We could successfully extend funding of the Collaborative Research Centre 960 (SFB960) ‘RNP biogenesis: assembly of ribosomal and non-ribosomal RNPs and control of their function’. To continue our ambitious research programs, we are now seeking highly motivated PhD students. We offer a highly competitive research environment and exciting research projects. For more information click here.
Mutations that alter the activity of RNA-binding proteins or their abundance have been implicated in numerous diseases such as neurodegenerative disorders and various types of cancer. This highlights the importance of RBP proteostasis and the necessity to tightly control the expression levels and activities of RBPs. In many cases, RBPs engage in an auto-regulatory feedback by directly binding to and influencing the fate of their own mRNAs, exerting control over their own expression.
Together with our colleagues Michaela Müller-McNicoll from the Institute of Cell Biology and Neuroscience at the Goethe University Frankfurt, Oliver Rossbach from the Institute of Biochemistry at the Justus-Liebig-University Giessen, and Jingyi Hiu at the State Key Laboratory of Molecular Biology (CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology), we have reviewed RBP-mediated autogenous feedback regulation in eukaryotic organisms. For this feedback control, RBPs employ a variety of mechanisms operating at all levels of post-transcriptional regulation of gene expression to either to maintain protein abundance within a physiological range (exerting negative feedback) or to enforce and stabilize cell fate decisions through generation of binary, genetic on/off switches.
The article has just been published in the Journal of Molecular Cell Biology – click here to read the full version.
Good news: the SFB 960 ‘RNP biogenesis: assembly of ribosomes and non-ribosomal RNPs and control of their function’ has been granted another funding period. We are grateful for the generous support by the Deutsche Forschungsgemeinschaft (DFG) that will allow us and our colleagues to continue and extend our scientific programs and to ensure further top-level education of PhD students within the Graduate Research Academy RNA Biology. We want to thank all reviewers that were involved in the selection process and we are looking forward to another four years of exciting science!
A news article on this topic can also be found on the homepage of the University of Regensburg (in German): DFG verlängert Regensburger Sonderforschungsbereich zur Ribosomen-Entstehung
New manuscript published – RhoA regulates translation of the Nogo-A decoy SPARC in white matter-invading glioblastomas
A collaborative effort lead by Björn Tews and supported by the research consortium ‘Systems Biology of the Unfolded Protein Response in Glioma’ (SUPR-G, generously funded by the BMBF in the framework of the e:med initiative) has resulted in a recent publication in Acta Neuropathologica that demonstrates a function of the peptide SPARC in migration and infiltrative growth of glioblastoma cells. SPARC production and secretion is enhanced via regulation of the UPR sensor IRE1 via AKT. SPARC secretion then prevents Nogo-A from inhibiting migration via RhoA. Advanced ultramicroscopy in undissected mouse brains reveals that gliomas require SPARC for invading into white matter structures and its depletion reduces tumor dissemination which significantly prolongs survival and improves response to cytostatic therapy. The discovery of a novel RhoA-IRE1 axis now provides a druggable target for interfering with SPARC production and underscores its therapeutic value. The full publiation can be accessed here.
New manuscript published – Purification of cross-linked RNA-protein complexes by phenol-toluol extraction (PTex)
We are happy that the collaborative effort spearheaded by Benedikt Beckmann at the Integrated Research Institute (IRI) for the Life Sciences has now resulted in a publication. We have described the approach earlier (see here) which, in a nutshell, allows the purification of cross-linked ribonucleoproteins by a series of organic extractions. Access the full article here at Nature Communications.
New manuscript from the lab published in Nucleic Acids Research: Drosophila Sister-of-Sex-lethal reinforces a male-specific gene expression pattern by controlling Sex-lethal alternative splicing.
In a collboration with the labs of Stefan Schneuwly, Gunter Meister (both at the University of Regensburg), Michael Krahn (Westfälische Wilhelms-Universität Münster), and Oliver Rossbach (Justus-Liebig-University Giessen), we could demonstrate that the protein Sister-of-sex-lethal (Ssx) is required in male flies to suppress production of Sex-lethal (Sxl).
Genomic tagging of the Sex-lethal (Sxl) locus in flies to reveal Sxl protein mis-expression (arrowheads) in male flies mutant for Sister-of-Sex-lethal. Arrows mark expression of a Sxl isoform in neural cell bodies and projections.
