Skip to main content
. 2021 Dec 2;10:e71656. doi: 10.7554/eLife.71656

Figure 1. The core tRNA ligase complex consists of RTCB, DDX1, FAM98B, and CGI-99.

(A) Domain composition of tRNA-LC subunits. RecA, RecA-like domain; CH, calponin homology-like domain; CC, coiled coil; LR, leucine-rich region; NLS, nuclear localization signal. (B) Deletion analysis of tRNA-LC subunits. StrepTactin (upper left), Amylose (upper right), and Ni-NTA (bottom left) affinity pull-down assays using lysates of Sf9 cells expressing full-length tRNA-LC subunits (affinity tags as indicated in the figure). The input (bottom right) and bound fractions were analyzed by SDS-PAGE and visualized by in-gel GFP (middle gel) or mCherry (bottom gel) fluorescence followed by Coomassie Blue staining (upper gel). tRNA-LC = RTCB:DDX1:FAM98B:CGI-99:ASW, omitted subunits in the deletion constructs are indicated (Δ). (C) Size-exclusion chromatography interaction analysis of the core tRNA-LC (RTCB:DDX1:FAM98B:CGI-99) and Archease. SDS-PAGE analysis of the elution peak components is shown in the inset. The estimated molecular weights of the peaks based on their elution volumes are indicated.

Figure 1.

Figure 1—figure supplement 1. The recombinantly expressed tRNA-LC is catalytically active.

Figure 1—figure supplement 1.

(A) SDS-PAGE analysis of the purified human tRNA-LC containing RTCB, DDX1, FAM98B, CGI-99, and ASHWIN. (B) In vitro RNA circularization ligation assay. Recombinant full-length tRNA-LC (RTCB:DDX1:FAM98B:CGI-99:ASHWIN), core tRNA-LC (RTCB:DDX1:FAM98B:CGI-99), minimal tRNA-LC (RTCB:DDX1(696-740):FAM98B(200-239):CGI-99(102-244)), and RTCB were incubated with radiolabeled 21-nt single-stranded RNA in the presence of Archease. RNA circularization was analyzed by denaturing PAGE and visualized by autoradiography. (C) In vitro RNA circularization ligation assay as in (B) with or without ATP.