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Functional characterization of the chaperone network connected to the eucaryotic ribosome

Subject Area Biochemistry
Term from 2008 to 2014
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 50070218
 
Final Report Year 2014

Final Report Abstract

The mechanism of protein synthesis is highly conserved in all kingdoms of life. This does not only apply to the core translational machinery, but also to the network of ribosome-bound protein biogenesis factors (RPBs), which coordinate the early steps of protein biogenesis. For example, many of the RPBs of the yeast Saccharomyces cerevisiae possess close homologs in mammalian cell. However, on closer inspection there is significant differences with respect to the nature of RPBs, as well as with respect to the mechanism of RPB action. In the course of FOR 967 we have performed a comparative analysis of RPBs, which are conserved from yeast to man. First, we compared properties and function of the yeast dimeric ribosome-associated complex (RAC) with mRAC, its mammalian counterpart. Second, we analyzed the interplay of signal recognition particle (SRP) with nascent chain-associated complex (NAC) in yeast and the interplay of SRP with the argonaute protein Ago2 in mammalian cells. Yeast RAC and mammalian mRAC interact with ribosomes directly and affect the biogenesis of newly synthesized polypeptide chains. However, on a mechanistic level RAC and mRAC differ significantly. While RAC is a cochaperone partner of a specific ribosome-bound Hsp70 termed Ssb, mRAC is a cochaperone partner of cytosolic Hsc70 and Hsp70, which do not bind to ribosomes directly. We found that one function of mRAC in mammalian cells is to recruit specifically Hsp70 to translating ribosomes. Such a role is not performed by RAC, because Ssb binds to ribosomes independently of RAC. We found that one subunit of mRAC, termed MPP11/ZRF1, serves an additional, extra-ribosomal function in the nucleus of mammalian cells. Here, MPP11 cooperates with the histone H2A deubiquitinase USP21 in the regulation of polycomb-repressed genes. This role of MPP11 in transcriptional regulation strongly suggests that in mammalian cells the MPP11-subunit of the mRAC complex acquired novel, important functions during evolution. We found that a signal anchor (SA) sequence inside the ribosomal tunnel folds into α-helical conformation. Ribosomes, which contain such α-helical SA sequences, recruit SRP to the tunnel exit even before the SA sequence becomes accessible from the outside. We analyzed the interplay of yeast NAC and SRP on translating ribosomes and found that ribosomes carrying nascent chains containing SA sequences interact with NAC and SRP in a coordinated manner, giving rise to NAC•RNC•SRP complexes, which are targeted to the ER membrane. In the mammalian system we discovered a functional interplay between SRP and the argonaute protein Ago2, which is not conserved in Saccharomyces cerevisiae. In mammalian cells the two RPBs compete for ribosomes carrying secretory nascent chains. While SRP binds to ribosomes carrying nascent chains containing a wild type signal sequence with higher affinity, Ago2 preferentially binds if mutations within the signal sequence hamper the interaction with SRP. Ago2 binding then triggers the decay of the corresponding mutated mRNA molecule. By that Ago2 not only prevents the translation of proteins with defective targeting information but also prevents further rounds of translation of the defective mRNA.

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