Identification and Characterization of Alternative Splicing Variants and Positive Selection Genes Related to Distinct Growth Rates of Antlers Using Comparative Transcriptome Sequencing

Abstract

The molecular mechanism underlying rapid antler growth has not been elucidated. The contrast of the wapiti and sika deer antler provides a potential model for comparative studies for the identification of potent growth factors and unique regulatory systems. In the present study, reference transcriptomes of antler RM tissue of wapiti and sika deer were constructed using single molecule real time sequencing data. The expression profiling, positive selection, and alternative splicing of the antler transcripts were compared. The results showed that: a total of 44,485 reference full-length transcripts of antlers were obtained; 254 highly expressed transcripts (HETs) and 1936 differentially expressed genes (DEGs) were enriched and correlated principally with translation, endochondral ossification and ribosome; 228 genes were found to be under strong positive selection and would thus be important for the evolution of wapiti and sika deer; among the alternative splicing variants, 381 genes were annotated; and 4 genes with node degree values greater than 50 were identified through interaction network analysis. We identified a negative and a positive regulator for rapid antler growth, namely RNA Binding Motif Protein X-Linked (RBMX) and methyltransferase-like 3 (METTL3), respectively. Overall, we took advantage of this significant difference in growth rate and performed the comparative analyses of the antlers to identify key specific factors that might be candidates for the positive or negative regulation of phenomenal antler growth rate.

Keywords: alternative splicing variants; antler growth rate; comparative transcriptome; positive selection genes; single molecule real time sequencing.

Figure 1

Gene expression and enrichment analyses of RM tissue in the antler growth centre of wapiti and sika deer: (A) Sika deer and wapiti antlers with the same growth stage. Tissues were sampled from the growing tips of antler (arrows). (B) Box drawing of all transcripts in the RM tissue of after FPKM data standardization. (C) Functional enrichment analysis of the highly expressed genes between wapiti and sika deer. Enriched GO terms (represented using blue columns) and signalling pathways (represented using orange columns) were highly correlated with translation, endochondral ossification and ribosome.

Figure 2

Differential expression, positive selection and alternative splicing events in RM tissue in the antler growth centre of wapiti and sika deer: (A) Heatmap of 3899 differential expression transcripts. The map was drawn through comparison of read count on each transcript of wapiti and sika deer. Of these transcripts, 1882 were up−regulated in the wapiti antler and 2017 up−regulated in sika deer antler. (B) The functional enrichment analysis of differentially expressed genes (DEGs). Note that most of the significantly enriched biological processes were found to all be related with the metabolism. (C) Distribution of Ka and Ks of 1531 orthologous transcripts. Of these transcripts, 315 were found to be divergent transcripts (Ka/Ks > 1). (D) Venn diagram of DEGs, positive selection genes (PSGs) and alternative splicing genes (AS genes). (E) The type and distribution of the alternative splicing events: 448 intron retention (RI) events accounting for 69.67% of the total alternative splicing events, 91 alternative 3’ splice site (A3), 80 alternative 5’ splice site (A5), 11 exon skipping (SE), 3 alternative first exon (AF) and 10 alternative last exon (AL), respectively.

Figure 3

Gene interaction and sub-module analyses of DEGs in the RM tissue in the antler growth centre of wapiti and sika deer: (A) Interaction of DEGs in the antlers of wapiti and sika deer, the red sub module represents the most significant sub module (p value < 0.01). (B) The most significant sub module contains RBMX, which has a potential interaction with METTL3 through the transcription initiation factor (GTF2F1) and RNA polymerase II (POLR2E, POLR2K, POLR2C and POLR2B).

Figure 4

The exon–intron structures and expressions of 13 transcripts of RBMX gene. The exon-intron structures of RBMX and expression of the 13 transcripts of RBMX were compared between wapiti and sika deer antlers and transcript 6664 was found to be expressed specifically in wapiti antler, while transcript 3824 was expressed specifically in sika deer antler.

Figure 5

Characterization of RBMX and METTL3 in growing antlers: (A) Expression levels of RBMX in seven segments of growing antlers and 12 types of control deer tissue: main branch RM zone, second branch RM zone, main branch cartilage zone, second branch cartilage zone, middle part antler, first branch antler, proximal part antler, cerebrum, cerebellum, heart, liver, kidney, rumen, lungs, spleen, small intestine, compact bone, cartilage and periosteum. (B) Sampling diagram of seven segments of growing antlers. (C) Expression levels of RBMX and METTL3 in the antlers with fast and slow growth rate. (D) Expression levels of GTF2F1 in the antlers with fast and slow growth rate. (E) Expression levels of POLR2E, POLR2K, POLR2C and POLR2B genes in the antlers with fast and slow growth rate. * p < 0.05; ** p < 0.01; *** p < 0.001; blue colour, antler with slow growth rate; red colour, antler with fast growth rate.