Monday, April 29, 2019

Wait, what do you do?
I Study RNA Processing in Mitochondria

Hello all! It's been awhile and this is my first blog post that isn't an assignment for JRN 504. I have really wanted to blog more about my research but it can be really hard to make the time to really sit down and write for fun (is there even such a thing?) when you have all this mandated writing to do. But I thought hey, I have this poster that I spent a lot of time writing, why not turn that into a quick and dirty blog post. Plus, I wanted to have this poster in a text format so that it could be more accessible.

I recently presented this poster at the Graduate Women in Science and Engineering (GWiSE) Women in STEM Research Showcase (you can check out my post on the event on the GWiSE blog). The audience for this poster was a general audience so I focused on background and my approaches and less so on what my results have been. Also my data is unpublished and I am paranoid about getting scooped so at this time I am not sharing it widely.

My poster as a whole: click to zoom in or scroll to have it broken down in paragraph form

Current Questions

What are RNA granules?
We know that RNA granules (RGs) are punctate, membraneless mitochondrial structures in the matrix that contains nascent (newly transcribed) RNA [ref 3]. We know that RGs are enriched with components of RNase P and other RNA processing enzymes [ref 4-8]. We do not fully understand the composition of RGs or how this composition changes. We also do not understand how RGs form or stay together. I am interested in determining and defining different subsets of RGs. I am also interested in how phase separation may contribute to RG formation.

Does mitochondrial RNA need to be completely processed at the RNA granule?
It is thought that RGs are dynamic platforms and through spatiotemporal regulation control RNA processing as well as mitoribosome assembly. I’m interested in studying which RNA processing events must occur at the RG and which, if any, are allowed to occur at locations outside of the RG.

Understanding RNA processing in mitochondria and how aberrant processing can lead to mitochondrial dysfunction may help us better understand complex diseases such as Parkinson's, Alzheimer's, diabetes and/or cancer. 

Abstract

Mitochondria are double-membrane organelles found in humans, plants, animals and essentially all eukaryotic organisms. Mitochondria contain their own DNA (mtDNA) that works in coordination with nuclear DNA (the DNA you usually think of) to build the respiratory complexes, which are responsible for the energy production of the cell necessary for life. Processing of RNA within the mitochondria is different from the processing of RNA in the rest of the cell. Mutations in nuclear genes leading to incorrect processing and maturation of mitochondrial RNAs are cause of most human mitochondrial diseases. Furthermore, mitochondrial dysfunction is involved in many common diseases such as Parkinson’s disease, Alzheimer’s disease, diabetes and cancer. I am interested in studying nuclear-encoded RNA-binding and cleaving proteins in human mitochondria, how they are involved in RNA processing and how they are organized.

Background

Mitochondria are energy producing, double-membraned organelles containing five compartments: outer membrane (OM), inner membrane (IM), intermembrane space (IMS), cristae and matrix (Fig. 1). Mitochondria take advantage of their structure to produce over 90% of the cell’s energy, in the form of adenosine triphosphate (ATP), through oxidative phosphorylation (OxPhos). The process of OxPhos is carried out on the IM through an electron transport chain (ETC); which consists of five respiratory complexes: Complexes I-V. The ETC causes protons to build up in the IMS and generate an electrochemical gradient across the IM. The energy in this potential is then used by Complex V to produce ATP.

Figure 1. Mitochondrial Structure Helps with Energy Production Efficiency



Mitochondria contain their own DNA (mtDNA). mtDNA is circular DNA, consisting of a heavy and light strand, and is tightly packed in structures called nucleoids that reside in the matrix.

mtDNA either undergoes replication, which is making copies of itself, or it gets transcribed into RNA (transcription), which are instructions and tools to make proteins (Fig. 2). The messenger RNA (mRNAs) are then translated by processing machinery called ribosomes into proteins. Mitochondria have their own ribosomes, called mitoribosomes, that differ from the ribosomes in the cytoplasm. Like ribosomes, these mitoribosomes use the transfer RNAs (tRNAs) as tools to build the proteins. And lastly, there are the ribosomal RNAs (rRNAs) which are part of the mitoribosome itself, with the proteins for the mitoribosome being imported from the cytoplasm.

