The broad goal of our research is to understand the fundamental mechanisms of RNA processing and the machineries that carry out these essential steps in gene expression. It is becoming increasingly clear that defects in components of these machineries underlie numerous diseases, ranging from neurodegenerative disease to cancer. A complete biochemical understanding of the RNA processing machineries will provide a rational path forward for identifying therapeutic strategies for disease. Recently, we identified an important new area of investigation concerning the RNA binding proteins associated with Amyotrophic Lateral Sclerosis (ALS). We discovered that these proteins interact with each other and also with RNA polymerase II (RNAPII) and the splicing factor U1 snRNP. Moreover, we identified a set of proteins, which we designated PALs (Partners of ALS), that associate with the ALS RNA binding proteins and with RNAPII and U1 snRNP. We now plan to determine both the normal and ALS-causative roles of these proteins using a combination of CRSPR- edited proteins in conjunction with in vitro systems for RNA processing that we have developed. Among these are transcription-coupled splicing, transcription-coupled primary microRNA processing, and a robust method for preparing active small-scale nuclear extracts. These and other in vitro studies will be pursued in both HeLa cells and motor neurons, which will be differentiated from CRSPR-edited stem cells. RNA processing dysfunction and RNA targets of ALS/PALs proteins within the motor neurons will be identified by RNA-seq and iCLIP, respectively. The planned analysis of the ALS/PALs proteins will yield valuable information on the functions of these proteins and their associated molecular pathways, which may lead to new avenues for understanding ALS pathogenesis. Recently, several reports revealed that mutations in the U2 snRNP protein, SF3B1, are associated with a variety of cancers. Another key goal of our work is to determine the molecular mechanisms by which these mutations cause the widespread mis-splicing that was reported in RNA-seq studies. We plan to prepare active nuclear extracts from isogenic cell lines containing CRSPR-edited mutant or wild type SF3B1 for assays of U2 snRNP assembly and function. In addition, we plan to use a powerful quantitative mass spectrometry approach to examine protein expression alternations in the isogenic B cell lines. This analysis may reveal specific pathways that are disrupted due to SF3B1 mutation that might otherwise be difficult to detect at the RNA level. Finally, we recently discovered a protein, HNRNPUL1, which links the conserved TREX mRNA export machinery to the ALS-causative proteins. We now plan to determine the functional significance of these interactions via an analysis of both mRNA export and the pathways involving the RNAPII/U1 snRNP/ALS/PALs complex(es). Together, our proposed studies will provide important new insights into the biology of key proteins and pathways involved in diseases for which rapid progress is of the essence.
RNA processing is an essential step in gene expression, and it is now clear that RNA processing underlies numerous human diseases, ranging from Amyotrophic Lateral Sclerosis (ALS) to numerous cancers. Therefore, it is essential to understand both the normal and the disease-causing biology of RNA processing and that is the goal of our research. We have developed robust systems and resources for this work, which will lead to rapid progress towards solving these diseases.
|Yin, Shanye; Iocolano, Alexander; Yu, Yong et al. (2018) In Vitro System for Coupling RNAP II Transcription to Primary microRNA Processing and a Three-Way System for RNAP II Transcription/Splicing/microRNA Processing. Methods Mol Biol 1823:43-50|
|Chi, Binkai; O'Connell, Jeremy D; Yamazaki, Tomohiro et al. (2018) Interactome analyses revealed that the U1 snRNP machinery overlaps extensively with the RNAP II machinery and contains multiple ALS/SMA-causative proteins. Sci Rep 8:8755|
|Paolella, Brenton R; Gibson, William J; Urbanski, Laura M et al. (2017) Copy-number and gene dependency analysis reveals partial copy loss of wild-type SF3B1 as a novel cancer vulnerability. Elife 6:|
|Yin, Shanye; Lopez-Gonzalez, Rodrigo; Kunz, Ryan C et al. (2017) Evidence that C9ORF72 Dipeptide Repeat Proteins Associate with U2 snRNP to Cause Mis-splicing in ALS/FTD Patients. Cell Rep 19:2244-2256|