My week on @RealScientists

#SBUPharm Feature

I was featured on my department's social media today!

Today’s #FeatureFriday: Caitlyn Cardetti (@CaitlynCardetti), third year student and @SBUGWiSE president! Find out more about Caitlyn: https://t.co/G9fc94J3Da
Blog: https://t.co/ReQpNxjFSl#PhDchat #PhDlife #WomenInSTEM #AcademicTwitter pic.twitter.com/Cyk0HXctvK

— SBU Pharmacology (@SBUPharm) January 24, 2020

"Research focus: RNA processing in human mitochondria
Outside of lab, Caitlyn enjoys trail running, board games, promoting women in science, and science policy."

You can follow my department here:
Twitter: @SBUPharm
Facebook: https://www.facebook.com/SBUpharm/
Instagram: @SBUPharm

What is a #RoCur?

As I mentioned in My Goals for 2020, I am trying to tweet more and to help achieve that goal I will be curating for @RealScientists and @IAmSciComm. But what I failed to mention is that I'll be curating this week. In fact, I start @RealScientists tomorrow for the week and @IAmSciComm the following starting on the 27th.

Mitochondrial Mysteries: Caitlyn Cardetti curates real Scientists https://t.co/yMxNtJGtGL pic.twitter.com/y8X36WVfde

— RealScientists Mods (@RealSciMods) January 19, 2020

What is a #RoCur?

#RoCur or a rotation curation is just the concept of having different people run one account in a rotation system. That's it. It's that simple. It's fairly common on Twitter but has also been done on Instagram and I'm sure other platforms as well.

I think they are incredibly fun to follow because you get exposed to new people all around the world and learn about their experiences and perspectives. I'm personally a huge fan of the science themed accounts.

But there are rocurs for plenty of other topics. For example, @WeRWorld has hosts tweeting from around the world.

And if a rocur doesn't exist for that you want then why not create one? I did. Back in 2015, I started @Neurotweeps which hosts various scientists, grad students, clinicians and other neurophiles to tweet about the brain and nervous system. Although I will warn you it can be incredibly difficult to find people to agree to curate. PLEASE CONTACT ME IF YOU WANT TO CURATE FOR NEUROTWEEPS!

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Science/Academia Themed #RoCurs to Check Out

@Neurotweeps

@RealScientists

@Biotweeps

@Astrotweeps

@DatSciTweeps

@PsyTweeps

@SciParty

@IAmSciComm

@IAmSciArt

@RecoveringAcademic

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Please tell me in the comments below what you think about the idea of a #RoCur. Would you curate one? Do you follow any? 

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If you liked this post, stay tuned for future posts on what it's like running a rocur, how to curate and about my experiences on @RealScientists and @IAmSciComm.

What's in a name? [The Why Behind My Blog Title]

Why did I name my blog the Wicked Witch of the Western Blot?

Well that's simple, I'm a witch and I run a lot of westerns.

Kidding. Kind of.

I do run a lot of westerns but I'm not a witch - well maybe I am if I'm hungry #hangry. I am, however, a feminist and what symbol is more feminist than a witch?! 

Actually the quote is a phrase from Author Tish Thawer's 2015 novel The Witches of BlackBrook

Witches are a symbol of female empowerment. YES TO FEMALE EMPOWERMENT! We need more of it! We need more witches!

Witches personify the fear of assertive women. DOWN WITH THE PATRIARCHY!

Witches have inundated the media. We love witch-themed everything - think Sabrina, American Horror Story: Coven (that was the best season btw - I mean who doesn't love Stevie Nicks?). Witchy aesthetics for style are in. Witch-themed self-help is in. And while I don't believe in witchcraft I do buy into the strength of the power of within which is what witchcraft kind of centers around, right?

Also I find science magical.

Plus, I thought my title was hella catchy! :)

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If you want to read more about witches as a symbol of feminism check these out:

From Circe to Clinton: why powerful women are cast as witches (The Guardian)

Women are invoking the witch to find their power in a patriarchal society (QZ)

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I would love to hear what's behind the title of your blog. Tell me in the comments below.

