State-of-the-art and preparatory work

ATP‐dependent chromatin remodelers play an important role in regulating gene transcription (1). It is becoming increasingly clear that chromatin remodelling, transcription and RNA processing occur simultaneously and that all three are intimately linked.

During the past funding period we have characterised the novel ATP‐dependent chromatin remodeler dChd3 of Drosophila (2, 3). Recently, we have found that both dChd3 and the related dMi‐ 2 remodeler are recruited to heat shock (HS) genes. HS gene activation is impaired in transgenic flies expressing reduced levels of dMi‐2 or a dominant‐negative dMi‐2 mutant, arguing that dMi‐2 plays a positive role during HS gene transcription. dMi‐2 is recruited to the transcribed region of the hsp70 gene within minutes following HS. Several observations suggest that dMi‐2 is brought to HS genes via an interaction with nascent RNA: (i) Recruitment is abrogated in the presence of the RNA pol II inhibitor α‐amanitin, (ii) dMi‐2 binds RNA in vitro and in vivo, and (iii) dMi‐2 association with actively transcribed genes is sensitive to RNase treatment.

Our findings raise the possibility that chromatin remodelers are directly involved in RNA transcription and processing. Indeed, identification of dMi‐2 interacting proteins by immunoaffinity purification has identified two proteins with a predicted role in splicing (CG6418: DEAH‐box RNA helicase; CG5931: DExH‐box RNA helicase, homologue of the yeast U5 snRNP associated Brr2) and one protein with a predicted role in 3’ end formation (CG7185: homologue of human CFlm‐68K, a component of the 3’ processing machinery). More importantly, transgenic flies expressing reduced levels of dMi‐2 show a defect in 3’ processing of hsp70 RNA. To uncover this defect we have used a system established in the lab of our collaborator from Moscow, S. Georgieva (4).

References

1. Kunert, N. & Brehm, A. (2009) Novel Mi‐2 related ATP‐dependent chromatin remodelers. Epigenetics 4, 209‐211.

2. Murawska, M., Kunert, N., van Vugt, J., Langst, G., Kremmer, E., Logie, C. & Brehm, A. (2008) dCHD3, a novel ATPdependent chromatin remodeler associated with sites of active transcription. Mol Cell Biol 28, 2745‐2757.

3. Kunert, N., Wagner, E., Murawska, M., Klinker, H., Kremmer, E. & Brehm, A. (2009) dMec: a novel Mi‐2 chromatin remodelling complex involved in transcriptional repression. Embo J 28, 533‐544.

4. Kopytova, D .V., Orlova, A. V., Krasnov, A.N., Gurskiy, D. Y., Nikolenko, J. V., Nabirochkina, E. N., Shidlovskii, Y. V. & Georgieva, S. G. (2010) Multifunctional factor ENY2 is associated with the THO complex and promotes its recruitment onto nascent mRNA. Genes & Dev 24, 86‐96.

Aims

The overall aim of this project is to define the roles of dMi‐2 in RNA processing. In particular, we aim to identify dMi‐2‐interacting components of the RNA processing machinery and to define the role of dMi‐2 during splicing and 3’ end formation.

Work programme and methods

Interaction of dMi‐2 with RNA processing components – candidate approach

We will raise antibodies against the three dMi‐2‐interacting RNA processing proteins described above and verify their association with dMi‐2 by coimmunoprecipitation. In addition, we will use available antibodies against RNA processing components. Towards this end we have obtained five monoclonal antibodies directed against nuclear RNP components from H. Saumweber (Berlin). In particular, we will collaborate with S. Georgieva (Moscow) to assess if subunits of THOC bind dMi‐2. THOC is involved in RNP biogenesis. S. Georgieva has recently shown that ENY2 is a common subunit of a chromatin regulating complex (SAGA) and THOC, highlighting the tight link between chromatin regulation and RNA processing. Her lab has raised antibodies against THOC subunits that we will use. In a complementary approach we will purify RNPs and test preparations for the presence of dMi‐2.

Interaction of dMi‐2 with RNA processing components – systematic approach

We have developed highly specific monoclonal antibodies against dMi‐2 and dChd3. We will use these for immunoaffinity purification of dMi‐2‐ and dChd3‐associated proteins. We will use state‐ofthe‐art mass spectrometry methods to identify interacting proteins.

Role of dMi‐2 in 3’ end processing

Our preliminary results show that hsp70 3’ end processing in flies expressing reduced amounts of dMi‐2 is disturbed. We will extend this study to other HS and non‐HS genes. We will also analyse if an involvement in hsp70 3’ end formation is unique to dMi‐2 or shared by dChd3 and other remodelers. We will make use of established transgenic fly lines and cell lines treated with RNAi.

Role of dMi‐2 in splicing

HS genes do not contain introns and are not suited to investigate splicing. Like the HS genes, four intron‐containing metallothionein (Mtn) genes are dramatically activated following (metal ion) stress. We will first verify that dMi‐2 is recruited to Mtn genes upon activation. Then, we will use RT‐qPCR to determine the ratio of correctly and incorrectly spliced Mtn transcripts in RNAi‐treated cells and transgenic flies. If successful, these studies will be extended to other chromatin remodelers. If a role of dMi‐2 in splicing can be firmly established, we will determine the effect of RNAi‐mediated dMi‐2 depletion on splice site selection by RNA Seq. For this, we will collaborate with A. Bindereif.

Titles for dissertations (prospective)

• Characterisation of RNA binding and processing functions of dMi‐2

Relationships/connections within the research training group

Georgieva: Analysis of dMi‐2 – THOC interactions

Bindereif: dMi‐2 and splicing

Hartmann: dMi‐2 RNA binding activity

Benefits of the scientific exchange

Georgieva: Exchange of reagents and expertise in analysing RNA processing in Drosophila

Bindereif: Expertise in acquiring and interpreting RNA Seq splicing data

Hartmann: Expertise in studying RNA‐protein interactions in vitro