Lab Research Focus
Our long-term objective is to understand the role of post-transcriptional regulation of gene expression in embryonic development and disease pathogenesis. Specifically, we focus on understanding the role of the minor spliceosome, which is responsible for the splicing of a rare type of intron called the U12-type or minor intron. In the mammalian genome <0.5% of the introns require the minor spliceosome and are often found embedded as single introns in genes that otherwise consist of introns spliced by the major spliceosome. Thus, regulation of these minor intron-containing genes (MIGs) requires coordinated action of both the major and the minor spliceosome. We aim to study how this coordination between the major and minor spliceosome controls expression of MIGs in stem cells. Specifically, we want to understand how minor intron splicing and MIG-expression inform self-amplification versus differentiative cell division. To study this, we primarily use mouse as the model organism. We employ molecular and biochemical methodologies in the developing mouse embryo combined with bioinformatics to understand splicing and alternative splicing of MIGs.
This project focuses on understanding the role of the minor spliceosome in regulating stem cells, called radial glial cells, in the developing cortex. We have modeled microcephaly observed in the disease . The disease microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1) is caused by mutation in U4atac snRNA, a component of the minor spliceosome. Using our conditional knockout mouse for U11, another crucial component of the minor spliceosome, we have modeled microcephaly as observed in MOPD1. Using this mouse we are able to test the effects of inactivation of the minor spliceosome in cortical development.
Alternative splicing across minor introns
We have discovered that in the absence of U11 snRNA, a subset of MIGs show alternative splicing, which circumvents the need for minor intron splicing. We have employed RNAseq and developed bioinformatics approaches to determine that these alternative splicing events are executed by the major spliceosome and are regulated by the minor spliceosome. We are currently exploring the mechanism by which the minor spliceosome regulates these alternative splicing by the major spliceosome.
Progenitor cell-specific regulation of MIGs
This project is focused on the effects of the inactivation of the minor spliceosome in the progenitor cells of the ventral diencephalon. We are investigating the distinct effects of minor spliceosome inactivation on the progenitor cells of the ventral diencephalon compared to those of the dorsal telencephalon. In doing so, we have found that the minor spliceosome plays a role in hypothalamic development and obesity.
Despite a systemic mutation in U4atac snRNA, the developing cortex and the limbs are most affected in MOPD1 patients. We are trying to identify the common molecular pathway between these tissues, by investigating the MIGs that are affected upon minor spliceosome dysfunction. To this end, we have crossed our U11 conditional knockout mouse to a limb-specific Cre line.
Amyotrophic lateral sclerosis
P525L mutation in FUS has been shown to lead to juvenile-onset ALS. This mutation causes FUS to sequester U11 snRNA in the cytoplasm, thereby inactivating the minor spliceosome. The long-term objective of this project is to use our U11 conditional knockout mouse to test that the ALS in the case of FUS mutation is caused by loss of minor spliceosome function.
We aim to use and develop novel bioinformatics approaches to analyze RNAseq data. Using these, we focus on detecting gene expression changes in a developing system, where we are interrogating not only changes between control and experimental samples, but also across time. Our analyses include detection of novel alternative splicing events as well as characterization of gene networks dynamics. Together, we explore and design ways to analyze RNAseq data produced by the Kanadia lab and our collaborators.