BIOLOGICAL OVERVIEW

The quest for genes expressed in the fly's brain is currently one of the major preoccupations of Drosophila geneticists. The gene roX1, previously known as yang, was identified by two different laboratories using different approaches, both designed to identify genes expressed in the brain. In one approach, an enhancer detector screen was carried out for mushroom body expression of a reporter gene. This study found a reporter expressed in females and not males, but an adjacent gene (roX1) was expressed in males but not females (Meller, 1997). A second study sought male specific genes expressed in mushroom bodies. A line of flies expressing green fluorescent protein in the mushroom body cells was used to obtain purified mushroom body cells using a fluorescence-activated cell sorter. A male cDNA library from GFP-positive cells was constructed and differentially screened with male- and female-specific mushroom body cDNA probes. Two genes were identifed: roX1 and roX2 (Meller, 1997). As will be described later, the RNA transcripts of both genes decorate the X chromosome of male flies, and thus the origin of their name: RNA on the X (roX) (Meller, 1997).

roX1 is expressed during embryonic development in a non-sex delimited fashion. Both males and females show expression of roX1 in neuroblasts, but this expression fades in females. Expression of roX1 is controlled by the Male specific lethal-2 (MSL-2) directed dosage compensation system. MSL-2 is responsible for directing the relatively high level of transcription from the single X chromosome of males, compared with a lower level of transcrition from the two X chromosomes of females (Kelly, 1995). Absence of any one of the four genes responsible for dosage compensation, completely eliminates roX1 expression. Interestingly, fading of the roX1 RNA in females takes place long after the association of MSL proteins (Meller, 1997 and Amrein, 1997).

Presence of functional Sex lethal is responsible for the absence of roX1 RNA in females. As presence of functional Sex lethal protein in females is responsible for a blocking of functional MSL-2 splicing in females, mutation of Sxl results in functional MSL-2 splicing in females and production of roX1 levels comparable to those observed in wild-type males (Meller, 1997).

What is the function(s) of roX1 and the distantly linked roX2 gene, both of which are expressed in adult males and are absent from adult females? Neither RNA codes for a protein, and roX1 RNA is found associated with the single X chromosome of pupae. Mutation of roX1 results in no apparent phenotype, a rather distressing result for geneticists who look to mutation to define gene function. Two facts are known: (1) Both roX1 and roX2 are closely linked to genes that are expressed only in females. Perhaps roX1 and roX2 regulate the expression of these female specific genes. Unfortunately, no evidence is provided for or against this possibility. (2) A similar sex delimited non-transcribed RNA species is involved in X chromosome inactivation in mammals (Penny, 1996 and Lee, 1996). XIST RNA, produced by the inactive X chromosome concentrates near the inactive X in an association with chromatin. XIST RNA remains with the nuclear matrix fraction after removal of chromosomal DNA but is released from its association with the inactive X during mitosis (Clemson, 1996). Incredably, a multicopy Xist transgene located on an autosome appears to produce RNA that binds to and inactivates autosomal chromatin in cis (Lee, 1996).

Might roX1 and roX2 function similarly in Drosophila? It is possible that the RNA of chromatin has a structural role that engenders gene silencing of proximal female specific genes on the male X chromosome of Drosophila. Such a function would have to be carried out by several genes with redundant function, since there is no phenotypic effect associated with roX1 mutation. It is proposed that roX1 and its family members associate along the entire X chromosome to help change chromatin conformation and achieve hypertranscription in the male, or, by their absence, change specific gene activation in the female by changing chromatin coformation, perhaps by associating with the MSLs, histone acetyltransferase, or other constituents of chromatin. This model is analogous to that for Xist, although Xist RNA achieves the opposite goal, that of condensing an X chromsome (Meller, 1997 and Amrein, 1997).

Perhaps no phenotypic effect to Xist mutation exists because the function of Xist in neurons (presumably the activation or repression of genes involved in determining male specific behaviors through modification of the phenotype of neurons) is in fact regulated by multiple backup pathways, ensuring a tight regulation of this important function. There is precedence for this type of redundancy. For instance, misexpression of Fasciclin 3 results in misdirection of certain neurons but does not alter targeting of neurons that normally express Fas3 (Chiba, 1995).

While the hypothesis that the rox RNAs are involved in dosage compensation is attractive, some of the data with regard to the rox RNAs are either lacking or appear to be in conflict with previous knowledge about dosage compensation. (1) Only rox1 has been shown to be associated with the male X chromosome; whether the rox1 RNA is bound to the same sites as the MSLs or is bound in some other pattern is unkown. (2) Some features of the reported temporal and spatial expression patterns of the rox RNAs seem to be incompatible with what is known about the msl-based dosage compensation system. rox1 RNA was reported to be present in both sexes up to germ band retraction with predominant staining in the CNS; a reduction of expression in female embryos occurred late in embryogenesis (Meller, 1997). However, the MSL proteins, known to be involved in dosage compensation, are associated with the X chromosome in all somatic cells of males from early gastrula stages on and not associated with the Xs in female embryos, as has been reported for rox1 (Meller, 1997). Data has been obtained in the current study that contradicts some of the observations of Meller, especially the finding of rox1 RNA with female chromosomes (Franke, 1999).

