Cytoplasmic localization of mRNAs is a widely distributed process and contributes to protein sorting, asymmetric cell division, or neuronal function. In budding yeast, several mRNAs are asymmetrically distributed between mother and bud. Localisation of one of them, ASH1 mRNA to the bud cortex is essential for the asymmetric distribution of the encoded transcription factor Ash1p that regulates mating type switching in budding yeast. mRNA localisation depends on cis-acting signals (localisation elements) in the RNA, on a functional microfilament system, and a number of specific proteins. A myosin V, Myo4p is acting as the molecular motor for cytoplasmic translocation. Association of the motor with the translocated cargo requires the adaptor She3p, which also serves as RNA-binding protein, and a second RNA-binding protein, She2p. Three additional RNA-binding proteins, Khd1p, Puf6p, and Loc1p have auxiliary roles during translocation or down-regulate translation of the mRNA during transport.
In general, RNA-binding proteins are known to regulate different aspects of RNA physiology like RNA stability, translation, or localization. Genome sequencing efforts in recent years revealed a large number of RNA-binding or -regulating proteins whose function we still do not understand. Therefore, several projects in the lab deal with the identification of mRNA targets of novel RNA-binding proteins in yeast.
Localization of messenger RNAs and local protein synthesis contribute to asymmetric protein distribution not only of cytoplasmic but also of membrane or secreted proteins. Since synthesis of the latter protein classes occurs at the rough endoplasmic reticulum (ER), mRNA localization and distribution of ER should be coordinated. However, this coordination is not yet understood. In yeast, a subfraction of localising messenger ribonucleoprotein (mRNP) particles co-migrate with tubular ER structures to the bud. This requires the RNA binding protein She2p, which can directly bind to membranes.
We want to study this novel model for mRNA localization that involves association of the RNA-binding protein She2p and localizing mRNPs with ER tubules and myosin dependent co-transport of tubules and localized mRNPs. To that end, we are combining advanced imaging approaches, in vitro lipid-protein and RNA-protein binding assays and yeast genetics to identify new components of this pathway and to characterize their function.
Translation is one of the most energy-consuming processes in the cell, yet the basis for its efficiency and regulation is still open to investigation. Also, the reasons for the prevalence of different synonymous codons in genomes in spite of no apparent benefit by popular codons of translational speed haven’t been thoroughly explained. A step towards a more mature understanding is the substrate channeling hypothesis, which holds that the ribosome’s propensity to only use “selfmade” aminoacyl-tRNAs is due to recycling on or near active ribosomes. Substrate channeling is the likely basis for the newly discovered effect of “autocorrelation”, whereby eukaryotic transcripts fine-tune their translation for efficiency by clustering codons read by the same tRNA, presumably aiding recycling or preventing diffusion. We want study the function of RNA-binding and ribosome-associated proteins that support the ribosome in ensuring efficient translation and to react to varying environmental conditions. To this end, we have developed qRT-based tRNA quantification protocols to determine the amount of individual tRNAs eluted from affinity-purified ribosomes to follow their usage during translation.
The molecular function and fate of mRNAs are controlled by RNA-binding proteins (RBPs) that assemble with the mRNAs to messenger ribonucleoprotein particles (mRNPs). However, the identification of all interacting proteins of a specific mRNA is still very challenging. Based on the widely-used RNA tagging with MS2 aptamers for RNA visualization, we developed a novel method called RNA proximity biotinylation (RNA-BioID). Here, a modified biotin ligase (BirA*) is tethered to the 3’-UTR of endogenous MS2-tagged RNAs. RNA-associated proteins can then be biotinylated in vivo and subsequently isolated via the biotin label. We have demonstrated the feasibility of this approach by characterizing the dynamic interactome of the conserved β-actin mRNA in mouse embryonic fibroblasts.