Autoregulation of a Noncoding RNA Gene Figure 3. roX1 RNA is needed to sustain endogenous roX1 transcription in males. X chromosomes from neighboring cells display a mosaic pattern in which the MSL complex either succeeded (arrowhead) or failed (arrow) to paint the X from roX2; msl2; H83M2 female (A) and male (B) salivary glands. (C) Endogenous roX1 and H83roX1D39 transcripts (Orange) and antisense riboprobe recognizing only full length roX1 (green). Whole mount roX1 FISH using the internal probe on salivary glands from (D) wild type male, (E) wild type female, (F) roX1+ roX22/Y; msl2; H83M2 mosaic male, (G) roX1+roX22/Y; msl2; H83M2 H83-roX1D39/+ male, (H) roX12 roX2 2/Y; msl2; H83M2 H83-roX1D39/+ male. The X chromosomes in G are fully painted in all cells with MSL complex relying upon roX1-D39 RNA (Figure S1D), but the truncated roX1 RNA is not recognized by the internal probe. doi:10.1371/journal.pgen.1002564.g003 animals also making the shorter transgenic H83roX1-D39 RNA (Figure 3D, 3H and Figure S1C). The roX2; H83M2 males (roX1+ roX22/Y; msl22; H83M2/+) displayed the same mosaic roX1 pattern (Figure 3F) typically seen in polytene squashes (Figure 3A and 3B). When we introduced H83roX1-D39 into these roX2 H83M2 males, the expression of full-length roX1 RNA made from the endogenous locus was restored to all cells (Figure 3G and Figure S1B, lane 3). In addition, while the distribution of roX1 RNA was often limited to a single band or several Mbp around 3F in nuclei from the mosaic H83M2 males (Figure S1E–S1F), the endogenous, full-length roX1 RNA coated the entire length of the X chromosome in nearly all cells when the constitutively expressed H83roX1-D39 transgene was also present, (Figure S1G). The H83roX1-D39 transgene was ineffective in H83M2 females (Figure S1B, lane 4), perhaps because females have two X chromosomes, depressed MSL1 expression [36] and so require more stimulatory activity than H83roX1-D39 can supply. We conclude that the unexpected mosaic pattern found in roX2 H83M2 animals results from a failure of the wild type roX1 gene to respond to MSL proteins. Providing a constitutive source of roX1 RNA is sufficient to reliably drive transcription of the endogenous wild type roX1 gene. Taken together, these results support an autoregulatory model where new transcription of the wild type roX1 gene requires pre-existing RNA, either roX1 or roX2, in addition to MSL proteins. We postulate that the early MSL- independent burst of roX1 RNA made at blastoderm normally assembles the first MSL complexes needed to set up the future maintenance of roX1 transcription in adult males. PLoS Genetics | www.plosgenetics.org 5 March 2012 | Volume 8 | Issue 3 | e1002564