My Goals for 2020

Courtesy of PhDComics.com
It's okay, I don't plan on graduating this year anyway

Personal Goals

• Blog - If you remember this blog was originally started as a class assignment but I've decided I'd actually like to invest time into it and keep it going
• Post at least once a week
• Create a content schedule
• Personally tweet more, not just retweet content
• I signed up to curate for the #RoCurs @RealScientists and @IAmSciComm
• Also be personal in my tweets
• See my tweet thread below - I got personal about the slump I went through in 2018 and honestly, it was hard to open up so publicly like that

[Thread] I've been having conversations with a lot of other grad students lately about that third year slump. Did you experience it? When? How'd you get through it? I'll tell you about mine if you tell me about yours. #PhDLife #PhDChat #AcademicChatter pic.twitter.com/h5kk8ylW9y

— Caitlyn Cardetti (@CaitlynCardetti) January 10, 2020

• Keep reading - I've been really good at this especially with listening to audio books on my commute
• Journal more
• Get back into running - I fell off in November
• I currently signed up for the 2020 Long Island Winter Run series which involves four 5ks over the next few Sundays, I've already completed one
• Get back into yoga - I fell off this in November too, can I blame it on my thesis proposal?
• I will rejoin my weekly Monday class starting in February
• Keep a budget
• I started observing my spending last year by manually writing everything down, I'll keep doing this because it makes me highly aware of where my money goes
• I will cut back my spending on coffee out by limiting myself to one Starbucks run per week and by making cold brew at home (recipe to come in future post)
• Exception: I'm allowed to purchase coffee out in the following situations: coffee date with my SO or a friend, if I physically stay at the coffee shop and relax or do some writing (hoping this also serves as motivation to keep up with writing)
• Pay off some debt
• Learn more about finances in general through pfforphds.com 
• Learn some new recipes
• I have a whole list but I want to start with Yakamein (this is the recipe I want to try)
• Try the group led meditation on campus - this has been on my to do list for awhile, I just need to make time to go

PhD-Related Goals

• Theme my days to increase productivity
• Be better about keeping my lab notebook updated - my labmate is graduating soon and has been reminding me that I must keep proper notes now or I'll suffer later
• Get a fellowship
• I plan to apply for the F31 April deadline
• If that doesn't work out, I will apply for F99/K00 and/or AAUW Dissertation Fellowship
• Submit an abstract to present at a conference
• Get into a regular writing practice
• I think theming my days will help with this
• I will focus on writing fellowships in the spring
• In the fall, I will focus on writing introduction chapters for my dissertation and hopefully a publication 
• Take 1 course each semester towards an Advanced Graduate Certificate in Data and Computational Science
• If possible (depends on finances, timing and acceptance), attend CSHL writing retreat (date not currently posted but likely in fall)
• Learn protein purification - this is already in progress (see photo below)

Me getting to work on those 2020 goals

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If you liked this post, you might also like:
Why Do Resolutions When You Can Use A Compass? [A Rave Review for YearCompass]

Why do resolutions when you can use a compass? [A Rave Review for YearCompass]

Step 2 is to prepare a hot drink so I hope it's okay I let me local cafe make one for me - delicious cappuccino by the way 

For those who don't know I love Twitter - I LOVE IT! Anyway, I came across this tweet (gotta give credit where credit is due) and decided to try Year Compass out for myself (see this is exactly why I love Twitter - you find great things!). This is my summary and rave review of this awesome FREE tool! I think it's perfect for EVERYONE not just grad students.

