 30 min (unless otherwise noted).

Techniques: Knockdown, In Vivo, Transfection, Isolation, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Control, Staining

Journal: Nucleic Acids Research

Article Title: Reconstitution of the human tRNA splicing endonuclease complex: insight into the regulation of pre-tRNA cleavage

doi: 10.1093/nar/gkaa438

Figure Lengend Snippet: Co-expression of catalytically active CLP1 with a tricRNA reporter in HEK cells reduces tricRNA formation. HEK 293 cells were transfected with a human tRNA-Tyr reporter containing Broccoli in the intron. Co-expression was conducted using the TSEN proteins and wildtype or kinase dead CLP1. Total RNA was isolated from HEK cells. (A) RNA was run on TBE-Urea gels and visualized for Broccoli containing RNAs using DFHBI-1T. Gels were washed and re-staned with SYBR Gold to visualize total RNA, which is shown as a loading control. (B) For the co-expression experiments involving wildtype and kinase dead CLP1 with the tricRNA reporter and the TSEN proteins, the respective tricBroc band was normalized to a band from the SYBR Gold (see ‘*’ in A) and quantified. The average and standard error for two biological replicates and a technical replicate are shown, a student's t-test was performed. (*P < 0.05).

Article Snippet: tRNA cleavage reactions were carried out in RNA Cleavage Buffer (50 mM KCl, 50 mM Tris pH 7.5, 5 mM MgCl 2 , 1 mM DTT and 1 unit/ml RNAsin (Promega)) at room temperature for 30 min (unless otherwise noted).

Techniques: Expressing, Transfection, Isolation, Control