6436 Nucleic Acids Research, 2011, Vol. 39, No. 15 Figure 6. Spliced and poly-adenylated msl2 RNA is associated to a non-chromatin bound MSL complex. (A–C) Fractionation and RIP experiments of three biological replicates using total RNA extraction (A and B) and extracted poly(A)+ RNA (C). The circle diagrams specify the relative proportion of the RNAs in the nucleoplasm (gray) and the chromatin fractions (dark gray). The majority of the roX1 and roX2 RNAs are located in the chromatin fraction while msl2 and the negative control Rpl32 are mainly found within the nucleoplasm. As illustrated in the histograms, enrichment of msl2 RNA in the MSL complex is conﬁrmed by RIP experiments on the nucleoplasmic fractions using MSL2 antibodies. Displayed are the fold enrichments relative to actin RNA. Immunoprecipitated msl2 in experiments A, B and C correspond to 3, 4 and 10% of input, respectively. Gel-electrophoretic analyses of ampliﬁed genomic DNA (gDNA), immunoprecipitated (NP RIP) and total RNA from the nucleoplasm (NP) and from chromatin (Chr) fractions show that msl2 RNAs associated to the MSL complex are spliced. The PCR product sizes of the spliced msl2 amplicon and the unspliced amplicon are indicated by arrowheads and stars, respectively. The minus reverse transcriptase controls (-RT) are shown below. (D) Model for feedback regulation of MSL complex via the association of a non-chromatin MSL complex with the msl2 mRNA. When free nuclear MSL complex is present this non-chromatin MSL-complex titrates msl2 RNA and thus reduces the amount of msl2 transcript available for export and translation, which in turn regulates the complex production as a feedback mechanism. complexes. It is well established that the MSL complex is restricted to males by a translational block caused by Sxl protein binding to the msl2 RNAs. However, it has been suggested that Sxl binds not only all msl2 transcripts, but also to a subset of msl1 transcripts and prevents their translation in females (19). This reﬂects the potential of these two RNAs to be regulated through speciﬁc protein interactions. These two RNAs encode the core of the dosage compensation complex. Here, we provide evidence that a non-chromatin bound fraction of the MSL complex has high afﬁnity for nuclear msl2 mRNA and also afﬁnity for nuclear msl1 mRNA. Based on this, we propose a novel mechanism for ﬁne-tuning the MSL complex concentration by feedback. We have used both formaldehyde ﬁxed nuclear extracts and native nuclear extracts in our RIP experiments. In both these sample types, we detected strong enrichments of roX1, roX2 and msl2 transcripts. In the native extracts, we could not detect signiﬁcant enrichment of RNA transcribed from genes targeted by the MSL complex nor any enrichment of RNA molecules transcribed from MREs. Although, we found robust enrichment of roX1