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Genetics, Vol. 177, 1429-1437, November 2007, Copyright © 2007
doi:10.1534/genetics.107.071001
Department of Cell Biology and Molecular Medicine, UMDNJ—New Jersey Medical School, Newark, New Jersey 07103
4 Corresponding author: Department of Cell Biology and Molecular Medicine, UMDNJ—New Jersey Medical School, 185 S. Orange Ave., Newark, NJ 07103.
E-mail: parky1{at}umdnj.edu
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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4.3} (PARK et al. 2002). The P{w+ 44.3} element is required to supply essential genes lost in Df(1)roX252 (MELLER and RATTNER 2002).
After crossing y w/Y; [roX2 transgene] males to y w roX1ex6 Df(1)roX252 P{w+ 44.3} females, male rescue frequencies of the roX2 transgenes (Figure 4F) were calculated by ratio of the male (y w roX1ex6 Df(1)roX252 P{w+ 44.3}/Y; [roX2 transgene]/+)/female (y w roX1ex6 Df(1)roX252 P{w+ 44.3}/y w; [roX2 transgene]/+) progeny from adult flies collected during 10 days after the first day of adult eclosion. For immunostaining of Figure 4, D and E, polytene chromosomes of salivary glands were used from y w roX1ex6 Df(1)roX252 P{w+ 44.3}/Y; [roX2 transgene]/+ male larvae acquired from the cross described above. Immunostaining of MSL proteins and RNA in situ hybridization of roX RNAs were performed as previously described (KELLEY et al. 1999).
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RT–PCR analysis:
To check the male-specific expression of roX1 and roX2 genes in other Drosophila species (Figure 1B), oligo(dT)-primed cDNAs were made from 5 µg total RNAs of male and female adults. For roX1, the 5' primers (D. melanogaster, 5'-ACCAGCAGTTGATTTGCG-3'; D. simulans and D. erecta, 5'-TCTATTGGCCTTGATTATTAAC-3'; D. yakuba, 5'-ACTGGGCGCCTACAATGCG-3'; D. ananassae, 5'-CGAGCCGCTCATGTTCGCA-3'; D. pseudoobscura, 5'-CCCTCTGTTGGTCAATCGTTC-3'; D. mojavensis, 5'-GAGGGCACTTAGAGTGTCAAC-3'; D. virilis, 5'-ACCTGCTGCGTCCCTCTGC-3') and the 3' primers (D. melanogaster, 5'-ATTTCGATTTTCTTTTTATAGTTTGGG-3'; D. simulans and D. erecta, 5'-CGGCTCAGGCGTATAACGAT-3'; D. yakuba, 5'-CGGCTCAAGCGTATAACGATT-3'; D. ananassae, 5'-CGGCACAGGCGTATAACGG-3'; D. pseudoobscura, 5'-GCTCAGACGTATAACGTTTCC-3'; D. mojavensis, 5'-CGGCTCAGACGTATAACAGTT-3'; D. virilis, 5'-CGGCTCGGACGTATAACGTT-3') were used for PCR. For roX2, the 5' primers (D. melanogaster, 5'-TATATCATAAGTCGAGCGTTTAG-3'; D. yakuba, 5'-CGGCCTGGTCACACTGAGCT-3'; D. ananassae, 5'-ACCCTCTCTAGATCTTACGAC-3'; D. pseudoobscura, 5'-CTTTTCCCGCTAAAAATAATTCAG-3'; D. mojavensis, 5'-GTTCTTGCATCAGATAGTTAGG-3'; D. virilis, 5'-GTTCATCATCAGACAGCTAGG-3') and the 3' primers (D. melanogaster and D. yakuba, 5'-ACTGGTTAAGGCGCGTAAAAC-3'; D. ananassae, 5'-CTGGTTAAGGCGCGTAAAAC-3'; D. pseudoobscura, 5'-GGCTCGTAAAACGTTACCATTG-3'; D. mojavensis, 5'-ATTGTTAAGGCGCGTATAACGT-3'; D. virilis, 5'-GTTAAGGCACGTATAACGTTAC-3') were used during PCR.
