Details of transgene construction determine effective siRNA production
Authors: Sudeshna Biswas; Victoria Meller
Affiliation: Wayne State University
Keywords: i. dosage compensation; s. siRNA/RNAi
Flies correct for imbalance of X chromosome dosage between the sexes by increasing X-linked gene expression in males. The Male Specific Lethal (MSL) complex, composed of proteins and roX RNA, localizes to the male X and participates in gene up-regulation. How the X is identified remains unclear. Loss of roX RNA causes mislocalization of MSL proteins and male lethality. Small interfering RNAs (siRNA) and satellite repeats enriched on the X contribute to X recognition. Our studies showed that ectopic expression of siRNA from 1.6883F (cytological position 3F) partially rescues roX1 roX2 males and localization of the MSL complex, but siRNA from other repeats with similar sequence, including 1.6881A, did not. We hypothesize that details of transgene construction could determine function. To test this, I engineered an siRNA-producing transgene with 1.6881A sequence but with size, phasing and orientation identical to the biologically active 1.6883F transgene. In accord with my hypothesis, the new 1.6881A transgene rescued roX1 roX2 males to the same level as a 1.6883F transgene. This suggests that details of transgene construction play a crucial role in production or processing of double stranded RNA. In the future, I will take advantage of siRNA-producing transgenes that differ in their ability to promote X recognition to determine if epigenetic marks at 1.688X repeats are enhanced only when transgenes that promote X recognition are present. My studies will serve to disentangle the role of siRNA and chromatin modification in dosage compensation.