Data source:

The dataset used for this project was downloaded from a previous study which investigated the roles of mitoribosomal S10 protein (RPS10) in the translation and splicing in the mitochondria of Arabidopsis (1). 24 mitochondrial genes are chosen. WT group refers to the wild-type Arabidopsis thaliana. The P3 group alludes to transgenic Arabidopsis thaliana with RNAi-silenced expression of the RPS10 gene (2).

Selection of differentially expressed genes (DEGs)

We decided to use “edgeR” package for the differential expression analysis since no biological replications of WT and P3 groups are acquired from the original paper. The biological coefficient of variation (BCV) of 0.1 is employed, yielding results denoted as 'DEG_edgeR'. The thresholds for the p value and the absolute value of log2FoldChange to select DEGs are 0.05 and 1, respectively.

Function enrichment analysis of DEGs of Arabidopsis

The Gene Ontology (GO) enrichment analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway exploration of DEGs of Arabidopsis are conducted by utilizing g:Profiler (https://biit.cs.ut.ee/gprofiler/gost).

Matching to human orthologs

After the differential expression analysis performed on Arabidopsis genes, the human genes orthologous to DEGs in Arabidopsis are searched using eggNOG (version 5, http://eggnog5.embl.de/#/app/home).

Gene annotation of human orthologs

The package “org.Hs.eg.db” is employed to seek for the Entrez IDs and the gene names of the human orthologs.

Function annotation analysis of human orthologs

We apply “clusterProfiler” package to conduct GO function analysis to determine the cellular components, biological pathways, molecular functions and pathways in which the human orthologs are enriched. The “KEGGREST” package is utilized to provide a client interface to the KEGG REST server.

 Selection of DEGs in Arabidopsis

A total of 24 mitochondrial genes were processed for their expressions in WT group and RPS10 knockdown group.  DEGs are present using a volcano plot (Figure 1). The knockdown of RPS10 are found to profoundly affect the expression levels of MATR, RPL16, RPS4, RPS12, TATC and RPL5, most of which are related to ribosomes.

 GO enrichment analysis of DEGs in Arabidopsis

To investigate the underlying biological effects of RPS10 silencing, we turned to an online tool (g:Profiler) to carry out the function annotation analysis (Figure 6). The results elucidated that the attenuation of RPS10 expression significantly altered the expression levels of genes located in proton-transporting ATP synthase complex, mitochondrial membrane, and ribosome. In the matter of biological processes, proton transmembrane transport and oxidative phosphorylation were affected by knockdown of RPS10. With regard to molecular functions, DEGs were enriched in proton transmembrane transporter activity, oxidoreduction-driven active transmembrane transporter activity.

Function enrichment analysis of human orthologs

To reveal the potential biological effects of silenced RPS10, we matched DEGs in Arabidopsis to their human orthologs and conducted a functional enrichment analysis using R script. The results demonstrated that the majority of human orthologs are enriched in mitochondrial protein−containing complex, mitochondrial inner membrane, and inner mitochondrial membrane protein complex. Furthermore, the genes enriched in ribosomal subunit and ribosome are all down-regulated (Figure 3). In terms of biological processes, the analysis revealed a bulk of the human homologies are related to ribose phosphate biosynthetic process, proton motive force−driven mitochondrial ATP synthesis, aerobic respiration and oxidative phosphorylation, all of which pathways were found to be up-regulated (Figure 4). Then, the molecular function annotation pointed out that the knockdown of RPS10 might be involved in oxidoreduction−driven active transmembrane transporter activity, proton transmembrane transporter activity, proton−transporting ATP synthase activity, rotational mechanism, structural constituent of ribosome and rRNA binding (Figure 5).

KEGG analysis on human orthologs

Next, we explored the potential signaling pathways that might be affected by the aberrant RPS10 expression. By mapping to the KEGG pathway database, it was suggested that oxidative phosphorylation, diabetic cardiomyopathy, chemical carcinogenesis−reactive oxygen species, thermogenesis, ribosome, parkinson disease, prion disease, huntington disease, amyotrophic lateral sclerosis, and Alzheimer disease were markedly associated with the human homologies (P < 0.05) (Figure 6).

RNA_Seq Analysis result

 Selection of DEGs in Arabidopsis

This is a volcano plot generated from RNA-seq data. The volcano plot displays genes with expressions significantly differentiated, including CCMB, RPL16, RPS4, and CCMFN2, indicating that the knockout of rps10 in Arabidopsis mitochondria also affects the downregulation of ribosomal genes. Additionally, the gene expression differences shown in the plot are much lower compared to Ribo-seq data. This is because RNA-seq data only consider transcriptional levels of genes, whereas Ribo-seq data directly measure the rate of protein synthesis. The difference in protein synthesis rate/translation efficiency results in Ribo-seq being more sensitive to detect differences in protein expression.