Identification and Expression Analysis of Ribosome Biogenesis Factor Co-orthologs in Solanum lycopersicum

Abstract

Ribosome biogenesis involves a large inventory of proteinaceous and RNA cofactors. More than 250 ribosome biogenesis factors (RBFs) have been described in yeast. These factors are involved in multiple aspects like rRNA processing, folding, and modification as well as in ribosomal protein (RP) assembly. Considering the importance of RBFs for particular developmental processes, we examined the complexity of RBF and RP (co-)orthologs by bioinformatic assignment in 14 different plant species and expression profiling in the model crop Solanum lycopersicum. Assigning (co-)orthologs to each RBF revealed that at least 25% of all predicted RBFs are encoded by more than one gene. At first we realized that the occurrence of multiple RBF co-orthologs is not globally correlated to the existence of multiple RP co-orthologs. The transcript abundance of genes coding for predicted RBFs and RPs in leaves and anthers of S. lycopersicum was determined by next generation sequencing (NGS). In combination with existing expression profiles, we can conclude that co-orthologs of RBFs by large account for a preferential function in different tissue or at distinct developmental stages. This notion is supported by the differential expression of selected RBFs during male gametophyte development. In addition, co-regulated clusters of RBF and RP coding genes have been observed. The relevance of these results is discussed.

Keywords: MACE; next generation sequencing; orthologous prediction; qRT-PCR; ribosome biogenesis; tomato.

Figure 1

NGS and MACE comparison. Comparison of MACE and NGS data for leaf tissue. The line indicates the Spearman correlation for both leaf expression datasets using RNA-seq and MACE. The inset gives the number of genes assigned in the genome, detected by MACE and detected by NGS, and depicts the higher coverage achieved by MACE.

Figure 2

Prediction of RBF coding genes. (A) Phylogenetic relation of the 14 plant species used for the analysis. (B) Discovery rate of RBFs based on 255 yeast RBFs in the genomes of 14 plant species in percentage of the number found in yeast given for the different families. The different colors indicate the phylogenetic relation (green: green algae; black: moss; light green: monocots; dark green: eudicots). (C) The percentage of plant RBFs encoded by the indicated number of co-orthologs. Atha: A. thaliana; Bdis: B. distachyon; Crei: C. reinhardtii; Gmax: G. max; Ljap: L. japonicus; Mtru: M. truncatula; Osat: O. sativa; Ppat: P. patens; Ptri: P. trichocarpa; Sbic: S. bicolor; Slyc: S. lycopersicum; Stub: S. tuberosum; Vvin: V. vinifera; Zmay: Z. mays.

Figure 3

Prediction of RP coding genes. The distribution of the number of co-orthologs identified for RPSs (A) and RPLs (B) in the 14 analyzed plants as shown in Figure 1.

Figure 4

Prediction of co-orthologs to RBF coding genes. (A) Comparison of the total number of sequences in yeast (black bar), A. thaliana (red bar), and tomato (green bar). (B) The percentage of RBFs assigned to the specific complexes encoded by the number of co-orthologs shown on the right.

Figure 5

RBF co-orthologs with alterations in domain architecture. Alignments given in Supplemental Alignment 1 are shown as bar diagrams including the Pfam domains assigned. See Supplementary Table 3 for visualization of differences. Italics indicates the genes excluded from further analysis; italics and underlined indicates the gene found in unigene database, but not yet represented by a Solyc ID.

Figure 6

The overall expression profile of RBF and RP (co-)orthologs in tomato. (A) Tissues used for NGS RNA-seq analysis (left) and the number of RBFs for which expression is detected in a least one tissue (beside) or in the individual tissue (below). On the right, the tissues analyzed by MACE in this study are indicated. (B) Relation of the TPM expression value in leaves and anthers for all genes (black circles), for all RBF genes (red circles), and for all RP genes (yellow star). Gray line indicates identical expression in leaves and anthers, the long dashed gray line expression with two-fold change, and the short dashed gray line expression with four-fold change (45% genes with less than two-fold change; 64% with less than four-fold change of expression). Indicated are RBF genes not expressed (top), expressed only in leaves (left) or anther (right). Inset on the right shows the distribution of the expression difference between leaf and anther. The gray section indicates a pool of genes with significantly higher expression in anthers, and the red line shows the least square fit analysis to a Gaussian equation.

Figure 7

Clustering of RP and RBF genes based on their expression. (A) Mean of the expression values of clustered genes normalized to the TPM determined for MACE leaf sample. Clusters were generated by k-means analysis. Error bars indicate the standard deviation. The clusters are ordered according to the number of factors present (first value) and the median of the cluster for the leaves (second value) is indicated. The two clusters on the top represent the set of genes that could not be related to a specific profile. (B) The Spearman correlation between the cluster profiles of RPs and RBFs was calculated, and the highest value for each column and each row is shown. Only values with strong correlation (above 0.5) are highlighted.

Figure 8

Comparison of expression in pollen and anthers. The ratio of the values listed in Tables 4 and 5 normalized to EF1α expression was calculated.

Figure 9

The expression profile of RBF encoded by multiple genes. The expression values (Supplementary Table 4) for genes coding for RBFs with multiple co-orthologs have been normalized to the individual maximal value given next to the panel and the expression profile is shown (scale on the right). The order of samples is indicated on the right, legend in bold indicates the MACE results; the rest of the samples are derived from the NGS data. Abbreviations: fmg: fruit mature green; fb: fruit breaker; fr: fruit ripe.