Mitochondria on Twitter

I wanted to post this yesterday but had some struggles (re: Not a Real Post) because I thought it was a really nice Twitter thread on mitochondria. Also it went well with my Mother's Day post from the day before. Twitter and meme culture love the mitochondria and for that (and other reasons but mostly that) I love Twitter and meme culture.

Anyway, please note that this Twitter thread was not created by me but @NakedCapsid. I'm just sharing it because I thought it was nicely done and encompasses how cool my beloved mitochondria are.

Oh and if you're curious why I was struggling yesterday, it was because I couldn't figure out how to revert back to classic Blogger and was in some very weird viewing format where I could not figure out how to input html for the life of me. Or maybe I couldn't figure it out because I had drank an entire bottle of wine. That was probably the real issue. I mean really, the revert back to classic Blogger was pretty obvious in the bottom left-hand corner of my screen today. Oh and if you must know what kind of wine it was, it is Beringer White Zinfadel. Is it my favorite? No, but it is cheap and it obviously goes down well.

Happy Mother's Day & Cheers to Maternal Inheritance of the Mitochondria

Happy Mother's Day! 

Don't forget to thank her for all she has done for you including giving you your mitochondrial DNA (mtDNA).

Let's talk about maternal inheritance of mtDNA (This post is slight adaptation from my original post which can be found here).

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.

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References

[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

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If you liked learning about mitochondria, you might also like the following posts:

Mitochondria: The Powerhouse of the Cell & Wait, what do you do? I study RNA processing in the mitochondria

My Week on @IAmSciComm

My week on @RealScientists

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. 

________________________________________

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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If you liked this post, you might also like:

Mitochondria: The Powerhouse of the Cell

Mitochondria: The Powerhouse of the cell

This is an assignment for JRN 504 (re: this post) where I am supposed to post a photo and video.

Mitochondria, the powerhouse of the cell! We've all likely heard this and/or have seen the many memes. I'm in training as a mitochondrial biologist so I obviously care about them. But why should you care? Well, check out this quick (less than 2 minutes) chalk talk by the National Science Foundation to find out why and get a general overview of mitochondria.

Here are the main points of the video:

• Mitochondria produce energy for most complex living things including people, plants and animals. This energy is produced in the form of adenosine triphosphate (ATP). TL;DR mitochondria = energy, no energy = death!
• Mitochondria contain their own DNA. Mitochondrial DNA (mtDNA) is inherited from only your mother (there are exceptions, there are always exceptions but it is rare), which also let's us trace back human origins.

Ready to move on?

Okay, let's dive a little deeper. Since we are talking about DNA, let's take a minute to refresh ourselves on the central dogma of molecular biology. And if you didn't know it before, you will now and you'll learn the secret - molecular biology really isn't all that hard. ;)

Central Dogma of Molecular Biology

DNA either undergoes replication, which is making copies of itself, or it gets transcribed into RNA (transcription), which are instructions and tools to make proteins. The messenger RNAs (mRNAs) are then translated by processing machinery called ribosomes into proteins. These ribosomes also use the transfer RNAs (tRNAs) as tools to build the proteins. And lastly there are ribosomal RNAs or rRNAs which are parts of the ribosome itself. This basic overview of the central dogma applies to both the cell and the mitochondria. See?! I told you molecular biology is not that hard!

But let's focus on just the mitochondria. The DNA in the mitochondria (mtDNA), encodes for 13 mRNAs, 22 tRNAs and 2 rRNAs. All of these help build only part of the respiratory complexes, or factories that produce ATP (energy). The mitochondria and the respiratory complexes obviously need a lot more than 13 proteins for it's complex structure/job, so all the other proteins (thousands!) are supplied or transported into the mitochondria by the rest of the cell. The mitochondria have their own ribosomes, called mitoribosomes. My PI (principal investigator aka boss) studies the assembly of mitoribosomes. I, however, am interested in studying mitochondrial RNA its processing. I'll go more into detail on what I study in future posts but for now, I want to introduce one of my favorite tools to study mitochondria.

The SIM!

The SIM is a really high powered microscope (SIM = super illumination microscopy) that let's me not only see the mitochondria but look at the organization within them. Mitochondria are really freaking tiny so that is one heck of a zoom. To put that in perspective, a human hair is about 75 microns (micrometers, um) wide and I'm looking at a 5um level. Here is what this bad boy looks like:

SIM Microscope at Stony Brook University Core Facility

And here is an image of two human cells with nuclei out of frame (that's how zoomed in we are, a whole cell doesn't even fit in the frame). The green outlines the mitochondria by staining for TOM20, a protein that is all over the outer membrane of the mitochondria (mitochondria have two membranes - inner and outer). The red is DNA (remember mitochondria have their own DNA).

Mitochondria with SIM

Please comment below with any questions or  suggestions on what you'd like to learn about mitochondria.