Functional analysis of human RPS14 null alleles

Abstract

Previously we described a large collection of cloned human DNAs that encode chemically defined missense mutations within the ribosomal protein S14 sequence. We determined that biologically inactive (i.e. null) alleles resulted primarily from point mutations targeted to two internal segments of the S14-coding sequence and designated these functionally critical regions as domains B and D. Further, we inferred that structural determinants within domains B and D are required for proper incorporation of the S14 protein into nascent 40 S ribosomal particles and/or for the normal function of mature cytoplasmic ribosomes. In this study we have used immunofluorescence to monitor the intracellular trafficking of epitopically labeled human S14 protein isoforms transiently expressed by cultured Chinese hamster cells. Data obtained distinguish null alleles of RPS14 which encode proteins that are not incorporated into pre-ribosomal subunit particles from null alleles whose products are compatible with normal ribosome assembly processes but result in functionally inactive cytoplasmic 40 S ribosomal subunits. Mutations assigned to the first allele class involve amino acid replacements located within S14 domains B and D; whereas mutations assigned to the second class are distributed throughout the S14 protein-coding sequence.

Cell‐based analysis of pairwise interactions between the components of the multi‐tRNA synthetase complex

“…Although the specific targets of the compounds are not the same, they all effectively stall the ribosome. Since the CHO‐K1 cell line is resistant to emetine, which has mutations on ribosomal protein S14 (RPS14), 40,41 emetine was used as a control. Interestingly, puromycin, cycloheximide, and harringtonine reduced the same binary interactions of the system (AIMP1:AIMP1, AIMP1:AIMP2, AIMP1:IARS1, AIMP1:KARS1, AIMP1:MARS1, AIMP2:AIMP2, IARS1:KARS1, IARS1:LARS1, IARS1:MARS1, IARS1:QARS1, KARS1:LARS1, and KARS1:MARS1) to a similar extent (> 0.55‐fold decrease) (Figure 4A), and the endogenous MSC showed comparable swelling to the system under puromycin treatment (Figure 4B‐D).…”

“…Although the specific targets of the compounds are not the same, they all effectively stall the ribosome. Since the CHO-K1 cell line is resistant to emetine, which has mutations on ribosomal protein S14 (RPS14), 40,41 emetine was used as a control. Interestingly, puromycin, cycloheximide, and harringtonine reduced the same binary interactions of the system (AIMP1:AIMP1, AIMP1:AIMP2, AIMP1:IARS1, was observed simultaneously.…”