Function and regulation of TIS granulesWe are currently identifying the mRNAs that are enriched in TIS granules in order to find new functions of TIS granules. Furthermore, we are investigating how the formation of TIS granules is regulated. Another focus of our research is to examine what roles mRNAs play in defining TIS granule properties. One of our goals is to find additional membraneless organelles that compartmentalize the cytoplasm.
Functions of constitutive 3′UTRsIn the last few years, our focus was to identify functions of alternative 3′UTRs. However, as every mRNA has a 3′UTR, we wondered about general functions of 3′UTRs. To approach this, we currently study the functions of constitutive 3′UTRs (meaning that the mRNA only produces a single 3′UTR isoform). To do so, we use CRISPR technology to delete 3′UTRs of mRNAs at the endogenous locus to study 3′UTR functions in vivo (Mayr, 2018a). For selecting the genes of interest, we focus on 3′UTR functions that go beyond the regulation of protein abundance.
How widespread is the function of 3′UTR-mediated protein complex formation?
To address how widespread is the regulation of 3′UTR-mediated protein complex formation, we are performing a screen using the yeast Saccaromyces cerevisiae. Regulation by 3′UTRs has largely been disregarded by yeast researchers. One reason is that it is thought that 3′ end processing in yeast is rather imprecise and that the variation of sequence at the 3′ end is probably inconsequential. Another reason is that 3′UTRs in yeast are quite short (median length, 120 bp), and thus, may not contain many regulatory elements.
Our interest in yeast 3′UTRs started when we obtained strong evidence showing that 3′UTRs do not need to be long in order to have important functions (Ma, 2018). 3′UTRs that are only 42 nucleotides long can have very strong effects if they contain the correct sequence elements or structure elements.In order to map 3′UTR boundaries in yeast with high confidence, we performed 3′-seq of yeast in various stress condition. Surprisingly, we found that there is substantially less heterogeneity in 3′ end formation than previously thought. Together with the fact that short 3′UTRs can have important function, we think that it is highly likely that yeast 3′UTRs have important regulatory roles. We are currently using the information on yeast 3′UTR boundaries to set up a large scale screen to assess how widespread 3′UTR-mediated protein complex formation is in yeast. We are also mapping alternative 3′UTR isoforms in yeast and are identifying genes whose alternative 3′UTR ratios change in different stress conditions.
Cell atlas of 3′UTR isoform usageWe previously established a quantitative tag-based sequencing protocol to map 3′UTR boundaries and 3′UTR isoform usage in several tissues, cell types, and cell lines (Lianoglou, 2013). Our study was chosen by Science Signaling as one of the Signaling Breakthroughs of 2013 (Science Signaling). Following up on this initial study, we examined intronic polyadenylation (IPA) isoform expression in 60 diverse samples, including a large number of normal and malignant immune cell types (Singh, 2018, Lee, 2018a). As 3′UTR isoform expression is highly cell type-specific, we are currently expanding our atlas to include various datasets derived from single cell sequencing approaches. Our goal is to obtain a comprehensive cell atlas of 3′UTR isoform usage and to learn about the distribution of alternative 3′UTR isoforms in single cells to obtain insights into the regulation of alternative cleavage and polyadenylation.