Figure 2. Central Dogma of Molecular Biology Applies to Mitochondria



In humans, mtDNA encodes for 13mRNAs, 22 tRNAs and 2 rRNAs. All of these help build up only part of the respiratory complexes in coordination with many proteins produced by the nucleus. The transcription of mtDNA results in two long polycistronic RNA strands, one for the heavy strand and one for the light strand. These two polycistronic RNA strands undergo unique endonucleolytic processing as described by the tRNA punctuation model (Fig. 3) [ref 1,2]. The tRNAs punctuate, or flank the mRNAs and rRNAs. These tRNAs are then recognized by the endonucleolytic (cleaving) enzymes, RNase P and RNase Z which cut them out on the 5’ and 3’, respectively. The cleavage and release of the tRNAs allow the mRNAs and rRNAs they flank to then also be freed. The release of the individual RNAs does not mark the end of their maturation process but rather just the beginning.

Figure 3. tRNA Punctuation Model



Adapted from Ferreira et al. 2017 

Results

Figure 4. SIM Imaging of Nucleoids and RNA Granules


Structured illumination microscopy (SIM) imaging of HeLa cells, an immortalized human cell line, showing the punctate and diffuse nature of nucleoids and RGs in the mitochondrial matrix. Tom20 (green) is a marker for the OM of mitochondria. A. DNA (red) is a marker for nucleoids. B. FastKD2 is a marker for RGs7.

Figure 5. Labeling Nascent RNA with BU


SIM imaging of HeLa cells with DNA (green) as a marker for nucleoids, Tom20 (white) a marker for the OM and 5-Bromouridine (BU) (red) as a marker for nascent (newly transcribed) RNA. A. and B. are the same image showing that only a subset of nucleoids have adjacent RGs containing nascent RNA. B. OM layer is removed for clarity of visualizing organization of nucleoids with nascent RNA.

Figure 6. Nucleoid & RG Organization in Knockdown of MRPP3

Figure 7. Bloated Mitochondria Phenotype


Mitochondrial RNase P protein 3 (MRPP3) is the protein in RNase P responsible for the 5’ end tRNA cleavage. The above is SIM imaging of HeLa cells with DNA (red) as a marker for nucleoids and FastKD2 (green) for RGs. The objective was to determine if there were changes in the organization of the nucleoids and RGs such as bloating (Fig. 7). Bloating is a phenotype observed when other RG-associated proteins are knocked down (unpublished data). In the case of MRPP3 knockdown (KD), no changes in phenotype were observed. A. Scrambled siRNA as a negative control. B. KD of MRPP3 with 80% efficiency of siRNA.

Continuing Efforts

  • Continue imaging organization of nucleoids and RGs with RNA-binding and cleaving proteins at normal levels, reduced levels (knockdown) and complete knock outs (CRISPR/Cas9).
  • Continue BU studies to follow how nascent RNA traverses the mitochondria as it matures.
  • Utilize CRISPR/Cas9 (gene editing) to knock in epitope tags on various mitochondrial RNA-binding and cleaving proteins. Epitope tags allow us to do various experiments without the need and limitations of antibodies. One such experiment is co-immunoprecipitation (co-IP); using the epitope tag to pull down the protein of interest and what it binds to – allowing us to identify binding partners.
  • Perform RNA-sequencing (RNA-seq) on both mitochondrial RNA and whole cell RNA after knocking down various RNA-binding and cleaving proteins. This allows us to understand how the various RNA -binding and cleaving proteins affect the RNA maturation process by measuring abundance of different RNA transcripts at different stages in their maturation. 

