Periodic Reporting for period 2 - RNAloc (Epithelial mRNA localization in homeostasis and pathophysiology)
Reporting period: 2022-08-01 to 2024-01-31
Messenger ribonucleic acid, or mRNA, is generated in the cellular nucleus by transcribing genetic information from DNA and is then transported into the cytoplasm where it is translated into functional proteins. Asymmetric distribution of mRNA within the cytoplasm can be widely observed across cell types and species, and we recently described it for the specialized epithelial cells of the digestive system. This mRNA asymmetry appears to parallel the characteristic apical/basal polarization seen in epithelial cells, which encompasses cytoskeletal components, proteins, and phospholipids. However, the functional significance of mRNA localization in physiology and cancer, as well as the molecular regulation of this process remains unclear. We aim to unravel the intricate mechanisms behind this phenomenon and understand its implications for cellular functions, particularly in the liver and intestinal tract. These organs are especially of interest as they are key players in metabolism, play crucial roles in our overall health, and frequently become sites of tumorigenesis. To achieve this, we employ cutting-edge bioengineering and imaging techniques, creating an extensive spatial map that details the subcellular location of both mRNA and proteins within digestive epithelia. These maps will provide insights into how the distribution of these molecules relates to organ function and the development of cancer. A key objective is understanding how mRNA molecules are transported within cells and how their specific locations are maintained. This process typically involves RNA-binding proteins (RBPs), which bind to specific mRNA transcripts and guide them to their designated positions. However, many details about this process, such as which RBPs bind to which transcripts and why, remain poorly understood. Our research delves into both mRNA localization and the functions of RBPs. We are particularly interested in a protein called Adenomatous polyposis coli (APC), known to be frequently mutated in colorectal cancer and likely serving as an RBP. We are investigating APC's role in mRNA binding and localization and its potential connection to colorectal cancer development. By leveraging advanced techniques and interdisciplinary approaches, we seek to contribute to our understanding of cellular dynamics and disease mechanisms.
We first set out to create a spatial map for mRNA transcript within intestinal epithelia to understand which transcripts are predominately apical (= facing the intestinal lumen). To this end, we adopted multiplexed error-robust fluorescence in situ hybridization (MERFISH) an image-based method to determine the localization of mRNA transcripts at subcellular resolution. In addition, we carried out a high-throughput, sequencing-based approach. To this end, we create a fusion protein of an APEX2, and an enzyme that can label mRNA transcripts in its immediate vicinity, and DPP4, a protein that localizes to the apical domain of intestinal epithelial cells. With this approach, we identified mRNAs that are enriched in the apical domain in different murine epithelial cells. These transcripts were often associated with metabolic processes related to nutrient sensing and digestion, which indicates a functional importance for their asymmetric distribution.
In parallel, we studied which specific nucleotide sequence motifs of mRNAs mediated their transport and localization. To this end, we used cultured neuronal cells as a model system and devised a high-throughput screening approach for nucleotide sequences that promote localization to neurites. We uncovered localizing motifs, generated a computational model to predict localization for artificial nucleotide sequences, and identified RBPs that can bind such sequences and may therefore mediate localization. We are applying what we learned from this neuronal model to intestinal epithelial cells and trying to find RBPs that bind to specific mRNA sequences and may mediate apical localization. We are also expanding our work to tumor models. To this end, we are using organoids, which are self-assembling spheroid cultures of cells that mimic tissues, to simulate colorectal cancer tissues. With this model, we have already discovered that the localization of some RNA species is perturbed in colorectal cancer compared to a healthy colon. We will delve deeper into the mechanism and consequences of this disturbance in cellular structure in the upcoming work of this project.
In parallel, we studied which specific nucleotide sequence motifs of mRNAs mediated their transport and localization. To this end, we used cultured neuronal cells as a model system and devised a high-throughput screening approach for nucleotide sequences that promote localization to neurites. We uncovered localizing motifs, generated a computational model to predict localization for artificial nucleotide sequences, and identified RBPs that can bind such sequences and may therefore mediate localization. We are applying what we learned from this neuronal model to intestinal epithelial cells and trying to find RBPs that bind to specific mRNA sequences and may mediate apical localization. We are also expanding our work to tumor models. To this end, we are using organoids, which are self-assembling spheroid cultures of cells that mimic tissues, to simulate colorectal cancer tissues. With this model, we have already discovered that the localization of some RNA species is perturbed in colorectal cancer compared to a healthy colon. We will delve deeper into the mechanism and consequences of this disturbance in cellular structure in the upcoming work of this project.
Our project pushes the boundaries of our understanding of mRNA localization, RNA-binding proteins, and their roles in cellular processes. We are advancing the current knowledge of mRNA localization and RNA-binding proteins, shedding light on fundamental cellular processes that were previously poorly understood. Understanding the physiological function of intestinal epithelia may have broader implications for human health, particularly in metabolic diseases and digestive disorders. Beyond this, our work in tumor models may have translational implications. By investigating how RNA localization is linked to tumorigenesis, such as in colorectal cancer, we may illuminate the mechanisms that trigger the transformation from a normal epithelial cell to a malignant one. Colorectal cancer ranks as the world's second most common cause of cancer-related deaths, and its incidence is on the rise in developed countries. Therefore, gaining a better understanding of colorectal cancer biology, its prevention, and its treatment has significant potential for socioeconomic impact.