The Am-tra2 Gene Is an Essential Regulator of Female Splice Regulation at Two Levels of the Sex Determination Hierarchy of the Honeybee

Abstract

Heteroallelic and homo- or hemiallelic Complementary sex determiner (Csd) proteins determine sexual fate in the honeybee (Apis mellifera) by controlling the alternative splicing of the downstream gene fem (feminizer). Thus far, we have little understanding of how heteroallelic Csd proteins mediate the splicing of female fem messenger RNAs (mRNAs) or how Fem proteins direct the splicing of honeybee dsx (Am-dsx) pre-mRNAs. Here, we report that Am-tra2, which is an ortholog of Drosophila melanogaster tra2, is an essential component of female splicing of the fem and Am-dsx transcripts in the honeybee. The Am-tra2 transcripts are alternatively (but non-sex-specifically) spliced, and they are translated into six protein isoforms that all share the basic RNA-binding domain/RS (arginine/serine) domain structure. Knockdown studies showed that the Am-tra2 gene is required to splice fem mRNAs into the productive female and nonproductive male forms. We suggest that the Am-Tra2 proteins are essential regulators of fem pre-mRNA splicing that, together with heteroallelic Csd proteins and/or Fem proteins, implement the female pathway. In males, the Am-Tra2 proteins may enhance the switch of fem transcripts into the nonproductive male form when heteroallelic Csd proteins are absent. This dual function of Am-Tra2 proteins possibly enhances and stabilizes the binary decision process of male/female splicing. Our knockdown studies also imply that the Am-Tra2 protein is an essential regulator for Am-dsx female splice regulation, suggesting an ancestral role in holometabolous insects. We also provide evidence that the Am-tra2 gene has an essential function in honeybee embryogenesis that is unrelated to sex determination.

Similar but not the same: insights into the evolutionary history of paralogous sex-determining genes of the dwarf honey bee Apis florea

“…In addition to these domains and in contrast to fem, csd is characterized by the hypervariable region (HVR), forming repeated structures of species-specific amino acid motifs that are highly variable in length (Hasselmann et al, 2008b). Heterozygous csd is required to induce the female pathway by interacting with transformer 2 (Nissen et al, 2012), leading to a female-spliced fem transcript; this decision is maintained throughout the development by a positive feedback loop. Homozygous or hemizygous csd induces the male pathway, mediated by a truncated Fem protein, which results from an early stop codon in the male fem mRNA (Beye et al, 2003;Gempe et al, 2009).…”

To “Bee or Not to Bee” Male or Female? An Educational Primer for Use with “The Am-tra2 Gene Is an Essential Regulator of Female Splice Regulation at Two Levels of the Sex Determination Hierarchy of the Honeybee”

“…However, csd and fem are paralogs specific to the honeybee lineage (Hasselmann et al 2008) that have evolved and acquired distinct roles in the sex determination pathway. To alleviate confusion when discussing orthologs from different species, genes from the honeybee will be designated with an "Am-" prefix (for A. mellifera, as in Nissen et al 2012). Gene names are written in lowercase italics (e.g., Am-dsx) and protein names in non-italics with the first letter of the protein capitalized (e.g., Am-Dsx).…”

“…A sequence homology search of the honeybee genome (Dearden et al 2006) previously identified a tra-2 ortholog; however, whether this gene is expressed or produces functional transcripts has not been demonstrated. Therefore, since tra-2 and Am-tra-2 are orthologs, the objective addressed by Nissen et al (2012) was to determine whether Tra-2 and Am-Tra2 share the same function (i.e., regulating sex-specific splicing of dsx) in the fruit fly and in the honeybee.…”

“…Therefore, to characterize RNA sequences, mRNA is converted into complementary DNA (cDNA) using the enzyme reverse transcriptase. Together with cDNA synthesis, the RACE technique was used by Nissen et al (2012) to determine the 59 and 39 sequences of a mature mRNA.…”

“…To apply this method, a suggested sequence of articles is the following: Beye et al (2003) (the finding that csd heterozygosity is the primary sex-determining signal) followed by Hasselmann et al (2008) (which characterizes the Am-fem and csd paralogs) and Gempe et al (2009) (which explores the regulation and function of Am-fem and csd). After reading these articles, the methods and questions of Nissen et al (2012) might seem more rational and logical to students. In addition, review articles on sex determination in Hymenoptera (Heimpel and de Boer 2008) and other insects (Gempe and Beye 2010;Verhulst et al 2010a) are valuable reading.…”