Acknowledgments

The Bogenhagen Lab, especially Anne Ostermeyer-Fay
NIH Training Grant in Pharmacological Sciences T32GM007518

References

  1. Ojala D, Montoya J, Attardi G. tRNA punctuation model of RNA processing in human mitochondria. Nature. 1981;290:470–474.
  2. Ferreira N, Rackham O, Filipovska A. Regulation of a minimal transcriptome by repeat domain proteins. Semin Cell Dev Biol. 2017;76:132–141
  3. Iborra, FJ, Kimura H, Cook PR. The functional organization of mitochondrial genomes in human cells. BMC Biol. 2004;2:9. 
  4. Lee K-W, Okot-Kotber C, Lacomb JF, Bogenhagen DF. Mitochondrial Ribosomal RNA (rRNA) Methyltransferase Family Members Are Positioned to Modify Nascent rRNA in Foci near the Mitochondrial DNA Nucleoid. J Biol Chem. 2013;288:31386–31399.
  5. Jourdain AA, Koppen M, Wydro M … Martinou J-C. GRSF1 Regulates RNA Processing in Mitochondrial RNA Granules. Cell Metab. 2013;17:399–410.
  6. Bogenhagen DF, Martin DW, Koller A. Initial Steps in RNA Processing and Ribosome Assembly Occur at Mitochondrial DNA Nucleoids. Cell Metab. 2014;19:618–629.
  7. Jourdain AA, Koppen M, Rodley CD … Martinou J-C. A Mitochondria-Specific Isoform of FASTK Is Present In Mitochondrial RNA Granules and Regulates Gene Expression and Function. Cell. 2015;10:1110–1121.
  8. Antonicka H, Shoubridge EA. Mitochondrial RNA Granules Are Centers for Posttranscriptional RNA Processing and Ribosome Biogenesis. Cell Rep. 2015;10:920–932.
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Monday, April 8, 2019

Supporting Women (and other minorities) in STEM

Last week, I touched a bit on the isolation you feel when in grad school (re: this post) which brings me to this week's topic: Women in STEM


Being a woman in STEM can come with it's own set of feelings of isolation and feelings that you don't belong (i.e. sexism, microaggressions, harassment, discrimination, etc). While we could talk for days upon days on these issues, I want to only talk about one aspect of being a woman in STEM today - and that's finding your support network.

I am very fortunate because I am in a department where my peers are fairly equal in number for being males and females which is extremely important. Why is this important? Well, studies have shown that women in Ph.D. programs that have more women are more likely to graduate (read more about that here). I am also fortunate because my peers are very supportive of each other; males and females.


But I know many grad students out there are not so lucky, in which case I want to point them to resources outside of their department.

Here at Stony Brook University, we have an amazing organization and community, the Graduate Women in Science and Engineering (GWiSE).

The GWiSE mission is to enhance the scientific, professional, and personal development of graduate women pursuing STEM degrees at Stony Brook University and provide a space for dialogue on issues unique to women in science. To sum it up, it is about amazing women doing amazing things. We're all about supporting each other, socializing and science. But this group is so much more than just women supporting other women, we support everyone. We have male allies in our club, we have non-STEM majors in our club. We want STEM and quite frankly, our world to be a space where everyone is welcome so that is what we strive for and hope to reflect in everyone of our events.


Many campuses, have their own form of GWiSE in some form or another. But if you don't have this group on your campus, start one! It will be a lot of work but I promise you, if you build it they will come.

If you need assistance forming a club, many national organizations (see list of resources below) will assist you with form a chapter. SBU GWiSE is not-associated with any national chapters as this allows us to not need to collect dues, we are funded through the graduate student fees through the Graduate School Organization (GSO). SBU GWiSE is also a pretty new organization, we've only been here about four years but every year we are growing and gaining traction. If you're at SBU, join us (check out our upcoming events on Facebook or join our mailing list).