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Northern analysis:
For Northern analysis (Figures 1C and 4C), total RNAs from adult flies were prepared using TRIzol Reagent (GIBCO-BRL, Carlsbad, CA) and 20 µg of total RNAs were loaded in each lane. In Figure 1C, specific probes for several Drosophila species were prepared by random priming (Invitrogen), using PCR products purified from Figure 1B. In Figure 4C, roX2 whole genomic sequence (1380 bp) was used to make the probe. Following overnight hybridization at 42° in hybridization solution (30% formamide, 1 M NaCl, 100 mM NaPO4 pH 7.0, 7% SDS, 10x Denhardt's, 100 µg/ml ssDNA-fish), the membrane was washed two times in 2x SSC, 0.1% SDS at 42°.
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RESULTS AND DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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1 kb of evolutionarily conserved 3' end regions) and roX2 (1 kb of the entire region except DHS) in distantly related fly species, we detected male-specific transcripts similar in size to roX1 (3700 nt) and roX2 (500 nt) of D. melanogaster (Figure 1C). One exception was roX1 RNA in D. mojavensis. Even though we used an additional roX1 probe from the 5' end of the sequence (2.5 kb), a roX1 transcript was not detected in Northern blot analysis (Figure 1C, top section, lane 9), suggesting that roX1 RNA of D. mojavensis might not be functional in the adult male.
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Previously, an 600-bp region in the 3' end of roX1 RNA was identified as an essential domain from serial deletion analysis of the roX1 gene (300 bp each) (Figure 3A) (STUCKENHOLZ et al. 2003). This region includes a stem–loop structure, which was shown to be critical for proper RNA function. Upon comparison of this stem–loop region found from different Drosophila species, we were able to identify conserved primary sequences and secondary structures (Figure 3E), suggesting that this region may be evolutionarily important for the function of roX1 RNA. So far we have not been able to identify this conserved region in more distantly related Drosophila species, D. virilis and D. mojavensis, both of which lost their roX1 RNA-binding activity along the entire X chromosome in polytene chromosomes as shown in Figure 2, G and H, respectively. It is possible that the lack of localization of roX1 RNA on their X chromosomes might be a result of the loss of this conserved stem–loop structure through evolution.
Alignment of nine roX2 RNAs from different Drosophila species revealed a stretch of conserved sequence (GUUNUACG box-1, GUb-1) in the 3' end of roX2 RNA, which is located at the 3' stem region of the putative stem–loop structure predicted using the mfold program (Figure 3, B and F). Further inspection of the alignment of the roX2 sequence allowed us to identify another conserved GUUNUACG sequence (GUb-2) downstream of GUb-1, which is located at the 3' minor transcript of roX2 RNA (Figure 3, B and F). The GUb-2 region was previously found as a small 25/30-nucleotide identity in both roX genes (FRANKE and BAKER 1999), but it was shown that the deletion of this region containing GUb-2 did not affect the function of roX2 RNA (PARK et al. 2003). This raises a possibility that multiple GUb's in the roX2 RNA might be functionally redundant, considering its conservation through evolution and multiple occurrences within roX2 (and roX1, see below). In D. pseudoobscura, D. mojavensis, and D. virilis we found an additional GUb (GUUNUACG) sequence upstream of GUb-1 (supplemental Figure S1 at http://www.genetics.org/supplemental/). Interestingly, the 3' stem region of the predicted stem–loop structure in roX1 RNA contains a GUUNUCCG sequence (Figure 3E), which is similar to the GUb (GUUNUACG) sequence of roX2 RNA. Although more experiments are required to test if these two stem–loop structures found in roX1 and roX2 RNAs have a similar function, it is possible that those two stem loops with similar nucleotide motifs (e.g., GUb at 3' stem) might explain the functional redundancy between roX RNAs despite no apparent resemblance otherwise.
