Knockdown of the Ribosomal Protein eL29 in Mammalian Cells Leads to Significant Changes in Gene Expression at the Transcription Level

Abstract

An imbalance in the synthesis of ribosomal proteins can lead to the disruption of various cellular processes. For mammalian cells, it has been shown that the level of the eukaryote-specific ribosomal protein eL29, also known as the one interacting with heparin/heparan sulfate, substantially affects their growth. Moreover, in animals lacking this protein, a number of anatomical abnormalities have been observed. Here, we applied next-generation RNA sequencing to HEK293 cells transfected with siRNAs specific for the mRNA of eL29 to determine what changes occur in the transcriptome profile with a decrease in the level of the target protein. We showed that an approximately 2.5-fold decrease in the content of eL29 leads to statistically significant changes in the expression of more than a thousand genes at the transcription level, without a noticeable effect on cell viability, rRNA level, and global translation. The set of eL29-dependent genes included both up-regulated and down-regulated ones, among which there are those previously identified as targets for proteins implicated in oncogenesis. Thus, our findings demonstrate that an insufficiency of eL29 in mammalian cells causes a significant reorganization of gene expression, thereby highlighting the relationship between the cellular balance of eL29 and the activities of certain genes.

Deficiency of the Ribosomal Protein uL5 Leads to Significant Rearrangements of the Transcriptional and Translational Landscapes in Mammalian Cells

“…Reverse transcription (RT) was carried out using 2.5 µg of RNA samples isolated from aliquots of the respective cell lysates or pooled polysome gradient fractions as indicated above, 100 pmol of random hexamer primer and 20 U of MMLV reverse transcriptase according to [40]. The resulting cDNA was then used for qPCR analysis, performed as described in [40] using appropriate gene-specific primers (Table S10). The experiments were performed in four biological replicates.…”

Reorganization of the Landscape of Translated mRNAs in NSUN2-Deficient Cells and Specific Features of NSUN2 Target mRNAs

“…In control experiment, the set of non-targeting siRNAs was used. To confirm the knockdown of the mRNA of NSUN2, the content of the protein itself in the cells was determined by Western blotting using specific rabbit polyclonal antibodies against NSUN2 as described in [ 38 ]. Rabbit antibodies against GAPDH were used as a reference.…”

“…To assess the ratio of 80S ribosomes in the polysome and monosome fractions (P/M), the integrated A 260 values of the respective peaks were calculated in the MS Excel program. All other manipulations, including the isolation of RNA from the samples of cells and polysomes lysed with the Trizol reagent, along with the RNA integrity index assessment, RNA quantification, and RNA polyA enrichment, were carried out in accordance with the described protocol [ 38 ].…”

Knockdown of the Ribosomal Protein eL38 in HEK293 Cells Changes the Translational Efficiency of Specific Genes

“…Oligoribonucleotides used as siRNAs were the same as given in [ 11 ]. HEK293 cells (ATCC CRL-1573) were cultured, transfected with the siRNAs and harvested as described in [ 26 ]. Cell lysis, the centrifugation of the lysates in a sucrose density gradient, and an analysis of the polysome profiles were performed in accordance with [ 27 ].…”

“…Cell lysis, the centrifugation of the lysates in a sucrose density gradient, and an analysis of the polysome profiles were performed in accordance with [ 27 ]. The contents of ribosomal protein eL38 and reference proteins GAPDH and eS4 in transfected cells and in sucrose gradient fractions were measured by western blotting using rabbit eL38-specific antibodies (#PA5-88313) (Thermo Fisher Scientific, Waltham, MA, USA), rabbit GAPDH-specific antibodies (#60004-1-Ig) (Proteintech, Rosemont, IL, USA) and home-made rabbit antiserum against human ribosomal protein eS4, as described in [ 11 , 26 ].…”