This was my first year using @YearCompass to reflect on 2019 and set goals for 2020. My word for the year ahead is DEDICATION. Wondering if anyone else on #AcademicTwitter has tried this and what your word will be for the new year? @AcademicChatter @MindfulAcademix pic.twitter.com/6FfbK9i72J

— Talia Nardo (@Talia_Neuro) January 2, 2020

What is Year Compass?
"YearCompass is a booklet that helps close your year and plan the next one. In the routine of everyday life it's easy to lose sight of your true goals and aspirations. And even though we all have dreams, only a few of us plan for them. Effectively, at least. YearCompass works simply. Using questions and exercises rooted in psychology it takes you through the past year, then helps you turn your dreams into achievable goals."
[Stolen directly from their website]

What's the story behind Year Compass?
Year Compass started out with a group of friends at Budapest University who made a booklet to do for NYE, it went viral in 2013 and is now a global movement. Year Compass has more than 100 volunteers in 25 countries and the FREE booklet is available in 22 different languages.
[Paraphrased from their website Press section]

What was it like doing the compass?
Hard. Self-reflection is hard. Being honest with yourself is hard. It also is a bit time consuming. I mean, they do warn you that it will take a few hours. And it does. But I divided it up into a few sessions and I don't see any issue with that.

What are some of my best moments in 2019?
I moved in with my partner after we had barely been dating (seriously, barely dating like not quite 5 months) and it paid off nicely (hopefully they agree?). I love them . I love having a reason to keep good hours for grad school (can you say [forced] work life balance) because I have someone I love coming home to - also they kind of expect me to be home because like what's the point of living together if I'm not there? I also really appreciate their outlook on life and reminders that there is a lot more to life than grad school. And their reminders that I don't get paid enough to put up with all the B.S. that grad school sends my way so the best thing to do is literally not put up with it. Honestly, they make me feel more grounded which has helped a lot with my anxiety. Also for my Ph.D. I finished all my required coursework, passed my qualifying exam, successfully gave a lecture (my TA requirement) without any anxiety (shocking!), successfully defend my thesis proposal and advanced to candidacy (well technically I don' think the paperwork has gone through yet but close enough).

What is my word for 2020?
Positivity

Why Positivity?
In 2019, I took great strides in learning how to and practicing how to reduce my anxiety. One of the biggest lessons was learning that I can control my attitude towards things and that greatly helps. I still need to work on this and honestly probably will for life but I'm happy about it. Also I just have a lot to look forward in 2020 and that's exactly what I'm going to do - look forward to it!

Will I do this again for 2021?
Absolutely!

Is it too late to do my Year Compass for 2020?
Absolutely not.

I would love to hear about your thoughts on YearCompass in the comments. Did you use it? What did you like/dislike about it? What is your word for 2020? Do you use some sort of other tool/strategy to reflect on the past year/plan for the new year? Tell me everything [and anything]!

[And no, I am in no way affiliated with YearCompass. Just here to share the love because honestly it is a great tool and deserves this support.]

Summer in the Lab: A PSA To Not Be Overzealous

This post is to remind you IT IS OKAY TO ENJOY YOUR SUMMER!

Sunset at the beach? How frivolous!

Last week, I was feeling really guilty and "behind" in my lab work. Why? Because the week before I took Thursday off for the Fourth of July to go to a pool party/BBQ/watch fireworks with my significant other. Actually, I didn't even take the day off - I stopped into lab for a few hours in the morning. But then I also didn't come in that weekend because I went to two other BBQs/pool parties. And then I took this last weekend off and went to the beach BOTH days. Also I plan to go to the beach both days this upcoming weekend.

Sounds nuts right?

But does it sound nuts that I'm spending so much time at the beach? Or does it sound nuts that I'm taking so much time off of lab? And why do we see not coming in on weekends counting as "taking time off" from the lab?!

Why is it I feel this way? Probably because we all are guilty of perpetuating the idea that we must be working at all times. Well you know what I have to say to that? Whatever! I work late into the evening plenty of days during the week so when the weekend forecast is for 90+ degrees F I am going to take my weekends off and enjoy them at the beach as they should be. 

