PASt projects

Here you will find brief descriptions of the projects I have worked on in the past, in roughly reverse chronology. For a more comprehensive overview, and deep dives into any of the projects listed here, check out my list of publications.

Snf2 regulates meiotic splicing

Snf2 regulates meiotic splicing

SWI/SNF chromatin remodeling complex, pre-mRNA splicing and meiosis.

The primary interest of the Tracy Johnson lab is the interplay between pre-mRNA splicing and the chromatin environment in whose context spliceosome assembly and splicing catalysis occurs. During my investigations into potential roles for the chromatin remodeling SWI/SNF complex in regulating splicing, I discovered an unexpected mechanism by which Snf2. the core ATPase subunit of the complex, regulates the availability of the spliceosome. By promoting the transcription of Ribosomal Protein Genes (RPGs), which encode for the bulk of introns within the yeast cell, Snf2 limis the availability of the spliceosome, thus preventing the splicing of sub-optimal introns.

This regulation is particularly important during the process of meiosis in budding yeast, since it depends on the expression and splicing of a number of sub-optimal intron containing genes. Nutrient-dependent downregulation of Snf2 in turn downregulates RPG mRNAs, leading to the redistribution of spliceosomes towards meiotic transcripts, allowing for their splicing and progression of meiosis.

However, before downregulation, Snf2 is also required for initiation of the expression of a specific enhancer of meiotic splicing, called Mer1. Snf2 is relocalized to the MER1 locus through exquisite co-ordinate control of its acetylation state and histone H3 lysine 9 acetylation at the MER1 locus itself. 

Snf2 exerts systems level control of meiotic gene expression through two temporally distinct mechanisms, demonstrating that it is a key regulator of meiotic splicing in S. cerevisiae.

Click on the image to view the manuscript!

Snf2, PTC7 splice isoforms, and Coenzyme Q biosynthesis during respiration.

When yeast have access to their preferred source of nutrition, glucose, they utilize that glucose primarily via a metabolic process called fermentation (beer, wine, BREAD!!!, yum...). However, when glucose is unavailable, or when they've used it all up, other carbon sources are used via a process called respiration, that occurs partly within the mitochondria of the cell.

Similar to meiosis, nutrient-deprivation induced downregulation of Snf2 also occurs during batch growth in culture, as the growing yeast deplete glucose from the medium. As this occurs, RPG levels go down, and the spliceosome is rendered no longer limiting, increasingthe splicing of the suboptimal intron containing PTC7 transcript. The spliced form of PTC7 encodes a mitochondrial phosphatase regulator of biosynthesis of coenzyme Q6 (ubiquinone or CoQ6), a mitochondrial redox-active lipid essential for electron and proton transport in respiration. Increased splicing of PTC7 increases CoQ6 levels.  

Curiously, the non-spliced isoform of the PTC7 transcript also encodes for a protein, making it one of the few known examples of functional alternative splicing in yeast.  This protein encoded by the nonspliced isoform of PTC7 actively represses CoQ6 biosynthesis

Taken together, these findings uncover a link between Snf2 expression and the splicing of PTC7 and establish a previously unknown role for the SWI/SNF complex in the transition of yeast cells from fermentative to respiratory modes of metabolism.

Click on the image to view the manuscript!

Snf2,  PTC7,  and Coenzyme Q biosynthesis during respiration

Snf2, PTC7, and Coenzyme Q biosynthesis during respiration

Snf2 deacetylation during meiosis

Snf2 deacetylation during meiosis

Regulation of the SWI/SNF complex during meiosis.

I have previously shown that coordinated deacetylation and degradation of Snf2 during the early hours of sporulation is important for the proper progression of meiosis in budding yeast. However, very little is known about how this precise temporal dance is choreographed. I in multiple avenues of Snf2 regulation during meiosis, including but not limited to the levels of transcription, RNA stability, translation, and protein degradation.  

Modeled Structure of Ptc7(ns)

Functional roles of a protein splice variant in budding yeast.

The PTC7 gene provides one of the few known functional examples of alternative RNA splicing in budding yeast. The intron within the PTC7 pre-mRNA lacks a premature termination codon, is divisible by 3, and is thus translatable in frame with the rest of the transcript. I have previously shown that the relative abundances of the two isoforms of the PTC7 transcript, as well as the proteins they encode, is controlled by Snf2 levels.

I have recently shown that the proteins encoded by the two PTC7 isoforms have opposing effects on the biosynthesis of coenzyme Q. However, the mechanism of action of the two isoforms still remain incompletely understood. 

I used a combination of high throughput techniques and analytical biochemistry to dissect the molecular functions of Ptc7 isoforms, and the roles they might play within the yeast cell.