Figure 5.

GTPBP5 directly interacts with the 16S rRNA and mtLSU proteins. (A, B) Knockdown (KD) of mitoribosome assembly factors and mitoribosome subunits in HEK293T cells using siRNAs for 3 days, verified by immunoblotting of whole-cell lysates. Lysates from HEK293T cells KO for GTPBP5, GTPBP10, MTG1, or DDX28 were also included in the study. (A) Representative image of immunoblot analysis of the steady-state levels of mitoribosome proteins after the silencing of target proteins. NTC is a non-targeting silencing control, and Mock consisted of transfection reagent only. Antibodies are listed on the right side, and ACTIN was used as a loading control. In the anti-GTPBP5 immunoblot, the asterisk marks the GTPBP5 protein. (B) Following analysis in panel (A), the densitometric data obtained on the abundance of mitoribosome proteins and assembly factors accumulated after silencing or knocking out of each target protein was used for cluster analysis (see Materials and Methods). The heat map, generated with the R studio software, represents the average value of log₂ scale of the ratio of the protein levels in knockdown or knockout samples to control (NTC) in three independent repetitions of immunoblotting analyses. 2-way ANOVA was performed followed by a Dunnett's multiple comparisons test: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (C) qPCR analyses of reverse-transcribed control or GTPBP5-FLAG co-immunopurified RNAs after 4-thiouridine treatment and either UV-mediated protein-RNA crosslinking (UV-CL) or not crosslinking. GTPBP5-FLAG was expressed under the control of either a standard CMV6 promoter or an attenuated CMV promoter (pCMV6Δ5) (25). In, input. FT, flow-through or unbound. B is bound. (D) Co-immunoprecipitation analysis of GTPBP5-FLAG and interacting mitoribosome proteins and assembly factors in mitochondrial lysates prepared in the presence of 1% digitonin, by using anti-FLAG agarose beads (FLAG) or plain beads as control (CTRL). In, input. FT, flow-through or unbound. B is bound.