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

Abstract

Protein uL5 (formerly called L11) is an integral component of the large (60S) subunit of the human ribosome, and its deficiency in cells leads to the impaired biogenesis of 60S subunits. Using RNA interference, we reduced the level of uL5 in HEK293T cells by three times, which caused an almost proportional decrease in the content of the fraction corresponding to 80S ribosomes, without a noticeable diminution in the level of polysomes. By RNA sequencing of uL5-deficient and control cell samples, which were those of total mRNA and mRNA from the polysome fraction, we identified hundreds of differentially expressed genes (DEGs) at the transcriptome and translatome levels and revealed dozens of genes with altered translational efficiency (GATEs). Transcriptionally up-regulated DEGs were mainly associated with rRNA processing, pre-mRNA splicing, translation and DNA repair, while down-regulated DEGs were genes of membrane proteins; the type of regulation depended on the GC content in the 3′ untranslated regions of DEG mRNAs. The belonging of GATEs to up-regulated and down-regulated ones was determined by the coding sequence length of their mRNAs. Our findings suggest that the effects observed in uL5-deficient cells result from an insufficiency of translationally active ribosomes caused by a deficiency of 60S subunits.

Mutation at the Site of Hydroxylation in the Ribosomal Protein uL15 (RPL27a) Causes Specific Changes in the Repertoire of mRNAs Translated in Mammalian Cells

“…The resulting lysate was centrifuged for 10 min at 14,000 g and 4°C, and the supernatant was layered on a linear gradient of 5–50% sucrose in 50 mM Tris–HCl (pH 7.5) buffer containing 100 mM KCl and 12 mM MgCl 2 . The sucrose gradient was centrifuged for 18 h at 17,000 g and 4°C in a SW-40 rotor, and fractionated as described in [ 23 ]. The content of the polysome fractions was precipitated with one volume of ice-cold EtOH and RNA from the pellet was isolated as in [ 23 ].…”

“…The sucrose gradient was centrifuged for 18 h at 17,000 g and 4°C in a SW-40 rotor, and fractionated as described in [ 23 ]. The content of the polysome fractions was precipitated with one volume of ice-cold EtOH and RNA from the pellet was isolated as in [ 23 ]. The integrated optical densities of the peaks of polysomes (P) and monosomes (M) were calculated from the file, with the readings of the UV absorption at 260 nm measured for 0.36 sec each in the flow cell of the Milichrom A-02 chromatograph (Econova, Novosibirsk, Russia), and the P/M ratio was calculated for each replicate.…”

“…The next-generation sequencing (NGS) of RNA (RNA-seq) is currently an advanced method for studying changes in the transcriptome and translatome landscapes of cells in response to various stimuli, including the deficiency of individual ribosomal proteins [ 23 , 24 , 25 , 26 , 27 ] or the introduction of specific mutations in them [ 28 ]. Being applied to the analysis of total cellular mRNA and mRNAs translated in polysomes, this method makes it possible to draw conclusions concerning the regulation of expression of individual genes at the levels of transcription and translation, and to evaluate their translational efficiencies.…”

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

“…The baseMean values obtained from the RNA-seq experiment with the total mRNA of HEK293T cells were used to plot the mRNA abundance in MS Excel. Differential expression analysis (DEA) was carried out using the DESeq2 (v. 1.30.0) package in general, as described in [ 39 ]. Raw counts were estimated using the Rsubread package; annotation was performed using the biomaRt package.…”

Deficiency of the ribosomal protein uS10 (RPS20) reorganizes human cells translatome according to the abundance, CDS length and GC content of mRNAs

“…Fastq files were deposited in GenBank under the study accession PRJNA976116. Downstream data analysis was performed as described in [15] with minor modifications. Briefly, the counts table was generated with the application of the Rsubread package (v. 2.10.5) using the featureCounts function with the GTF file (ensembl v. 104) as an annotation in the reverse stranded mode.…”