Plus, lots of workplaces have summer Fridays. So take a summer Friday, take a weekend off, heck take a day off to do whatever because we are already midway through summer and summer should be enjoyed.

Credit: PhDComics.com

Also be mindful of your expectations for what you're going to accomplish this summer. Don't set yourself up for failure. I think grad students get a little overzealous (why do I think this? Because I've personally experienced it) with what we think we will accomplish over the summer because we have less commitments - or so we think...

And while we do probably have less academic commitments (i.e. meetings, classes, etc); I don't know about you but I end up with a way more social commitments. Think graduation celebrations, pool parties, beach outings, camping trips, barbecues, etc. And you know what?  I am going to not only go to them but I am not going to feel bad about going to them instead of the lab (or at least I am going to lie to myself that I won't feel bad until I hopefully start believing it). Could I skip them and get more work done? Sure. But I already work hard and I am so much more than my work. Same goes for you - you work hard. We all work hard. So we're allowed some time to play hard too.

Don't get me wrong though, I'm not saying to not get any work done over the summer. I'm just saying to really check in with your expectations on what you are going to get done and make sure they are reasonable and allow for some fun too. Think of the summer like a non-academic employee, just another day on the job - not a time to catch up on what you didn't get done during the academic year.

If not, this is how you will feel:

me when asked how my summer research is progressing pic.twitter.com/4sJSjoDwe0

— Shit Academics Say (@AcademicsSay) July 12, 2019

P.S. Make sure to wear your sunblock. Also long sleeves/pants in the lab are not just an important part of lab safety but a great way to hide those tan lines from the boss. ;)

Call me Madam President

I can't tell you all how proud I am to be a part of this group! Also what better time than now to announce that I will be @SBU_GWISE President for the 2019-2020 academic year! #WomenInSTEM #WomenInScience #WomenLeaders https://t.co/OakzXNzBYg

— Caitlyn Cardetti (@CaitlynCardetti) May 7, 2019

Okay, so I already announced it on Twitter but I am more than happy to say it again. I am really looking forward to being the president of  Graduate Women in Science and Engineering (GWiSE) at Stony Brook University for the 2019-2020 academic year!

I cannot express in words how excited I am for this opportunity! Over my three years here at SBU, I have really seen GWiSE grow. And the rest of campus has noticed it too. All our hard work (e-board and general members) has paid off and GWiSE received the Jerrold L. Stein Student Life Award from the Graduate School Organization for being an Outstanding Organization Award last month.

On that note, I wanted to reflect on how I got involved with GWiSE and where I hope GWiSE is headed.

My first year here, I was a "general member" and would occasionally show up to some of the social events including a bike repair night that had pizza and a brown bag lunch. You know, the typical grad student event where you attend because you're hungry and they are serving food where the price is right, aka free. But somehow I ended up at the end of the year town hall meeting where they were looking for people to join the following year's executive board (e-board). I will be honest, I mainly went because they were once again having free lunch and I had a spare moment - but something possessed me to last minute throw my hat in the ring to be more involved. Literally last minute, as they were packing up the submissions and I was like wait, let me fill one out quick...

Anyway, I ended up getting the role of managing the GWiSE Twitter which was fine by me because I love Twitter! (P.S. You better follow me @CaitlynCardetti). But as this past year progressed, opportunities for more responsibilities came up and I took more and more on (some I even just purely made up) including event planning, Facebook, creating/sending out the newsletters, starting a book club, co-founding the SBU Grad Moms group, etc. And we had some really awesome events, my two favorites being hosting Dr. Elaine DiMasi, a personal friend and the physicist who ran for Congress in our district discuss her experience in politics (watch a video of her talk here) and then the Women in STEM Research Showcase.

