What we are working on
Gene expression begins with transcription, but pre-mRNA splicing determines the mRNA isoforms that are expressed and their stability. Recently, we showed that the efficiency of splicing controls the amount of RNA and protein produced by a gene, demonstrating that the spliceosome can influence gene expression as much as the strongest transcription factors. In addition, the Cajal body, a membrane-less organelle, controls the concentration of spliceosomal subunits (snRNPs) within the nucleus. Therefore, the 3D organization of the cell nucleus is instrumental in this mechanism. We study transcription and splicing regulation through two perspectives, represented by our two lab subgroups: “Splicing Group” and “Body Group”.
Our main research interests include:
Splicing group: Coordination of splicing and transcription
We are studying the in vivo kinetics of splicing, which occurs while the nascent RNA emerges from RNA polymerase II (Pol II). We have developed single molecule RNA sequencing methods that reveal whether the nascent transcript is spliced relative to the base-pair position of Pol II, which shows that the spliceosome can act on nascent RNA as soon as it emerges from Pol II. Thus, the spliceosome is physically close to Pol II for efficiently spliced introns. There are also inefficiently spliced introns, and we have found that splicing is coordinated among introns within a single transcript. For example, the globin pre-mRNA contains two introns, and it is either efficiently spliced and cleaved at the 3’ end or inefficiently spliced and uncleaved. This is significant because transcription rate can influence alternative splicing and failure to splice co-transcriptionally leads to transcriptional read-through and transcript degradation, thereby shaping expression levels. The latter features are characteristics of cancer cells and stressed cells. We are currently addressing the mechanisms underlying this level of gene resolution by identifying gene features (e.g., sequence or RNA structure), chromatin states, and trans-acting factors that determine the kinetics of co-transcriptional splicing.
Body group: Cajal Bodies and rnp assembly
We are studying the Cajal body, a membrane-less organelle located in the cell nucleus where the spliceosomal snRNPs assemble. Cajal bodies were discovered by Ramon y Cajal over 100 years ago in sections of vertebrate brain, yet their composition, assembly principles, and detailed mechanism of function remain unknown. We have shown that the scaffolding protein of Cajal bodies, coilin, is essential for zebrafish embryogenesis. Our model is that Cajal bodies promote the efficient assembly of snRNPs, allowing the rapidly dividing embryo to cope with the onset of zygotic transcription and splicing. We are currently identifying the comprehensive proteome of Cajal bodies in tissue culture cells and neurons to discover novel Cajal body functions, and we are working to gain insight into its nucleation and assembly. We are approaching a more mechanistic understanding of Cajal body assembly with our recent work that demonstrates that assembly occurs via complex interactions between the intrinsically disordered Nopp140 protein and oligomerized coilin. We have recently shown that the Spinal Motor Neurons (SMN) protein, the loss of which causes the fatal childhood disease Spinal Muscular Atrophy (SMA), is required together with coilin and snRNPs to assemble Cajal bodies, possibly through a liquid-liquid phase separation process. Liquid-liquid phase separation is a cellular process by which proteins and/or nucleic acids spontaneously separate into more dense and less dense liquid phases, which have diverse functions in the cell. We are currently conducting a structure-function analysis of Cajal body assembly processes, as we have discovered folded domains in SMN and coilin that mediate oligomerization and binding to an understudied post-translational modification, dimethylarginine (DMA).