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, 21 (1), 25-38

The Functional Role of pack-MULEs in Rice Inferred From Purifying Selection and Expression Profile

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The Functional Role of pack-MULEs in Rice Inferred From Purifying Selection and Expression Profile

Kousuke Hanada et al. Plant Cell.

Abstract

Gene duplication is an important mechanism for evolution of new genes. In plants, a special group of transposable elements, called Pack-MULEs or transduplicates, is able to duplicate and amplify genes or gene fragments on a large scale. Despite the abundance of Pack-MULEs, the functionality of these duplicates is not clear. Here, we present a comprehensive analysis of expression and purifying selection on 2809 Pack-MULEs in rice (Oryza sativa), which are derived from 1501 parental genes. At least 22% of the Pack-MULEs are transcribed, and 28 Pack-MULEs have direct evidence of translation. Chimeric Pack-MULEs, which contain gene fragments from multiple genes, are much more frequently expressed than those derived only from a single gene. In addition, Pack-MULEs are frequently associated with small RNAs. The presence of these small RNAs is associated with a reduction in expression of both the Pack-MULEs and their parental genes. Furthermore, an assessment of the selection pressure on the Pack-MULEs using the ratio of nonsynonymous (Ka) and synonymous (Ks) substitution rates indicates that a considerable number of Pack-MULEs likely have been under selective constraint. The Ka/Ks values of Pack-MULE and parental gene pairs are lower among Pack-MULEs that are expressed in sense orientations. Taken together, our analysis suggests that a significant number of Pack-MULEs are expressed and subjected to purifying selection, and some are associated with small RNAs. Therefore, at least a subset of Pack-MULEs are likely functional and have great potential in regulating gene expression as well as providing novel coding capacities.

Figures

Figure 1.
Figure 1.
Tissue Specificity of Transcription for Pack-MULEs, Their Parental Genes, and MULEs That Encode Transposases (Auto-MULEs). The percentage of cases was calculated using the number of MPSS tags detected for a particular sequence class (Pack-MULE, parental genes, or auto-MULEs) in each library divided by the number of total MPSS tags for the sequence class in question among all libraries. The abbreviations for the libraries are as follows: NPO, mature pollen; NCL, 14 d, young leaves stressed in 4°C cold for 24 h; NGS, 3 d, germinating seed; NCR, 14 d, young roots stressed in 4°C cold for 24 h; NYR, young roots; NSL, 14 d, young leaves stressed in 250 mM NaCl for 24 h; NCA, 35 d, callus; NST, stem; NIP, 90 d, immature panicle; NSR, 14 d, young roots stressed in 250 mM NaCl for 24 h; NDR, 14 d, young roots stressed in drought for 5 d; NGD, 10 d, germinating seedlings grown in dark; NYL, young leaves; NDL, 14 d, young leaves stressed in drought for 5 d; NSO, ovary and mature stigma; NL4, leaf; NME, 60 d, crown vegetative meristematic tissue; NR2, root. The pound sign indicates libraries in which difference in percentage of cases is significant (q < 0.05) between Pack-MULEs and parental genes. The asterisk indicates libraries in which difference in percentage of cases is significant (q < 0.05) between Pack-MULEs and auto-MULEs.
Figure 2.
Figure 2.
The Variation in Percentage of Expressed Pack-MULEs and Association with sRNA Signatures (All sRNAs) among Pack-MULEs with Different TIR Sequences. For each type of data (expression or small RNAs), if two columns have different letters (a, b, c, etc.), the difference between the relevant families is significant (χ2 test, P < 0.05); otherwise, the difference is not significant.
Figure 3.
Figure 3.
The Relationship between Percentage of Expressed Pack-MULEs and Sequence Identity between TIRs. For cDNA, only elements associated with fl-cDNA sequences are considered expressed. For all expression, elements with any type of expression evidence (cDNA, MPSS, peptide) are considered. For Os0037, all expression data are used. The letters on the columns indicate the significance of difference (see legend for Figure 2).
Figure 4.
Figure 4.
The Impact of Pack-MULE–Related Small RNAs on the Tissue-Specific Transcription of Their Parental Genes. (A) Transcription level and percentage of libraries with transcripts from parental genes vary with the presence and level of shared sRNAs. TPM, transcripts per million; TPQ, transcripts per quarter million. (B) A parental gene (Os07g37730) that is possibly influenced by sRNAs caused by the corresponding Pack-MULE (chr05-00030). Pack-MULE TIRs are shown as black arrowheads, and black horizontal arrows indicate target site duplications with their sequences shown underneath. Exons are depicted as purple boxes and introns as the lines connecting exons; other sequences are shown as horizontal lines. The gene structure was based on annotation provided by TIGR. Homologous regions are associated with solid or dashed lines. The long arrow above the gene indicates transcripts from the gene. Unique sRNAs are shown as solid triangles and nonunique sRNAs as empty triangles. The sizes of triangles are proportional to the level of sRNAs. Green triangles indicate sRNAs from stem, with red ones from immature panicle and blue from germinating seedlings. For the gene, all sRNAs in the relevant tissues are shown. For the Pack-MULE, only those in the acquired region are shown.
Figure 5.
Figure 5.
The Relationship between TIR Similarity and Abundance of Small RNAs. (A) Total sRNAs. (B) Shared sRNAs (with parental genes).
Figure 6.
Figure 6.
Purifying Selection on Pack-MULEs Reflected by Ka/Ks Values. (A) The comparison of Ka/Ks distribution between Pack-MULE–parental sequence pairs and human pseudogene-parental sequence pairs. (B) The distributions of Ka/Ks ratios for Pack-MULE–parental sequence pairs for Pack-MULEs with sense, antisense, and no expression. (C) Relationships between Ka/Ks and Ks values in the Pack-MULE and the parental gene lineages.

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