I was happy I was able to contribute at a higher level this past year as an e-board member and I believe I will be able to contribute even more as GWiSE President next year. My main mission as President will be sustainability. I believe we've really elevated our standing as a school club to a true, well recognized  organization and I want this high level to continue to be attainable long after the current active members are gone. So to do this I want to make sure we have well-written documentation of how to manage our different e-board roles, have regular meetings with the Dean of the Graduate School and create and maintain more relationships with other organizations and centers on campus. I'm also hoping to fight to get GWiSE a seat on the Graduate School Council because really they could use more student perspective on that panel and preferably a diverse one (right now there is one student on the panel).

But if I could only say one thing about being on the GWiSE e-board, it would be that it has given me an amazingly supportive group of friends, all of which started with just wanting a slice of free pizza.

Anyway, keep up with SBU GWiSE on our blog to learn about the other members of the GWiSE Executive Board. And of course, follow us on Facebook and Twitter since I will still be running that for the next month.

And if you're interested, I had mentioned GWiSE in this past blog post: Supporting Women (and other minorities) in STEM.

Thank you Mom for my Mitochondrial DNA (PSA to not forget Mother's Day)

Don't forget Mother's day is this Sunday! (Do people celebrate Mother's day outside of the U.S.? Is it on the same day as the U.S.? Okay, I just Googled it - 40+ countries recognize it and most celebrate this weekend but some celebrate on different days. Read more on Wiki.)

Since I don't live by my mother to celebrate with the typical brunch, I like to send her a card - the nerdier the better. I can't share the card I sent this year because I don't want to ruin it but I promise it is funny. Anyway, here is one of my favorites that I sent two years ago. Why is it my favorite? Because it mentions the mitochondria, specifically mitochondrial DNA (mtDNA). Go figure!

Side note: If you're looking for nerdy or niche cards I highly recommend checking out Etsy. 

Photo courtesy of my mom's FB, Card courtesy of Nerdy Words Inc

As this card implies, mtDNA is inherited maternally aka you get your mtDNA from your mom (Thanks Mom!). I also mentioned that mtDNA is maternally inherited in this past post. So let's talk about maternal inheritance of mtDNA. 

For most organisms (living things), including plants, animals, and fungi, mtDNA is inherited from a single parent (uniparental inheritance). In animals that reproduce sexually (make offspring/babies by... well I think you get it) the mtDNA is normally* inherited from the mother (maternal inheritance). 

*Like in most science, there are almost always exceptions. And in this case, there are examples of certain species having paternally inherited mitochondria such as Plymouth Rock chickens [1] or organisms that get "leakage" and have mtDNA from both mom and dad such as fruit flies [2], honeybees [3] cicadas [4], mice [5], sheep [6] and even humans [7, 8]. 

Back to human mtDNA, why does Mom's mtDNA beat out Dad's? There are two main ideas on how this happens; the dilution model and the active elimination model [9]. In the case of dilution, a human egg has ~200,000 mtDNA molecules whereas sperm has maybe 5 and I'm sure you can do that math (this model also allows for "paternal leakage" or some mtDNA from the dad to get through as seen in the above *exceptions). Also most mitochondria in the sperm are in the tail (mitochondria like to hang out where they are needed to make energy and the tail needs a lot since it is the motor for the sperm to swim) and the tail is often lost during fertilization. And lastly, there is evidence that mitochondria in mammalian sperm are destroyed by the egg after fertilization, active elimination [10].

[9] Carelli V. (2015).

Why does mtDNA usually only come from one parent? To be honest, we don't really know but there are plenty of theories out there and scientists are working on it (Possible future post? I don't know. Maybe. Tell me in the comments if you want me to write about this).

Why care where mtDNA comes from? Well, for genealogy (study of the family tree), it let's us trace back maternal lineage. We can do that for the paternal lineage using Y chromosome DNA. Also mtDNA is highly conserved with relatively slow mutation rates (doesn't change much generation to generation) so that also let's us study our evolutionary relationships to other species.

