Background

The central goal of our research is to map the positions of RNA elements in human genes and to determine their function. This problem is perhaps most relevant in the context of alternative pre-mRNA splicing, a mechanism that dramatically expands the protein coding complexity of human genes by generating multiple mRNA isoforms from a single locus. The major goals of our work are to understand (1) how alternative mRNA isoforms are produced and which isoforms are physiologically relevant under specific cellular conditions, (2) how the process of alternative pre-mRNA splicing influences downstream steps in gene expression and (3) how mechanisms of mRNA processing fail in human disease.

Mechanisms of aberrant splicing in human inherited disease

            In a conceptually related project, supported initially through the Ellison Medical Foundation, we developed an orthogonal approach to identify splicing regulatory elements in human genes using human genetic variation. By comparing two different sequence variation databases, one corresponding to deleterious rare mutations and the other corresponding to common variants from the 1000 Genomes Project, it became possible to pinpoint the locations of functional cis-acting RNA elements in protein coding genes. Remarkably, we found positional and sequence signatures that are indicative of the functional impact of DNA sequence variants on splicing of specific exons (Sterne-Weiler et al. 2011). The significance of this work rests in our innovative hypothesis that very different genetic diseases share a common underlying molecular mechanism. For example, we determined that a specific splicing silencer sequence, ACTAGG, is created by 83 different disease-causing mutations in 67 different genes.  Using splicing reporters, we determined that this class of mutations did indeed induce aberrant splicing in four out of five exons tested. We went on to identify trans-acting splicing factors that not only discriminate between the disease-associated and wild types sequences but also mediate the effects of the ACUAGG splicing silencer. Our ongoing research in this area includes collaborations with Drs. Sean Mooney (Buck Institute, Novato CA) and David Cooper, curator of the Human Gene Mutation Database (University of Cardiff School of Medicine). Together, we developed a computational tool for predicting the functional impact of DNA sequence variants on splicing. A manuscript describing this work was recently published in the journal Genome Biology (Mort et al. 2014). Our work also generated two patents (pending) on methods for identifying functional RNA elements using genomic sequence variants and on several hundred potentially therapeutic sites in the genome.

This project also provides an outstanding framework for undergraduate research. Over the last two years I’ve recruited three undergraduates to test the splicing sensitivity of mutations identified by Sterne-Weiler et al 2011. The students are focusing on mutations associated with ALS, Parkinson’s disease and Alzheimer’s disease. Their goal is to close sequences encompassing mutated exons in to a heterologous splicing reporter and determine the impact of the mutation of splicing efficiency. We think, with appropriate funding, this project could support a large cohort of students. It is my goal to develop this project into an undergraduate elective lab course.

Alternative splicing coupled translational control (AS-TC)

            The other major project in my laboratory is based in my long-standing interest in functional connections between different steps of mRNA processing. As a post-doctoral fellow with Dr. Javier Caceres I discovered a novel role for an alternative splicing factor in mRNA translation  (Sanford et al. 2004; Sanford et al. 2005; Michlewski et al. 2008; Sanford et al. 2008). The results of our work suggested an intriguing hypothesis: could alternative splicing generate mRNA isoforms with different translational fates? To discover human genes with coordinated regulation of splicing and translation, my laboratory developed a methodology based on subcellular fractionation and high throughput sequencing (Frac-seq). Using this approach, we discovered that ~30% of alternative splicing events generated mRNA isoforms with different translational fates (published in Genome Research, Sterne-Weiler et al. 2013). Our goal now, is to understand how AS-TC works by studying the functions of RBPs and characterizing cis-regulatory sequences associated with isoform-specific translation.

Molecular mechanisms of IGF2BP3-mediated gene regulation in cancer models

            Another exciting project focuses on roles of the Insulin-like Growth Factor 2 mRNA Binding Protein 3 (IGF2BP3) in solid tumors and leukemia. IGF2BP3 expression is strongly correlated with malignancy and lethality. Despite more than a decade of basic research a causal role for IGF2BP3 in oncogenesis has not been established and only two mRNA targets of IGF2BP3 have been identified. We are investigating the mRNA targets and functions of IGF2BP3 in pancreatic cancer cell as well as murine and human B-cell leukemia models. Using cell models my lab discovered that IGF2BP3 promotes mRNA decay by promoting association of the RNA induced silencing complex with mRNA transcripts (Ennajdaoui et al 2016). In collaboration with Dinesh Rao at UCLA, we contributed to the first study showing that enforced expression of IGF2BP3 in murine hematopoietic stem cells induces a leukemia-like phenotype (Palanichamy et al 2016). My lab used iCLIP to identify mRNA targets of IGF2BP3 and determined that these targets are aberrantly expressed in the mouse model in an IGF2BP3-dependent manner. Our findings demonstrate that IGF2BP3 regulates expression of Myc and CDK6. We propose that IGF2BP3 is an important part of the pathway through which Myc transforms cells.

Genomic analysis of protein-RNA interactions in living cells

In experiments led by former graduate student Jon Howard and Hai Lin (Yunlong Liu’s lab at Indiana University School of Medicine) we applied iCLIP-seq to address cause and affect relationships between RBPs. We would like to understand how perturbing the levels of a one RBP affects the binding specificity of other RBPs in living cells. This experiment will determine how two potentially antagonistic splicing factors, SRSF1 and hnRNPA1, influence the binding specificity of each other and another protein, U2AF65, which functions in early steps of pre-mRNA splicing. Based on in vitro data from other labs, we predict that high levels of hnRNPA1 will disrupt binding patterns of SRSF1 and U2AF65. This experimental design will allow us to determine how different combinations of splicing factors not only influence U2AF65 binding, but also reveal specific features of exons that are subject to combinatorial control. Jon’s work (manuscript in preparation) demonstrates that over expression of hnRNPA1 causes global changes in U2AF65 crosslinking patterns. This change is reflected primarily in depletion of U2AF65 within 200 nt of the 3’ss. However, Jon also observed a striking accumulation of crosslinks in exon proximal anti-sense Alu elements. These data suggest that Alu elements may have been coopted by the splicing machinery as regulatory RNA elements. We think these results could have interesting implications for the evolution of species-specific patterns of alternative splicing. I am quite enthusiastic about this line of investigation.

Collaborative research projects

            Since arriving a UCSC, our goal has been to establish a productive, diverse research program. I have been successful in this endeavor in part, by establishing successful collaborations both locally and abroad. Beyond the UCSC campus, I am working closely with several labs including Drs. Yunlong Liu (Indiana University School of Medicine), Dinesh Rao (UCLA), Andrew Smith (University of Southern California) and Luiz Penalva (University of Texas San Antonio Health Sciences Center) on methods for genomic analysis of protein-RNA interactions (Uren et al. 2012). Collectively, these collaborations advance my primary goal of understanding how RNA binding proteins regulate post-transcriptional gene expression in animal models of human disease.

We also seek to establish collaborations with Industry. Our collaboration with the Biotech firm BIOO Scientific (Austin, Texas) aims to develop an iCLIP-compatible library preparation kit. This led to a successful Phase I STTR grant from the NIH.

Curricular Undergraduate Research Experiences:

https://news.ucsc.edu/2017/10/undergrad-research-lab.html