Alternative 3’UTRs determine multi-functionality of proteins
We determined the 3’UTR-dependent protein interaction partners for several candidates using SILAC-based quantitative mass spectrometry. For each candidate we found up to 150 3’UTR-dependent protein interaction partners. We were able to validate a large fraction of them, which suggests that 3’UTRs can recruit many different protein interaction partners to the newly translated proteins. We are currently studying the different functions that are carried out by proteins that are in complex with different 3’UTR-mediated interaction partners.
Conservation of 3’UTR-mediated protein functions
We have evidence that alternative 3’UTRs can determine alternative functions of proteins despite having an identical amino acid sequence. We are currently asking if the 3’UTR-dependent protein functions observed in humans are conserved in other organisms.
Evolution of multi-functionality
It is largely unknown how biological complexity of organisms is achieved, as the number of protein-coding genes is similar between humans and worms. However, 3’UTR length correlates with organismal complexity, and human 3’UTRs are more than ten times longer than 3’UTRs in worms. We showed that 3’UTRs can determine protein functions and alternative 3’UTRs can accomplish protein multi-functionality. Therefore, we hypothesize that higher organisms use 3’UTR-mediated protein complex formation to increase their functional complexity. We will test this hypothesis by comparing mouse and human cells; hundreds of genes generate alternative 3’UTRs in humans, but their corresponding mouse genes only generate one 3’UTR isoform.
Regulation of 3’UTR-dependent protein complex formation with a focus on RNA granule biologyDuring 3’UTR-mediated protein complex formation, an RNA-binding protein binds to the long 3’UTR and recruits an effector protein. The effector protein is then transferred onto the newly made protein. We want to understand how the transfer of effector proteins is accomplished. The interaction between the effector protein SET and CD47-LU is based on electrostatic interactions, which can only take place if the two proteins are surrounded by a hydrophobic environment. We postulate that this hydrophobic environment is generated by an RNA granule (Figure 4). This is based on the observation that the interior of RNA granules is hydrophobic. Furthermore, mRNAs within RNA granules were shown to have long 3’UTRs, where mRNAs detected in the cytoplasm have short 3’UTRs. We are currently purifying the components of the co-translational complex that contains the ribosome, the mRNA with a long 3’UTR as well as the newly made protein.
CD47 reduces mitochondrial health and promotes cell death induced by oxidative stress
CD47 is known as a plasma membrane protein that acts as a “don’t eat me” signal and protects cells from phagocytosis by macrophages. Surface CD47 is predominantly generated from the CD47 mRNA with a long 3’UTR (CD47-LU). However, CD47 that was generated by the short 3’UTR (CD47-SU) is expressed in the endoplasmic reticulum and in mitochondria. We observed that CD47-SU makes cells susceptible to oxidative stress–induced cell death. Mechanistically, we found that CD47-SU decreases mitochondrial membrane potential and increases OPA1 cleavage, which impairs mitochondrial health. Thus, depending on the expression levels of the short and long CD47 3’UTR isoforms, cells are either protected or susceptible to oxidative stress–induced cell death.