Additional Reading/Resources:


Do you have a similar organization on your campus? Do have any other resources or interesting articles relating to this topic? Please share them below in the comments.

Monday, April 1, 2019

Moving, Grad School & Isolation

I took a poll on Twitter to see what topic people would like for my podcast assignment for JRN 504 and it was overwhelmingly in favor of me discussing PhD/Grad school life. So I thought I'd write a little bit about that today.

I went to Philly this weekend to run the Philly Love Half marathon with my good friend, D. We grew up together in Minnesota, lost touch during college but later reconnected when we both found ourselves on the East Coast (~5 years ago). She lives in Boston and me in New York. I can't say how great it is to have someone from back home out here. She gets it - we can really talk and understand about how different life can be from the two places (it's mostly the people). Also it's fun to discuss our childhoods and where everyone else ended up. When we hang out, it feels like a little slice of home, minus the cold winters. She did break the news to me on this trip that she plans on moving back. I'm happy for her and I understand because I'm also ready for the New York chapter of my life to close soon but I'm also a bit sad in a bittersweet sort of way.

Our fourth race in Philly

In 2014, I moved 1200 miles away for a job in NYC. It was really hard. I cried a lot. I questioned if I made the right choice. I did. But it was still hard. That summer, D and I reconnected and it helped. A lot.

Moving puts you in a new space and in a new way of life and while any change is hard, that isn't the hardest. The hardest part about moving is personal connections - your friends and family. As humans we need to feel connected to others. Like I said, change is hard. But changes in relationships are REALLY [insert profanity here] hard. But you should realize, friendships come and go; regardless of whether you move or not. And relationships/connections with friends and family also evolve throughout your lifetime even if you stay in the town you grew up in. But if you do move, these relationships are essentially forced to change all at once. And that's indescribably difficult.

And that's what happens in grad school too. [I realize that many people move far for grad school as well, thus facing the double whammy at once which I don't even want to imagine. But I feel for you, I really do.]

Moving and grad school can both be very isolating but they don't have to be. I promise! Although, like all good things, it will take work and it will take time.

In grad school, your friends and family are often not nearby and even if they are, you probably still feel like they just don't understand what you're going through. You know what this sounds like? Teenage angst. Do you remember that? I do and I don't know about you, but it was mostly just me being over dramatic. "Mom, you just don't understand!!" She did, you were just being ridiculous. It's silly to think people don't understand you, they do, especially if you take the time to communicate with them. While your friends and family likely can't 100% understand what you're going through or feeling, I promise you they want to be there and try to understand. So let them. Talk to them, try and explain what you can, and be vulnerable - the worst thing you can do is put up a facade about how amazing everything is going. Because it's not. And maybe your parents will never understand your science but they will understand and feel for your heartache, they will understand the feeling of tying your self-worth to something you shouldn't (this isn't something isolated to academia). And talk to your classmates, be vulnerable with them too - you'll be amazed how good it feels to just open up, and they will likely follow suit. They might never understand what back home is like for you (although I have found other Minnesotan grad students here, so maybe you can find someone from back home too) but they will understand how frustrating working with that one professor is or how the university raising fees is just too much.

Going full circle: look at this vintage thermocycler/PCR machine at the Science History Institute in Philadelphia I saw this past weekend [I highly recommend visiting it when in Philly, I totally nerded out there].  Look how it has evolved! This evolution took a lot of work, and I wouldn't be surprised if it took tears too, to get it to where it is today. And I bet it will be different 10 years from now as well. Which machine would you prefer to use? The one better suited for today or a nostalgic blast from the past? That's how you should think of your relationships, they might not be what they were, but if you put the work in you can have them evolve to what you need today.



Thermocycler/PCR

Speaking of PCR, I better get back to work and fail at it again ;)


Look what I found

And old blog of mine. Is writing about this meta? I guess it doesn't matter. Be prepared for this to just be a bunch of discombobula...