Prof. Dr. Ketting
Colaboration with Dr. Jean-Yves Roignant
PROJECT INFORMATION :
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 :
- Model systems: zebrafish and C. elegans
- Small RNA sequencing and other NGS approaches
de Albuquerque BF, Placentino M, Ketting RF. (2015)
Maternal piRNAs Are Essential for Germline Development following De Novo Establishment of Endo-siRNAs in Caenorhabditis elegans.
Dev Cell. 34(4):448-56.
de Albuquerque BFM, Luteijn MJ, Cordeiro Rodrigues RJ, van Bergeijk P, Waaijers S, Kaaij LJT, Klein H, Boxem M and Ketting RF (2014)
PID-1 is a novel factor that operates during 21U-RNA biogenesis in Caenorhabditis elegans
Genes Dev. 28(7):683-8
Kamminga LM, Luteijn MJ, den Broeder MJ, Redl S, Kaaij LJ, Roovers EF, Ladurner P, Berezikov E, Ketting RF (2010)
Hen1 is required for oocyte development and piRNA stability in zebrafish.
EMBO J. 29(21): 3688-700
van Wolfswinkel JC, Claycomb JM, Batista PJ, Mello CC, Berezikov E, Ketting RF (2009)
CDE-1 affects chromosome segregation through uridylation of CSR-1-bound siRNAs
Houwing S, Kamminga LM, Berezikov E, Cronembold D, Girard A, van den Elst H, Filippov DV, Blaser H, Raz E, Moens CB, Plasterk RH, Hannon GJ, Draper BW, Ketting RF (2007)
A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in Zebrafish