In search of more clues for functional redundancy between roX RNAs, we attempted to find GUUNUACG motifs in the roX1 RNA. Upon detailed analysis of several roX1 RNA sequences from different fly species, we identified three GUb's in the 3' end of roX1 RNA in eight Drosophila species (Figure 3, A and G). The second GUUNUACG (GUb-2) found in roX1 RNA is a previously identified 25/30-nt region (FRANKE and BAKER 1999). Similar to GUb-2 of roX2 RNA, deletion of this GUb-2 did not affect function of roX1 RNA (STUCKENHOLZ et al. 2003), which might also be attributed to the presence of other GUb's in the roX1 RNA. D. ananassae and D. virilis contain another GUb sequence (total of four GUb's) and D. mojavensis contains two more GUb sequences (total of five GUb's) in the more upstream region of roX1 RNA (supplemental Figure S1 at http://www.genetics.org/supplemental/). However, an evolutionarily conserved 5' stem sequence around the GUb regions of roX1 RNA has not yet been identified in eight Drosophila species. This suggests several possibilities. Alignment imperfection may have not enabled us to detect the conserved 5' stem sequence. It is also possible that this region functions as a sequence without secondary structure or that other distantly related Drosophila species (for example, D. mojavensis and D. virilis) have lost their secondary structure around the GUb regions during evolution, such as the stem–loop structure identified previously (Figure 3E) (STUCKENHOLZ et al. 2003). At this point we are not certain if these GUUNUACG sequences are necessary for the function of roX1 and roX2 RNAs. However, it is interesting that they are evolutionarily conserved in the 3' ends both of roX1 and of roX2 RNAs, which are functionally redundant in spite of low similarity between total sequences and a low homology between several Drosophila species.
A stem–loop region of roX2 RNA alone can induce the X chromosome-specific binding of the MSL complex and H4-Lys16 acetylation:
To determine the functional importance of the putative stem–loop region of roX2 including a GUb sequence at its 3' stem, we made tandem repeats of the stem–loop region of roX2 RNA (W-SL-6) under the control of constitutive promoter (hsp 83) (Figure 4A). The size of W-SL-6 RNA is 432 nt (72 nt x 6), similar to the major isoform of roX2 RNA (500 nt) (PARK et al. 2005). RT–PCR analysis confirmed that the W-SL-6 construct expresses a transcript including SL-6 RNA (Figure 4B, lane 4) in roX– mutant flies (see MATERIALS AND METHODS). However, the steady-state level of W-SL-6 RNA in the W-SL-6 transgenic flies was much lower than that of roX2 RNA from wild-type roX2 transgenic flies even though both transcripts are expressed from the hsp83 promoters (Figure 4C), suggesting that the other parts of roX2 might be required for the stability of the RNA.
Interestingly, in the polytene chromosome of W-SL-6 transgenic flies we found that all five MSL proteins were detected not only on the autosomal transgenic location (three different locations tested, arrow in Figure 4D), but also on the X chromosome, indicating that SL-6 itself is sufficient to attract MSL proteins to the site of its own transcription and then target the MSL complex to the X chromosome. Unlike the other MSL proteins (MSL1, MSL2, MSL3, and MOF), which showed strong and consistent signals on the X chromosome in all nuclei (Figure 4D), MLE showed heterogeneous staining with variable degrees of intensity (Figure 4D). To compare binding efficiency of W-SL-6 and wild-type roX2 RNAs with MSL proteins, we performed double staining of MSL3 with either MOF or MLE protein in the polytene chromosome of each transgenic fly (Figure 4E). First, the numbers of nuclei showing MSL3 staining were counted and next, double staining (MSL3 + MOF or MSL3 + MLE) from them was counted. The percentage of double staining was calculated [(MSL3 + MOF)/MSL3 or (MSL3 + MLE)/MSL3]. Even though it is slightly lower in W-SL-6 transgenic lines, the percentages of double staining of MSL3 + MOF and MSL3 + MLE were comparable in W-SL-6 and wild-type roX2 RNAs (Figure 4E), suggesting that all five MSL proteins are assembled with the W-SL-6 RNA. As shown in individual staining (Figure 4D), MLE binding to the X chromosome in W-SL-6 transgenic flies was heterogeneous with variable intensity unlike consistent binding of MSL3 within every nucleus in double staining (data not shown), which implies that weaker staining of MLE in some nuclei is not due to the weak binding of other MSL proteins. At this point we do not know yet whether a more efficient interaction between MLE (RNA helicase) and other MSL proteins may require other regions of roX2 RNA outside of the stem loop and then the instability of W-SL-6 RNA (Figure 4C) may be caused by heterogeneous binding of MLE. Interestingly, the MSL complex assembled with the SL-6 RNA was able to induce H4-Lys16 acetylation on the X chromosome and the autosomal transgene (Figure 4, D and E), although the percentage of double staining of MSL3 and H4-Lys16 acetylation [(MSL3 + H4lys16Ac)/MSL3] in W-SL-6 RNA (57%) was a little lower than that of wild-type roX2 RNA (80%).
