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 biology

During 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.

Regulation of CD47 3’UTR ratios during development, aging, and cancer progression

We showed that CD47-LU has a pro-survival role, whereas CD47-SU promotes cell death. All cells express the short and long CD47 3’UTRs, but at different ratios. Whereas normal cells predominantly express CD47 mRNA with a short 3’UTR, we observed a switch toward increased expression of the long 3’UTR isoform in different types of leukemia (Figure 5). Thus, cancer cells achieve a double advantage for cell survival through a single gene regulatory event. We are very interested in investigating how the switch in CD47 3’UTR isoform expression is accomplished. This has consequences for cancer biology, but also for the treatment of myocardial infarction or stroke, as loss of CD47-SU protects cells from cell death induced by oxidative stress. 

Regulation of 3’UTR-mediated membrane protein localization

We showed that long 3’UTRs enable efficient cell surface localization of several plasma membrane proteins (Figure 6). This is partially due to HuR-dependent recruitment of SET to the newly made proteins. We are interested in identifying other proteins recruited by 3’UTRs that enhance cell surface localization. This will have major implications for the biology of G-protein coupled receptors as well as for cancer immunotherapy.

Regulation of alternative 3’UTR ratios by promoters and enhancers

Alternative 3’UTRs are expressed in a cell type– and gene-specific manner. As promoters are known to regulate gene expression in a cell type– and gene-specific manner, we investigated the influence of promoters and enhancers on polyadenylation (pA) site usage. We established a reporter assay to investigate pA site usage in living cells in the context of transcription and found that regulatory elements located in the DNA and outside of mRNAs regulate proximal pA site usage (Figure 7). Thus, transcription factors are major regulators of pA site usage.  

Contribution of intronic polyA site usage to cancer biology

In more than 20 percent of human genes, intronic polyadenylation (IPA) sites are used to generate alternative 3’ ends. This results in the expression of truncated mRNAs contributes to proteome diversity in normal cells. However, cancer cells can take advantage of this gene regulatory mechanism. We found that leukemia cells (but not their corresponding normal cells) can express truncated mRNAs that result in dominant-negative versions of the wild-type protein (Figure 8). Several genes that generate IPA isoforms belong to the genes that have truncating mutations in leukemia. Whereas some patients have mutations, other patients express the corresponding IPA isoforms.  Interestingly, the functional outcome of the IPA isoforms is similar to the functional outcome of the truncating mutations, which suggests that IPA isoforms can phenocopy DNA mutations. As IPA isoforms can have similar functional effects as genetic mutations, this new type of cancer aberration increases substantially the genes involved in the pathogenesis of cancer; however, so far, they escape detection because they are silent at the DNA level.