Out with the Mold, in with the New

In a fungus, cell division meets mRNA transport

Why would anyone choose a common bread mold fungus to study the way cells work? –  in particular, to unravel the ways that living cells beget new cells by the process known as cell division?  In the James laboratory, we seek to understand “why normal cells get it right every time they divide, and why cancer cells get it wrong every time”.  A worst-case scenario, played out again and again in cancer, occurs when a normal cell “forgets” how to stop dividing, and begins to proliferate incessantly and relentlessly.  We address this fundamental problem in the fungus Aspergillus nidulans because basic features of cell division evolved early on, and proved so effective in promoting survival that they’ve been conserved with little or no change in increasingly complex creatures, including us.  In fact, fungi and humans are similar enough that we could literally swap many genes without harm to either organism, like trading sparkplugs between a Volkswagen and a Ferrari.

This summer the excitement is palpable as we close in on two genes, discovered by us, that harbor previously unreported roles in cell division.  Mutant cells lacking these genes let slip the brakes on cell division, allowing cells to divide more readily than normal.  By extension, this means that the normal versions of these two genes must act as restraints to keep cell division from getting out of control.

One of the two new cell division genes, called snxA, is a conserved gene in many other species, where it is best known for its role in escorting messenger RNAs (mRNAs) out of the confines of the nucleus, where mRNAs are made, and into the cytoplasm where these RNA messages are translated into proteins.  In this process, snxAparticipates with several other proteins that together govern the export of mRNAs out of the nucleus.  To better understand snxA activity, it is important to know how snxA works with these other proteins to influence mRNA export and cell division.  To this end, Julia Palmucci (’18) and Elliot Rodriguez (’18) are studying some of these other proteins, as they describe below.

The other new cell division gene, called GYF, is also conserved across the evolutionary spectrum from fungi to humans, but comparatively little is known about its function.  This summer, Morgan Brown (’19) is completing an initial study of this gene, as she explains below.

 

Elliot Rodriguez (’18)

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As mentioned above, our lab studies cell cycle control, specifically proteins involved in the shuttling of messenger RNA (mRNA) from the nucleus into the cytoplasm. Along with the star protein of our lab, snxA, we also study the THO/TREX protein complex. THO/TREX is an 8-protein complex responsible for binding nascent mRNA and escorting it through nuclear pores with the assistance of snxA. The THO/TREX complex is the focus of my research. Our lab has worked to characterize some of the subunits that comprise the THO/TREX complex. By characterizing each subunit of the THO/TREX complex, it will provide us with a better understanding of the role each subunit has in the complex as well as how the complex interacts with other proteins such as snxA. My research in particular involves characterizing THO complex subunit 5, or thoc5.

Screen Shot 2017-06-22 at 2.09.49 PMThoc5 is evolutionarily conserved in higher eukaryotes, however the exact roles of Thoc5 in transcription and mRNA export are still unclear. In adult mice, thoc5 is essential in the maintenance of hematopoietic stem cells and cytokine-mediated hematopoiesis. In Drosophila, thoc5 mutants are viable but have spermatogenesis defects. Thoc5 is present in fungi and mammals but is absent in the popular model organism budding yeast, Saccharomyces cerevisiae. Studies of thoc5 in fungal model systems are limited, which makes Aspergillus an especially relevant model system for studying thoc5. The main goals of my research are to complete the initial characterization of thoc5 mutants as well as to investigate potential thoc5 interactions with the snxA shuttling mRNA-binding protein that has been the long-term focus of our lab’s efforts.

I am also assisting Julia with her work with another THO/TREX subunit, thoc6. Our lab has shown that the absence of thoc6 causes a mislocalization of snxA. Normally snxA remains in the nucleus, but the absence of thoc6 delocalizes snxA to the cytoplasm through an unknown mechanism. I am adding a green fluorescent protein tag to thoc6. This will allow us to visualize thoc6 in Aspergillus using fluorescence microscopy. By tagging thoc6 and other proteins such as snxA, we hope to better understand the interactions between these proteins as well as the unknown mechanism responsible for the mutant phenotype.

 

Julia Palmucci (’18)IMG_1197-1

This has been my first week as a summer research student in the James Lab.  The earlier part of my summer was spent doing research of a much different sort in the Peruvian Amazon with Dr. Trillo’s Tropical Terrestrial Biology course.  The class brought us to the one of the most biodiverse areas in the world where we identified over 100 species of birds, observed numerous mammals—including nine species of monkeys, and learned how to characterize insects and amphibians. We were also given the opportunity to use the pristine natural resources of Cocha Cashu Biological Station in Manú National Park for independently designed research studies.  Utilizing the area’s untouched mature forest (500-700 years old) and the more successional forest (about 150 years old) formed as a result of the meanderings of the nearby Rio Manú. I compared soil composition and insect diversity across differently aged forests.