Most higher eukaryotes reproduce sexually, increasing the variability in the offspring. This allows e.g. rapid adaption to a new (or changing) environment or the cleansing of harmful mutations from a population. Sexual reproduction in higher eukaryotes usually involves individuals of different sex: males and females. Not surprisingly, the genetic programs that determine sex and control sexual differentiation need to be particularly robust in order to ensure survival of the population.
In Drosophila, a single protein, the master regulator Sex-lethal (Sxl), governs female development by controlling the expression of key factors involved in female morphology and behaviour. Once expressed, it engages in an auto-regulatory, positive feedback loop to ensure its sustained expression. This stably ‘flips the switch’ and commits to female development.
In contrast, in males Sxl expression needs to be shut-off which is achieved by alternative splicing that generates RNA isoforms encoding truncated, non-functional Sxl protein. Fluctuations inherent to gene expression can, however, produce small amounts of functional Sxl protein. When left unchallenged, this protein can trigger a self-enforcing cascade resulting in Sxl protein expression snowballing out of control. Until now, however, it remained unclear how males completely shut off the Sxl expression cascade and protect themselves against runaway protein production to ensure robust sex-specific development.
We have discovered a safeguard mechanism that prevents Sxl production in adult male flies. We identified the protein Sister of Sex-lethal (Ssx) as the first antagonist of Sxl-mediated auto-regulatory splicing that defines a precise threshold level for activation of the auto-regulatory, positive feedback loop that controls Sxl expression. We could show that Ssx exerts function by competing with Sxl for the same RNA regulatory elements thus preventing Sxl from triggering the self-enforcing expression cascade in adult male animals.
The advent of interactome capture has allowed the unbiased identification of RNA binding proteins (RBPs) dramatically expanding their number and yielding novel insights into RNA biology (see also our recent review).
For interactome capture, RBPs are photo-cross-linked to their RNA targets. Subsequently, oligo-dT resin is used to capture polyadenylated RNAs and to co-purify with them the covalently bound proteins. RNAs that lack a ploy(A)-tail can, however, not be captured by this approach, limiting its broad application. In particular, prokaryotic organisms that do not polyadenylate their mRNAs are not amenable to interactome capture.
Now three manuscripts have been uploaded to bioRxiv by the Lilley, Krijgsveld, and Beckmann labs (we contributed to one of them). In all cases extraction with organic solvents is employed to purify cross-linked RNPs (see figure) circumventing the requirement of a poly(A)-sequence for RNP capture. Moreover, this approach also captures RBPs that bind to RNA as short as 30 nt.
The manuscripts can be found here:
Nucleic Acids Research just accepted another manuscript for publication to which we have contributed. In an experimental effort headed by our colleague Sébastien Ferreira-Cerca (University of Regensburg, Biochemistry III), the function of the atypical Rio kinases in ribosomal smal subunit (SSU) biogenesis and maturation was addressed in Archea. This revealed activation of Rio2 by an ancient and conserved mechanism involving ribosomal RNA that stimulates release of the kinase from the nascent 40S particle. Watch out for the next NAR table of contents: there you should find a link to the manuscript, once it is out!
Good news: A manuscript from the lab has just been accepted for publication in the RNA Journal!
We have identified the protein Sister-of-Sex-Lethal (Ssx) as a novel repressor of translation. Ssx is a paralog of the master regulator of female development in Drosophila, Sex-lethal (Sxl), that acts as a repressor of male-specific lethal-2 (msl-2) mRNA translation. It employs two distinct and mutually reinforcing blocks to translation that operate on the 5’ and 3’ untranslated regions (UTRs) of msl-2 mRNA, respectively. While 5’ UTR-mediated translational control involves an upstream open reading frame, 3’ UTR-mediated regulation strictly requires the co-repressor protein Upstream of N-ras (Unr) which is recruited to the transcript by Sxl.