The strongest evidence that rox1 might be involved in dosage compensation is the fact that it is associated with the X chromosome in male larval salivary gland nuclei (Meller, 1997). Using a similar technique, the same has been found to be true for rox2 RNA. However, this technique does not resolve the distribution of these RNAs along the X chromosome because the extant methods for immunostaining of polytene chromosomes and RNA in situ hybridization are not easily compatible. A protocol that gives an MSL banding pattern without removing the rox RNA molecules was developed by using less concentrated acid solutions for relatively short fixation times. rox1 RNA and MSL-1 protein are colocalized to exactly the same chromosomal bands. No signals were detected that show one of the two molecules alone. The same double staining was performed with rox2 RNA and MSL-1 protein and gave an identical result. These results show that MSL proteins and rox RNAs are distributed on the male X chromosome in exactly the same pattern, providing strong evidence for a direct involvement of the rox RNAs in dosage compensation (Franke, 1999).

The rox RNA distributions were reexamined in embryos: both rox1 and rox2 are expressed in male embryos in patterns completely overlapping with that of the MSL proteins. Both RNAs are detected in a punctate pattern in every cell of the developing male embryo. This nuclear staining pattern is observed from early gastrula stages on. No staining is seen in female embryos, which were stained with anti-Sxl antibodies afterward to verify their sex. To prove that the nuclear pattern represents association with the X chromosome in these diploid cells, double labeling experiments with rox RNAs and anti-MSL-1 antibodies were performed. Both signals completely overlap in all of the embryonic cells, as shown for rox1 and MSL-1. These findings establish that rox RNAs are associated with the male X chromosome from early embryonic stages on and that their expression patterns and subnuclear localizations in embryonic cells correspond exactly to those of the MSL proteins (Franke, 1999).

The MSL protein distribution was examined in sections of adult flies and the pattern was compared to the rox RNA in situ hybridization pattern obtained from similar sections. On sections from male flies, MSL proteins and both rox1 and rox2 can be detected in nuclei in all body parts, most likely in every cell. In every case, the MSL proteins and the rox RNAs are restricted to an area covering about 20% of the nucleus. The distribution of signals obtained with the rox probes is virtually indistinguishable from the signal obtained with anti-MSL antibodies. That the subnuclear localization of the MSL1 protein in adult cells is due to its association with the X chromosome was verified by simultaneous in situ hybridization with a probe that painted the whole X chromosome and anti-MSL-1 antibody staining. To establish that the rox signals overlap with the MSL signal and therefore are associated with the X chromosome, double-labeling with an antibody to the MSL-1 protein and in situ hybridization to the rox RNAs was performed. The rox2 and MSL-1 signals completely overlap. The same result is obtained with rox1 probe, indicating that both rox RNAs are localized on the X chromosome in all somatic cells from adult males. Together with the preceeding results, these findings strongly support the idea that the rox RNAs are involved in msl-mediated dosage compensation beginning at early gastrulation. They continue to be used in all somatic tissues throughout all stages of development (Franke, 1999).

Simultaneous removal of rox1 and rox2 function leads to a loss of X Chromosome binding of the MSL proteins in male embryos. If the rox RNAs are integral members of a nucleic acid-protein complex that regulates dosage compensation, then one might expect that mutations in the rox genes could disrupt the assembly and/or functioning of such a complex. Experiments addressing this possibility have been limited to examining the effects of mutations at the rox1 locus that were shown not to affect MLE binding to the male X chromosome (Meller, 1997 ). The effects of rox2 deletions and rox1/rox2 double mutants were examined on the binding of the MSL proteins to the X chromosome in male embryos. Deletions in the 10B,C region of the X chromosome were used, where the rox2 gene is located (Amrein, 1997). Two deficiencies in the region that remove the rox2 gene [Df(1)DA622 and Df(1)HM456] and two deficiencies that do not remove the rox2 gene [Df(1)KA6 and Df(1)M259-4] were used. These deletions are embryonic lethals, but developed to late embryonic stages. Thus, MSL binding could be studied at earlier stages in these embryos. The rox1MB710 mutation has no effect on the distribution of the MSL-1, -2, and -3 proteins in male embryos, compared to wild-type embryos. The deletions Df(1)DA622 and Df(1)HM456 that remove the rox2 gene, as well as the deletions Df(1)KA6 and Df(1)M259-4 that do not, also have no effects on the MSL distribution. In contrast, double mutants of rox1 and rox2 show a clear lack of MSL association with the X chromosome in male embryos. Sibling control embryos, that are wild type with respect to rox1 and rox2 and are distinguished from the mutant embryos by the ftz-lacZ staining show normal MSL staining. The deletions in the 10B,C region that do not remove the rox2 gene do not show any abnormal MSL distribution when combined with the rox1 mutation. These data thus show that only simultaneous mutations in both rox1 and rox2 abolish MSL association with the X chromosome. These results strongly argue that the rox RNAs are redundant in their function and that they are essential components of the dosage compensation machinery (Franke, 1999).