The research I am undertaking for the remainder of this summer involves the interaction between snxA and the THO/TREX complex.  Specifically, I am studying a subunit of the THO/TREX complex—THOC6. This subunit is one of seven THO components in animals, and we believe Aspergillus nidulans may be the least complex organism that contains THOC6. Other common model eukaryotes used in molecular genetics research such as the budding yeast Saccharomyces cervisiae lacks thoc6.  Thoc6 is a fascinating protein, composed of multiple WD40-repeats that confer a β-propeller structure important in mediating its interactions with other proteins. A β-propeller protein (seen below) is composed of 5 to 8 “propeller blades” each with 4 to 8 β-pleated sheets.  A common misconception is

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Basic structure of the aptly named β-propeller protein (Fulhop and Jones, 1999)

that each WD40 repeat translates directly to one blade, but this is not the case. Instead, each repeat results in a majority of one blade and some fraction of the next, allowing for a gene with numerous WD40-repeats to have fewer blades. Additionally, the approximately 40 amino acid WD40 repeats are notoriously diverse in sequence between their starting ‘GH’ and ending ‘WD,’ and even these namesake characteristics can vary as well.

Previously, it was unclear if the gene we were studying, AN1056, is actually the ortholog of thoc6 in higher organisms. Much of the research on THOC6 focuses on the pathology of thoc6 mutations in humans. For example, several thoc6 mutations cause intellectual disability. In addition, thoc6 defects confer stress sensitivity in the fruit fly Drosophila melanogaster. This past year, however, our lab found that a deletion of AN1056 results in a cold-sensitive phenotype and delocalization of snxA into the cytoplasm, indicating that this protein may be associated with snxA and may be involved in cell cycle regulation. This summer we have begun accumulating answers to this puzzle in hopes to determine more conclusively if AN1056 is indeed THOC6, and how this protein interacts with snxA.  Using a new program that more accurately predicts WD40, we have discovered tantalizing commonalities between AN1056 and the sequences of thoc6 in flies and humans, including the same number of similarly arranged WD40-repeats.

My goal for the remainder of the summer is to determine the function of THOC6 and its interaction with snxA using fluorescence microscopy.  I will use strains carrying several fluorescent tags, including green-fluorescent snxA and red-fluorescent thoc6 to monitor defects in snxA localization during the cell cycle in wild type and Δthoc6 mutants using the Nikon Ti-U inverted epifluorescence microscope.  These tagged strains will be helpful for determining the role of THOC6 and clarify the reason for the defective phenotype exhibited by the Δthoc6 mutants.

 

Morgan Brown (’19)

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Morgan (left) and Dr. James (right)

While my labmates have been focusing on proteins that function alongside snxA, my research looks to characterize snxA itself. Much of our previous work has involved mutated strains of A. nidulans that suppress defects in regulators of the CDK1 mitotic induction pathway, which controls whether or not a cell can enter mitosis. Other phenotypes of these strains include extreme cold-sensitivity and decreased levels of mRNA, and we’ve been referring to these as snxA1 and snxA2, as though there were mutations within the snxA gene of these strains that caused the defects. However, we were stunned and flabbergasted to discover that, rather than a simple point mutation, the disruption of the snxA locus actually resulted from a reciprocal translocation! This means that the arm of Chromosome II where snxA is located, swapped with an arm of Chromosome I. Consequently, the snxA locus was broken within the first intron, separating the promoter region and first exon from most of the coding region. At the same time, the Chromosome I breakpoint occurred within the fourth exon (out of five) in a novel unstudied gene, AN6228. We know only that this gene contains a GYF domain, which is known to mediate binding to proline-rich motifs in certain other proteins. Now that we find  that the “snxA mutant” is actually two mutations in unrelated genes, I am recreating the GYF gene truncation and comparing it to a deletion of snxA to determine how each affects cell cycle control. This will allow us to tease out the contribution of each to the overall mutant phenotype.

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A diagram of the reciprocal translocation occurring between snxA and GYF.

To determine the contribution of each disrupted gene, snxA and GYF, to cell cycle regulation, I first deleted GYF from a wild-type background. It grew perfectly fine at cold temperatures, while strains in which snxA was deleted couldn’t grow at all, so our cold-sensitive phenotype was clearly a result of the snxA truncation. A deletion of snxA was shown to rescue cell cycle defects, leading us to believe that snxA (and not GYF) was responsible for cell cycle rescue. To our lasting surprise, a deletion of GYF also suppressed these defects. This suggests that the reciprocal translocation serendipitously occurred within two unrelated genes that both happen to play a role in regulating the G2-M transition of the cell cycle. It is important to note that a gene deletion completely eliminates its function, but since the translocation breaks GYF in the fourth exon, this truncated allele could retain all of its function, partial function, or no function. So, in order to resolve this question, I have engineered the truncated version of GYF into a wild-type strain, and am now testing the phenotype.

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Currently I am performing genetic crosses to generate strains carrying both the GYF deletion or truncation with mutations in G2-M cell cycle regulators. Furthermore, today I am tagging GYF with Green Fluorescent Protein (GFP) and other fluorescent tags in order to characterize the location and function of this novel gene. It’s been busy over here in the mold lab, but it’s been exciting as well!

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An image of the lovely protoplasts that we will be injecting our mutations into. Aren’t they adorable??

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