Ssx and Sxl have a comparable RNA-binding specificity and both proteins can associate with Uracil-rich RNA regulatory elements present in msl-2 mRNA. Moreover, both repress translation when bound to the 5’ UTR of msl-2. However, Ssx is inactive in 3’ UTR-mediated regulation as it cannot engage the co-repressor protein Unr. The difference in activity maps to the first RNA-recognition motif (RRM) of Ssx. Conversion of three amino acids within this domain into their Sxl counterpart results in a gain-of-function and repression via the 3’ UTR, allowing detailed insights into the evolutionary origin of the two proteins and into the molecular requirements of an important translation regulatory pathway.
Find the full text here. RNA. 2017 Oct 31. pii: rna.063776.117. doi: 10.1261/rna.063776.117. [Epub ahead of print], PMID: 29089381
Are circular RNAs (circRNAs) translated? It is very intriguing to speculate that (at least some) circRNAs might encode functional peptides. So far however, evidence in support of this theory is weak. One might hypothesize that by non-canonical initiation once in a while a ribosome might indeed translate an open reading frame encoded by circular RNA, whether this yields significant amounts of a functional protein still remains to be demonstrated.
In a collaborative effort with the Bindereif lab at the Justus-Liebig-University of Giessen, we have analyzed the sedimentation of circRNAs in sucrose gradients. If circRNAs are indeed translated this could be revealed by a change of sedimentation behavior after treatment with a drug that releases elongating ribosomes from RNAs (Puromycin). In our experiments however, no significant change in sedimentation behavior of selected abundant circRNPs from HeLa cells could be observed, suggesting that these circRNAs are not (or at best only weakly) translated.
CircRNAs do however associate with proteins to form ribonucleoproteins (RNPs). In our recent publication ‘CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs’ (published in Scientific Reports) we demonstrate that in HeLa cells, circRNAs form distinct, large RNPs. Moreover, we identify the RNA-binding protein and tumor marker IMP3 (IGF2BP3) as a protein component of numerous circRNPs.
(Picture taken from Schneider et al., 2016, Scientific Reports 6, 31313, doi:10.1038/srep31313, CC 4.0)
A special issue from Pflügers Archiv – European Journal of Physiology focusing on the role of RNA in physiology and disease has just been published. It features review articles – many of which are open access – that address diffenrent aspects of and the latest findings in RNA biology. So take some time and indulge yourself with some exciting reading!
From October 10th to 14th 2016, a methods course on ‘Analysis of NextGen RNA-Seq data for expression profiling and protein binding RNAs‘ will take place here in Regensburg. Insightful lectures will be deliverd by reknown experts in the field, including (in alphabetical order) Simon Anders (FIMM Helsinki), Markus Hafner (NIAMS/NIH Bethesda), Steve Hoffman (University of Leipzig), Stefan Kirsch (Fraunhofer ITEM, Regensburg), Charlotte Soneson (University of Zurich), Rainer Spang (University of Regensburg), Nicholas Strieder (University of Regensburg), and Grischa Toedt (EMBL Heidelberg). After the lectures, there will be ample time for hands-on training allowing the participants to gain some practical experience with the latest computational approaches. If you are interested in participating, please register before July 13th.
The course is generously supported by the Graduate Research Academy RNA Biology of the Collaborate Research Center SFB960 ‘Ribosome formation: principles of RNP biogenesis and control of their function’.
Soon a special issue with focus on ‘RNA biology in physiology and disease’ will be published by the European Journal of Physiology (Pflügers Archiv). Together with our colleagues Benedikt Beckmann (IRI for the Life Sciences, Humboldt University Berlin) and Alfredo Castello (University of Oxford) we have contributed a review article entitled: The expanding universe of ribonucleoproteins – of novel RNA-binding proteins and unconventional interactions.
We focus on the recent advances in the identification of novel RNA-binding proteins (RBPs) and the unexpected finding that many of the novel RBPs do not contain identifiable RNA-binding domains (RBDs), raising the question of how they interact with RNA. It is surprising that despite the many functions that have been attributed to RNA, our understanding of ribonucleoproteins (RNPs) is still mostly governed by a rather protein-centric view, leading to the idea that proteins have evolved to bind to and regulate RNA and not vice versa. However, RNPs formed by an RNA-driven interaction mechanism (‘RNA-determined RNPs’) are abundant and offer an alternative explanation for the surprising lack of ‘classical’ RBDs in many RNA-interacting proteins (which we discuss in detail in the review article).