Molecular characterization and expression of buffalo (Bubalus bubalis) DEAD-box family VASA gene and mRNA transcript variants isolated from testis tissue

• We isolate, sequenced, and characterized the VASA gene in buffalo testis.
• 5′-RACE identified four buffalo VASA transcripts with different TSSs.
• 3′-RACE identified four different buffalo VASA transcripts with different lengths.
• We found five VASA variants, one full length and four splice variants.
• The VASA1 and 6 are highly expressed in the testis tissues.

VASA is a member of the DEAD-box protein family that plays an indispensable role in mammalian spermatogenesis, particularly during meiosis. In the present study, we isolated, sequenced, and characterized VASA gene in buffalo testis. Here, we demonstrated that VASA mRNA is expressed as multiple isoforms and uses four alternative transcriptional start sites (TSSs) and four different polyadenylation sites. The TSSs identified by 5′-RNA ligase-mediated rapid amplification of cDNA ends (RLM-5′-RACE) were positioned at 48, 53, 85, and 88 nucleotides upstream relative to the translation initiation codon. 3′-RACE experiment revealed the presence of tandem polyadenylation signals, which lead to the expression of at least four different 3′-untranslated regions (209, 233, 239 and 605 nucleotides). The full-length coding region of VASA was 2190 bp, which encodes a 729 amino acid (aa) protein containing nine consensus regions of the DEAD box protein family. VASA variants are highly expressed in testis of adult buffalo. We found five variants, one full length VASA (729 aa) and four splice variants VASA 2, 4, 5, 6 (683, 685, 679, 703 aa). The expression level of VASA 1 was significantly higher than rest of all (P < 0.05) except VASA 6. The relative ratio for VASA 1:2:4:5:6 was 100:1.0:1.6:0.9:48.