Ordered assembly of roX RNAs into MSL complexes on the dosage-compensated X chromosome in Drosophila

Background: In the male Drosophila, the X chromosome is transcriptionally u pregulated to achieve dosage compensation, in a process that depends on ass ociation of the MSL proteins with the X chromosome. A role for non-coding R NAs has been suggested in recent studies. The roX1 and roX2 RNAs are male-s pecific, non-coding RNAs that are produced by, and also found associated wi th, the dosage-compensated male X chromosome. Whether roX RNAs are physical ly part of the MSL complex has not been resolved. Results: We found that roX RNAs colocalize with the MSL proteins and are hi ghly unstable unless the MSL complex is coexpressed, suggesting a physical interaction. We were able to immunoprecipitate roX2 RNA from male tissue-cu lture cells with antibodies to the proteins Msl1 and Mle, consistent with a n integral association with MSL complexes. Localization of roX1 and roX2 RN As in mutants indicated an order of MSL-complex assembly in which roX2 RNA is incorporated early in a process requiring the Mle helicase. We also foun d that the roX2 gene, like roX1, is a nucleation site for MSL complex sprea ding into flanking chromatin in cis. Conclusions: Our results support a model in which MSL proteins assemble at specific chromatin entry sites (including the roX1 and roX2 genes); the roX RNAs join the complex at their sites of synthesis; and complete complexes spread in cis to dosage compensate most genes on the X chromosome.