Study of the role of long non-coding RNA ROX in maintaining of the dosage compensation complex in Drosophila melanogaster

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Abstract

The proteins MSL1, MSL2, MSL3, MLE, MOF and non-coding RNAs roX1 and roX2 form the Drosophila dosage compensation complex (DCC), which specifically binds to the X chromosome of males. It is known that non-coding RNA roX are primary component of the DCC in the process of assembly and spreading of the complex among the X chromosome of males. However, it still remains unclear the role of this RNA in maintaining the structure of the already assembled complex. In this work, we have shown that the full-assembled complex of dosage compensation dissociates rather weakly when treated with RNases: the MLE helicase is effectively released from the complex, and the remaining protein components, MSL1, MSL2 and MSL3, undergo partial disassembly and continue to be part of subcomplexes. The results confirm the importance of the non-coding RNA roX2 not only in the processes of initiation of CDK assembly, but also at the stage of maintaining the structure of the already assembled complex.

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About the authors

V. A. Babosha

Institute of Gene Biology RAS

Author for correspondence.
Email: v.babosha@gmail.com
Russian Federation, Moscow

P. G. Georgiev

Institute of Gene Biology RAS

Email: v.babosha@gmail.com

Academician of the RAS

Russian Federation, Moscow

O. G. Maksimenko

Institute of Gene Biology RAS

Email: maksog@mail.ru
Russian Federation, Moscow

References

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2. Fig. 1. Identification of CDK components in a sucrose gradient of 10-40%. (a) Lysate without nuclease treatment , with VNC. (b) Lysate treated with RNase A. (c) Lysate treated with RNase III. Immunoblot analysis was performed with antibodies specifically recognizing the proteins MSL1, MSL2, MSL3, and MLE. The numbers of the collected fractions are marked from 1 to 13 (from "heavy" to "light").

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3. Fig. 2. (a) The results of immunoprecipitation of nuclear extracts isolated from S2 cell culture with antibodies specifically recognizing the MSL2 and MSL3 proteins, or with rabbit immunoglobulins G (negative control). The extracts underwent 3 types of treatment: 1) without nucleases, treated with a non-specific RNase inhibitor VNC, 2) with the addition of RNase A, 3) with the addition of RNase III. Immunoprecipitates were analyzed using immunoblot analysis for the presence of MSL1 protein in the samples. ‘input’ is the initial extract; IP–MSL2 is a sample after immunoprecipitation with antibodies specifically recognizing the MSL2 protein; IP-MSL3 is a sample after immunoprecipitation with antibodies specifically recognizing the MSL3 protein; IP–IgG is a sample after immunoprecipitation with rabbit immunoglobulins G. (b) Detection of roX2 RNA in fractions obtained after analytical ultracentrifugation in a sucrose gradient of 10-40% nuclear extract from S2 cell culture. The dotted line marks the background level. The standard deviations are based on the results of the

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