%0 Generic
%A Ilik, Ibrahim Avsar
%D 2011
%F heidok:12287
%K Dosage compensation
%R 10.11588/heidok.00012287
%T A biochemical study of roX2 interacting proteins
%U https://archiv.ub.uni-heidelberg.de/volltextserver/12287/
%X Dosage compensation is a biological phenomenon where a sexually dimorphic organism balances the inequality in gene expression that results from unequal distribution of sex chromosomes. Different organisms have invented different ways to carry out dosage compensation. For instance, in mammals females transcriptionally down-regulate one of the two X-chromosomes they possess to match the male gene expression. Flies, on the other hand, hyper-transcribe the single male X-chromosome to reach the transcriptional output generated by two X-chromosomes in females. A ribonucleoprotein complex, called the Male Specific Lethal (MSL) complex is essential for fly dosage compensation. It is composed of five proteins and two non-coding RNAs called the roX RNAs. The complex contains at least two enzymes: MOF, an acetyltransferase that specifically acetylates Histone 4 Lysine 16; and MLE, a DNA/RNA helicase. Both enzymatic activities are indispensible for dosage compensation. Evidence coming from genetic studies have shown that male flies lacking both roX RNAs die due to a failure in dosage compensation, although these ncRNAs are redundant in function and only one of the two is enough to rescue male lethality. MLE was shown to be required for the incorporation of these RNAs into the MSL complex although it is not clear if this is the only function of MLE in dosage compensation During the first part of my PhD I carried out a tandem affinity purification to reveal proteins that interact with MLE in an effort to understand its role in dosage compensation. In accord with previous observations, I have not been able to detect any protein that stably interacts with MLE under various purification conditions. This work thus supports the view that MLE is a lone RNA-helicase and is recruited to the X chromosome by its interaction with the roX RNAs. In the second part of my thesis I describe the biochemical purification and analysis of roX2 interacting proteins in vitro. With this approach I have identified MLE and two novel proteins; CG5787 and CG3613 that interact with roX2 RNA specifically. Interestingly, CG5787 and CG3613 were found to co-localize with each other and with MLE on chromatin. CG3613 was also shown to interact with roX2 RNA in vivo validating the initial in vitro approach. By using ChIP analysis I was able to detect CG3613 on high-affinity sites on the X-chromosome, which might indicate that it is recruited to the X-chromosome via roX RNA similar to MLE. CG3613 was further characterized and found to be a phosphoprotein in vivo. CG3613 also co-localizes with RNA polymerase II and is recruited to heat-shock loci after a brief heat-shock, indicating a strong relationship with transcription. Knocking-down CG3613 in flies was lethal, but in S2 cells reduction of its levels lead to the stabilization of intron-containing pre-mRNA suggesting a role in pre-mRNA processing. This work was the first attempt to biochemically define roX2 interacting proteins in flies and shows that the MSL proteins are not the only proteins that roX RNAs interact with. In fact, this study suggests that roX RNAs’ role may be to act as a platform that brings together various proteins in addition to the MSL complex to hyper-transcribe the male X chromosome.