Alu-Derived Alternative Splicing Events Specific to Macaca Lineages in CTSF Gene

Abstract

Cathepsin F, which is encoded by CTSF, is a cysteine proteinase ubiquitously expressed in several tissues. In a previous study, novel transcripts of the CTSF gene were identified in the crab-eating monkey deriving from the integration of an Alu element-AluYRa1. The occurrence of AluYRa1-derived alternative transcripts and the mechanism of exonization events in the CTSF gene of human, rhesus monkey, and crab-eating monkey were investigated using PCR and reverse transcription PCR on the genomic DNA and cDNA isolated from several tissues. Results demonstrated that AluYRa1 was only integrated into the genome of Macaca species and this lineage-specific integration led to exonization events by producing a conserved 3' splice site. Six transcript variants (V1-V6) were generated by alternative splicing (AS) events, including intron retention and alternative 5' splice sites in the 5' and 3' flanking regions of CTSF_AluYRa1. Among them, V3-V5 transcripts were ubiquitously expressed in all tissues of rhesus monkey and crab-eating monkey, whereas AluYRa1-exonized V1 was dominantly expressed in the testis of the crab-eating monkey, and V2 was only expressed in the testis of the two monkeys. These five transcript variants also had different amino acid sequences in the C-terminal region of CTSF, as compared to reference sequences. Thus, species-specific Alu-derived exonization by lineage-specific integration of Alu elements and AS events seems to have played an important role during primate evolution by producing transcript variants and gene diversification.

Keywords: Alternative splicing; Alu; CTSF; exonization; primate.

Fig. 1. Structural analysis of human, rhesus monkey, and crab-eating monkey CTSF gene transcripts

The antisense-oriented AluYRa1 element (blue arrow) is located on the 11^th intron of CTSF in rhesus monkey and crab-eating monkey. Open, black, and gray boxes represent exon’s untranslated region (UTR), open reading frame (ORF; protein-coding region), and putative protein-coding region, respectively. The horizontal line represents intron sequences. This figure is a structural illustration and is not drawn to scale.

Fig. 2. Integration of the AluYRa1 element in the CTSF gene during primate evolution

(A) PCR amplification of AluYRa1 in several primates. M indicates the molecular size marker. Primate DNA samples are abbreviated as follows: (1) HU: human; (2) hominoids: CH: chimpanzee; (3) Old World monkeys: JA: Japanese monkey, RH: rhesus monkey, CR: crab-eating monkey, PI: pigtail monkey, BO: bonnet monkey, MA: mandrill, AF: African green monkey, LA: langur; (4) New World monkeys: MAR: Marmoset, TA: tamarin; and (5) prosimian: RL: ringtailed lemur. (B) Schematic representation of the timing of AluYRa1 integration in Macaca CTSF genes. The blue arrow indicates the integration event of AluYRa1. Mya, million years ago.

Fig. 3

Structural analysis of the CTSF gene transcripts in rhesus and crab-eating monkeys using reverse transcription PCR and sequence analysis. In both species, the antisense-oriented AluYRa1 element is located on the 11^th intron of the CTSF gene and six transcript variants were identified from various tissues. Open, black, and gray boxes represent exon’s untranslated region (UTR), open reading frame (ORF; protein-coding region), and putative protein-coding region, respectively. Vertical dashed lines represent the 3′ (GT) and 5′ (AG) splice sites, and the horizontal line represents intron sequences. Black and green arrows represent validation primers and RT-PCR primers, respectively. The transposable element exon (TE-exon), 12a, 11a, 11b, 12b, and 12c, exons indicated in red font correspond to new AS-derived exons in the six variants compare with original transcript. This figure is a structural illustration and is not drawn to scale.

Fig. 4. Reverse transcription-PCR amplification of CTSF reference sequences

(A) Human, (B) rhesus monkey, and (C) crab-eating monkey. The GAPDH gene was used as the positive control (indicated in the 120 bp). M indicates the molecular size marker. Numbers indicate cDNA tissue samples. Human – 1: bone marrow; 2: brain (whole); 3: fetal brain; 4: colon; 5: small intestine; 6: heart; 7: kidney; 8: liver; 9: fetal liver; 10: lung; 11: placenta; 12: prostate; 13: skeletal-muscle; 14: spinal cord; 15: spleen; 16: stomach; 17: testis; 18: thymus; 19: trachea; 20: uterus. Rhesus monkey and crab-eating monkey–1: cerebellum; 2: cerebrum; 3: kidney; 4: large intestine; 5: liver; 6: lung; 7: pancreas; 8: small intestine; 9: spleen; 10: stomach; 11: testis. RT-PCR products were validated by sequence analysis.

Fig. 5. Reverse transcription-PCR amplification of reference sequence of the six transcript variants of the CTSF gene in rhesus and crab-eating monkeys

(A and B) V1 (157 bp) and V6 (266 bp), (C and D) V2 (145 bp), (E and F) V3 (186 bp), (G and H) V4 (234 bp), (I and J) V5 (187 bp), and (K and L) GAPDH (120 bp), used as the positive control. M indicates the molecular size marker. Numbers indicate rhesus and Crab-eating monkeys cDNA tissue samples. 1: cerebellum; 2: cerebrum; 3: kidney; 4: large intestine; 5: liver; 6: lung; 7: pancreas; 8: small intestine; 9: spleen; 10: stomach; 11: testis. Transcripts were validated by sequence analysis of the reverse transcription-PCR products.

Fig. 6. Multiple sequence alignment of CTSF amino acid sequences

The six transcript variants of CTSF identified from rhesus and crab-eating monkeys were aligned with CTSF reference sequences from human, rhesus monkey, and crab-eating monkey. Dots indicate amino acids are identical to those found in the human sequence. The gray, blue, red, and green box indicate the signal peptide, cystatin-like domain, I29 inhibitor, and mature cathepsin F, respectively. RH, rhesus monkey; CR, crab-eating monkey; V1–V6, CTSF variants.