To analyze the function of W-SL-6 RNA in flies, a rescue assay was performed by expressing W-SL-6 transgenes (four independent lines) in the roX– mutant and counting survival of male flies (Figure 4F). In contrast to the immunostaining results including positive histone H4 lysine 16 acetylation on the X chromosome (Figure 4, D and E), a rescue frequency of SL-6 was low (17%) compared to that of the wild-type roX2 transgene (80%). One explanation for this partial rescue efficiency of the W-SL-6 transgene is that it might be caused by low stability of W-SL-6 RNA (Figure 4C) due to the absence of other parts of roX2 RNA in SL-6, which are necessary for better interaction with MSL proteins.
To confirm the specific interaction between the stem–loop of roX2 RNA and MSL proteins, we tested other tandem repeats (hexamers) that have mutations in a GUb sequence (Figure 3F) of the 3' stem region (M-SL-6) or an antisense transcript of stem–loop (A-SL-6) (Figure 4A). These hexamers did not either attract MSL proteins or induce H4-Lys16 acetylation (Figure 4D). In addition, they showed low frequency for rescue (2.2%) of the roX– deficiency male, which is similar to no transgenic control (1.5%). Considering the moderate rescue frequency of wild-type hexamer (W-SL-6, 17%), this result suggests that a GUb sequence or stem–loop structure (or both) within roX RNA plays an important role in the interaction with MSL proteins.
Although roX1 (3700 nt) and roX2 (500 nt) RNAs are apparently different in size and primary sequence, they function redundantly in dosage compensation on the Drosophila X chromosome (FRANKE and BAKER 1999; MELLER and RATTNER 2002), suggesting that they share common functional domains. In several Drosophila species, male-specific binding to the X chromosome by roX RNAs is evolutionarily conserved (Figure 2), indicating that roX RNAs keep common functional domains despite evolutional change as noncoding RNA (40 million years apart, Figure 1A). Considering low homology in total sequences and no cross-hybridization between D. melanogaster and other distant Drosophila species (Figure 1), functional domains could be short primary sequences and/or the secondary structures. Using a comparative evolutionary approach, we successfully found stretches of conserved motifs (GUb) and putative stem–loop structures within the roX RNAs from several different Drosophila species (Figure 3).
roX1 and roX2 double-mutant males die from failure of dosage compensation on the X chromosome in contrast to females that suffer no harmful effects. In the roX-deficient male fly, MSL proteins show little to no ability to localize to the X chromosome and mostly mislocalize to the heterochromatic chromocenter (MELLER and RATTNER 2002). These observations suggest that roX RNAs are important for accurate targeting of the MSL complex to the X chromosome. It is unknown how MSL proteins interact with roX RNAs to make the MSL complex functional or if roX RNAs regulate enzymatic activity of MSL proteins. However, our data showed that the conserved stem–loop region of roX2 is a core functional domain sufficient to attract MSL proteins, assemble MSL complexes, and target them to the X chromosome, followed by subsequent acetylation of histone H4 lys16 on the X chromosome (Figure 4). This suggests a possibility that roX RNA is required not only to assemble the MSL complex, but also to regulate enzymatic activity of MSL proteins by the conserved stem–loop region. A more detailed study about the conserved functional domains of roX RNAs will reveal how noncoding RNA regulates protein components in a ribonucleoprotein complex for chromatin organization.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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FOOTNOTES |
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2 Present address: Doctoral Programs in Molecular BioSciences, Rutgers University, Piscataway, NJ 08854.
3 Present address: HHMI, Waksman Institute, Rutgers University, Piscataway, NJ 08854.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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