Figure 1: An example of cooperative, highly specific RNA recognition by two general but distinct RNA binding proteins, Sex-lethal and UNR, regulating the translation of msl-2 mRNA during Drosophila dosage compensation (Hennig et al., 2014).
Figure 2: Current model of how translation repression of msl2 mRNA in Drosophila females works to ensure normal transcription of both X chromosomes. Sxl recruits Unr and Hrp48 to the E and F site within the 3′ UTR of msl2. This tight protein-RNA complex prevents the preinitiation complex (43S) to assemble on the 5’UTR. This might occur due to the interaction of Hrp48 with eIF3d. If this fails, Sxl also blocks the 43S at the 5’UTR. Poly(A)-binding protein (PABP) also plays an essential role in this process and likely interacts with Unr.
Figure 3: Our current hypothesis how RNA regulates the ubiquitination function of TRIM25 (orange colours). RNA binds to the PRY/SPRY and coiled-coil domain of TRIM25 simulatenously and mediates ubiquitination efficiency of its substrate (here RIG-I) in antiviral defence. We also hypothesize that viral RNAs and proteins can outcompete endogenous RNAs and block substrate ubiquitination to downregulate the hosts immune response.
The Hennig group employs integrated structural biology (nuclear magnetic resonance (NMR) spectroscopy, X-ray, small-angle scattering and cryo-electron microscopy) to investigate the molecular mechanisms underlying translation regulation and ribonucleoprotein complex assembly.
Previous and current research
Dosage compensation is an essential molecular process in sexually reproducing organisms, which compensates the imbalance of number of sex chromosomes between the sexes. Although these processes can be quite different between species, recent research shows that all have common mechanisms. One of which is the involvement of complexes between proteins and different RNA molecules, mRNA and long non-coding RNAs, often involved in phase separation.
In Drosophila, we study both, the female and male side. In males, we want to understand how exactly the long non-coding RNAs RoX1 and RoX2 are remodelled to allow assembly of the dosage compensation complex (or MSL complex) on the single male X chromosome to achieve 2-fold hypertranscription. This hypertranscription would be lethal in females. Instead, the female-specific protein sex-lethal (Sxl) binds to the mRNA of the MSL complex’ rate-limiting component MSL2, to prevent its translation. The highly conserved protein Upstream-of-N-Ras (Unr) is recruited by Sxl to the same site on the mRNA (Figure1) and, together with Hrp48, essential for translation repression of msl2 mRNA (Figure 2). The male MSL complex is also conserved in humans, where it is regulating autosomal compensation. All RNA binding proteins regulating translation in female flies are conserved in humans, and Unr for example is highly expressed in certain cancer cell lines.
Another main project of the lab revolves around novel RNA binding proteins, meaning proteins, which do not feature a classical RNA binding domain (like RRM, CSD, KH or dsRBD domains), but have been shown to bind single-stranded RNA in mRNA interactome capture. Of special interest to us are RNA binding E3 ligases of the tripartite motif (TRIM) protein family. Here we want to understand how RNA binding is connected to these proteins’ main biochemical function: ubiquitination. Based on our data, we hypothesize that some TRIMs bind to mRNA and regulate translation by ubiquitinating components of translation complexes. Other TRIM proteins and their ubiquitination function seems to be actually regulated by RNA, which we could recently show for TRIM25 (Haubrich et al., BioRxiv, 2020, Figure 3).
Future projects and goals
Our ultimate goals are to obtain high resolution structures of these large protein-RNA complexes validated by biochemical and cell biological experiments to get a detailed molecular understanding of these essential mechanism. To this end, we employ all available structural biology methods (NMR, X-ray crystallography, cryo-EM and small-angle scattering) and more.
We also collaborate on many exciting projects within and outside of the EMBL, where we help out with our NMR and integrative structural biology expertise.