While on the topic of maternal inheritance of mtDNA, I should mention mitochondrial replacement therapy (MRT). MRT is an in vitro fertilization (IVF) technique where the mitochondria from a donor egg is moved to the mother's egg and results in a baby with mtDNA from a donor female and nuclear DNA from the mother's egg and father/donor's sperm - this procedure is used when a woman with genetically defective mitochondria wants to have a baby with healthy mitochondria but have the baby be genetically similar to her (she could also use a donor egg). Wait, so is this the three parent baby I hear about in the news? Yes. And you can read more about it here. Some people think it's controversial but I personally find it no more controversial than egg or sperm donation. mtDNA contributes such minimal DNA (37 genes, when there is an estimated 20,000 genes in the nucleus) to have a major impact on the child's identity (this is what most of the controversy centers on) other than allowing them to be healthy.

Honestly, I could easily write a whole blog post on the ethics and the different methods behind MRT - let me know if you're interested below in the comments. 

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Sources

[1] Alexander M et al. (2015). Mitogenomic analysis of a 50-generation chicken pedigree reveals a rapid rate of mitochondrial evolution and evidence for paternal mtDNA inheritance. https://doi.org/10.1098/rsbl.2015.0561

[2] Wolff JN et al. (2012). Paternal transmission of mitochondrial DNA as an integral part of mitochondrial inheritance in metapopulations of Drosophila simulans. https://doi.org/10.1038/hdy.2012.60

[3] Meusel MS, Moritz RFA. (1993). Transfer of paternal mitochondrial DNA during fertilization of honeybee (Apis mellifera L.) eggs. https://doi.org/10.1007/BF00351719 

[4] Fontaine KM et al. (2007). Evidence for Paternal Leakage in Hybrid Periodical Cicadas (Hemiptera: Magicicada spp.). https://doi.org/10.1371/journal.pone.0000892

[5] Gyllensten U et al. (1991). Paternal inheritance of mitochondrial DNA in mice. https://doi.org/10.1038/352255a0

[6] Zhao et al. (2004). Further evidence for paternal inheritance of mitochondrial DNA in the sheep (Ovis aries) https://doi.org/10.1038/sj.hdy.6800516

[7] Schwartz M, Vissing J. (2002). Paternal Inheritance of Mitochondrial DNA. https://doi.org/0.1056/NEJMoa020350

[8]  Luo S et al. (2018). Biparental Inheritance of Mitochondrial DNA in Humans. https://doi.org/10.1073/pnas.1810946115 

[9] Carelli V. (2015). Keeping in Shape the Dogma of Mitochondrial DNA Maternal Inheritance. https://doi.org/10.1371/journal.pgen.1005179

[10] Sutovsky P et al. (1999). Ubiquitin tag for sperm mitochondria. https://doi.org/10.1038/46466

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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Mitochondria: The Powerhouse of the Cell

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

Believe it or not I'm a woman in science everyday 👩🏻‍🔬 Happy #WomenInScience Day! #WomenInSTEM pic.twitter.com/MAjauQqMKi

— Caitlyn Cardetti (@CaitlynCardetti) February 11, 2019

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.

I honestly couldn't do grad school without my pharm fam. This is my cohort taking some much needed respite. We orally defended our qualifying proposals this past week after spending the last few months writing them. Love you all! #phdchat pic.twitter.com/AgfpBIwr32

— Caitlyn Cardetti (@CaitlynCardetti) January 27, 2019

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.

We're loving the turn out tonight at our Women In STEM Research Showcase. Thank you for supporting #WomenInSTEM here at @GradSBU pic.twitter.com/lU4nJrrM5Z

— SBU GWISE (@SBU_GWISE) April 4, 2019

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:

Most Successful Women Surround Themselves with Other Women

500 Women Scientists

American Association of University Women (AAUW)

American Indian Science and Engineering Society (AISES)

Association for Advocacy of Women in Science and Medicine (AAWSM)

Association for Women in Science (AWIS)

Graduate Women in Science (GWIS) National Organization

#MeTooSTEM

National Organization for the Professional Advancement of Black Chemists and Chemical Engineers (NOBCCHE)

Society for Women Engineers (SWE)

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.