Thus it has been established that the rox1 and rox2 RNAs function as integral components in dosage compensation. The findings that the rox1 and rox2 genes have an essential but redundant function in dosage compensation and that these RNAs colocalize with the MSL proteins suggest that they are components of an RNA-protein complex that brings about dosage compensation. This RNA-protein complex is here termed "compensasome" (Franke, 1999).

Several results suggest that the rox RNAs are redundant in their function. The absence of any phenotype in flies that are mutant for rox1 led to the initial suggestion that these RNAs might be members of a redundant gene family (Meller, 1997). The finding that embryos exhibit a normal MSL binding pattern with either one or the other of the rox genes mutated shows that neither of these RNAs alone is required for dosage compensation. The result that the MSL binding pattern is disrupted in rox1/rox2 double mutant male embryos shows that these RNAs are functioning as components of the dosage compensation machinery and that they are functionally redundant. However, the results do not address whether the rox1 and rox2 RNAs are present in a fixed stoichiometry in a compensasome or, alternatively, are present in an either/or manner. If as discussed, the rox RNAs are redundant in their function, one might expect similarities in their primary and/or secondary structures. A comparison of the two sequences reveals a region of homology within a 30 bp stretch. Screening the GenBank sequence database with these two 30 nt sequences did not detect other sequences with significant homology. The biological significance of this sequence is not obvious at this point (Franke, 1999).

Colocalization of the rox RNAs and MSL proteins on the X chromosome and the lack of MSL binding to the X chromosome in a rox1/rox2 double mutant background indicates that there is likely a physical interaction between the rox RNAs and the MSL proteins. Such a physical interaction offers a potential basis for the finding that the MLE protein (but not the other MSLs) can be removed from X chromosomal binding sites by treatment with RNAse (Richter, 1996). In contrast, in rox1/rox2 double mutants none of the MSL proteins bind to the male X chromosome. These results are not contradictory, but rather indicate that the rox RNAs are important for the association of all the MSL proteins with the X chromosome, perhaps by being required to form a stable compensasome, and also potentially required for the subsequent RNA dependent stable association of MLE with the X chromosome. Besides being important for the association of the compensasome with the X chromosome, it is possible that the rox RNAs have an additional role(s) in eliciting hypertranscription of the male X chromosome. For several other RNA-protein complexes (i.e., ribosomes, spliceosomes, and telomerases), it has been found that the RNA components of these complexes are not just structural components, but rather perform major functions within these complexes. There are several ways that the rox RNAs could potentially have direct roles in dosage compensation. (1) The rox RNAs may be important for contact between the X chromosome and the compensasome dosage compensation complex. Base pairing between the RNAs and the chromatin to physically connect the compensasome with its target sequences is attractive because the MSL proteins do not appear to have DNA binding activity. Thus, although the MLE protein has been shown to bind nucleic acids, removing this activity did not interfere with the association of MLE with the X chromosome (Lee, 1997). (2) Since an alteration in the X chromosome chromatin structure is certainly correlated with, and may be a prerequisite for, dosage compensation, the rox RNAs might be involved in this alteration of chromatin structure. (3) The rox RNAs may interact with RNA polymerase or other components of the chromatin remodeling and/or transcription machinery to more directly increase transcription rates. The recently discovered 'chromatin remodeling factors' like CHRAC, NURF, and ACF, among others, contain helicase-like components and are involved in the regulation of histone acetylation. More generally, the involvement of RNA molecules in dosage compensation raises the interesting possibility that these factors, like the dosage compensation machinery, might contain important functional RNA components, as well (Franke, 1999).

While the process of dosage compensation in both mammals and Drosophila appears to be quite different, two findings suggest that these two dosage compensation processes might have in common the basic biochemical mechanism through which they modulate X chromosome transcription. In both cases, dosage compensation involves opposite changes in the pattern of acetylation of histone H4. In addition, noncoding RNA molecules are integral components to the dosage compensation machinery in both systems. This suggests that dosage compensation in the two systems is likely mediated by similar biochemical machineries (Franke, 1999).

GENE STRUCTURE

Two transcripts of 3.6 and 3.8 are expressed in adult flies. Two cDNAs are similar in size and sequence, while a third contains a 68 base pair intron (Meller, 1997).

roX1 and roX2 are both linked to female-specific genes. roX2 is closely linked (within 1kb) to a female-specific gene, no distributive disjunction (nod). nod has been mapped to 10B on the X chromosome, the same chromosomal region in which roX2 resides. nod is a member of the kinesin family of genes shown to be essential for proper segregation of nonexchange chromosomes during female meiosis. roX1 is closely linked to oligo peptide transporter 1 (opt1), a gene that shows high homology to members of the class of 12 transmembrane domain oligopeptide transporters. Expression of OPT1 mRNA is largely restricted to nurse cells in the ovary (Amrein, 1997).

Exons - 2

date revised: 15 October 99  

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