Colaboration with Prof. Dr. Michael Sattler, Prof. Dr. Ketting and Dr. Jean-Yves Roignant
PROJECT INFORMATION :
RNA-binding proteins (RBPs) are critical players in the posttranscriptional control of gene expression and regulate various mRNA processing steps, including splicing, 3’ end processing and translation. Most often, multiple RBPs come together to form large messenger ribonucleoprotein particles (mRNPs), and the cooperative action of these mRNPs controls the fate and function of each transcript. However, the molecular features that define this so-called mRNP code in a given functional context remain poorly understood.
Here, we propose to combine transcriptome-wide approaches using in vitro and in vivo iCLIP with biophysical and structural studies to unravel the molecular mechanisms of mRNP assembly at cis elements in the 3’ splice site of human introns. A pioneering event in splice-site recognition is binding of the U2 auxiliary factor (U2AF) heterodimer with its subunits U2AF65 and U2AF35 to the polypyrimidine (Py)-tract and the invariant 3’ splice site AG motif (3’ AG), respectively. Intriguingly, the Py-tracts in human introns exhibit large sequence variations, but how these variations influence an intron’s splicing competence remains poorly understood. Py-tract recognition by U2AF65 is frequently modulated by other RBPs, including U2AF35 as well as splicing factor 1 (SF1), which binds to the upstream branch point sequence (BPS). Moreover, splicing fidelity is enhanced by proof-reading against binding of U2AF to cryptic Py-tracts without 3’ AG. Understanding the molecular mechanisms of 3’ splice-site recognition and specifically the role of U2AF35 is of high importance, since mutations in this RBP are associated with human disease.
The goal of this project is to dissect the dynamic interplay of RBPs at the 3’ splice site. In particular, we will address the following key questions: 1) What are the rules of U2AF binding at the highly diverse 3' splice sites in human introns? What is the impact of U2AF35 and its disease-linked mutations on mRNP assembly? 2) What is the contribution of SF1 binding and branch point recognition to U2AF recruitment during mRNP assembly? What renders a 3' splice site sensitive to the presence of SF1 and how is this modulated by SF1 phosphorylation? 3) What are the molecular mechanisms of proof-reading and the removal of erroneously assembled mRNPs at 3' splice sites?
Bridging the fields of structural biology and functional genomics, our approach will help to decipher the mRNP code of 3’ splice-site recognition and splicing regulation - from the mechanistic principles of combinatorial and dynamic RBP binding at the molecular level to the functional consequences of mRNP assembly in living cells. Beyond splice-site recognition, our results will offer a blueprint for understanding the assembly of other mRNPs. Given the unique combination of complementary methods employed, we expect numerous interactions with other researchers within the SPP1935.
Summary of your Project 2 (with Jean-Yves Roignant and Rene Ketting):
Gene expression during early development from fruit fly to human depends almost exclusively on posttranscriptional mechanisms. Maternally inherited mRNAs are translated during oocyte maturation, when the cell enters the meiotic division, and during embryogenesis. Many mRNAs in oocytes have short poly(A) tails and require specific mechanisms, such as cytoplasmic polyadenylation, to initiate translation. Moreover, maternal mRNA degradation serves as a crucial checkpoint during zygote genome activation. While the physiological functions of these posttranscriptional processes are well understood, the responsible RNA-binding proteins (RBPs) and their underlying mechanisms remain elusive.
An interesting RBP family in this context are the Makorin (Mkrn) proteins which are highly conserved throughout the animal kingdom. They are characterized by 1-3 RNA-binding C3H Zn finger modules combined with an E3 ligase domain. Previous studies and our preliminary work show that Mkrn expression is highly enriched in ovaries and early embryos of fruit flies, zebrafish and human. Strikingly, we found that mkrn1 knockout flies are viable but sterile and display severe oogenesis defects, while its partial loss of function affects early embryonic development. On the molecular level, Mkrn1 was shown to act as a ubiquitin ligase and suggested to function in mRNA metabolism. Our preliminary results confirmed the previously reported association of MKRN1 with the poly(A) binding protein PABPC1 in human cells and identified interactions with further regulators of translation and mRNA stability, including LARP1, IGF2BP1 and ELAVL1. Moreover, our initial iCLIP experiments show that MKRN1 specifically binds to internal A-tracts in the 3’ UTRs of distinct target mRNAs. We hypothesize that by forming a ternary complex with PAPB on internal A-tracts, Mkrn mRNPs could facilitate a polyA tail-independent PABP activity, impacting e.g. on mRNA translation or stability. The role of Mkrn mRNPs could be particularly important during oogenesis and in early developmental stages, when short polyA tails on maternal RNAs coincide with a peak in mkrn expression that is evolutionarily conserved across animals.
In this proposal, we want to unravel the function of Mkrn mRNPs in posttranscriptional regulation during oogenesis and early development. Combining well-established model organisms for developmental studies with state-of-the-art ribonomics and proteomics approaches, we will systematically address the following key questions: (1) What is the physiological role of Mkrn proteins in Drosophila and zebrafish? (2) What is the molecular function of human MKRN1 in posttranscriptional regulation? (3) What are the components and functional interactions within the MKRN1 mRNP? (4) Are Mkrn mRNP composition and function conserved across animals? Our approaches will enable us to obtain detailed insights into Mkrn mRNP assembly and function and its contribution to oogenesis and early embryogenesis.
KEY TECHNOLOGIES :
- iCLIP (in vivo and in vitro)
- Reporter gene assays
M. Tajnik, A. Vigilante, S. Braun, H. Hänel, NM. Luscombe, J. Ule, K. Zarnack$ and J. König$ (2015) Intergenic Alu exonisation facilitates the evolution of tissue-specific transcript ends. NAR doi: 10.1093/nar/gkv956 (Pubmed)
I. Huppertz, J. Attig, A. D’Ambrogio, LE Easton, CR. Sibley, Y. Sugimoto, M. Tajnik, J. König$, J. Ule$ (2014) iCLIP: Protein–RNA interactions at nucleotide resolution. Methods 65: 274-287 (Pubmed)
K. Zarnack*, J. König*, M. Tajnik, I. Martincorena, S. Eustermann, I. Stévant, A. Reyes, S. Anders, NM. Luscombe, J. Ule (2013) Direct Competition between hnRNP C and U2AF65 Protects the Transcriptome from the Exonization of Alu Elements. Cell 152: 453-466 (Pubmed)
J. König, K. Zarnack, NM. Luscombe, J. Ule (2012) Protein-RNA interactions: new genomic technologies and perspectives. Nat Rev Genet 13: 77-83 (Pubmed)
J. König*, K. Zarnack*, G. Rot, T. Curk, M. Kayikci, B. Zupan, DJ. Turner, NM. Luscombe, J. Ule (2010) iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol 17: 909-915 (Pubmed)