Let's talk more about RNA today! RNA is similar to DNA as they are both nucleic acids and made up of a string of nucleotides (ATCGU). But unlike DNA, RNA is usually single stranded and it uses uracil instead of thymine. 
The 5-carbon sugars ribose & deoxyribose are important components of nucleotides found in RNA & DNA, respectively. The difference is ONE oxygen atom. This difference is important because it allows for enzymes to differentiate between RNA & DNA.
The major function we think of for RNA is it's role in protein synthesis (transcription). A really simplified idea of this process is that a strand of RNA gets fed into the ribosome which then puts out a chain of amino acids which will fold up into a protein.
The nucleotides A, U, C, G code the different amino acids. You can decipher the code by looking at a codon table. It's important to note that mitochondria have a slightly different codon table - the codons in red indicate the codons that do not follow the universal code.
Speaking of it being important to note: there can be considerable differences between yeast, fruit fly, protozoan, mammalian mitochondria so I want to make it clear I am only talking about mammalian mitochondria.
But RNAs do so much more than just make protein. They are also involved in other regulatory processes like interfering with expression of genes (e.g. microRNAs, small interfering RNAs) or enhancing expression (enhancer RNAs).
There are RNAs called riboswitches that detect & respond to environmental or metabolic cues & then affect gene expression accordingly. It's unknown if these exist in mitochondria but that isn't saying mitochondria aren't affected by the outcomes of riboswitches in the nucleus.
I could keep listing types of RNAs & I wouldn't be surprised if there are types we haven't discovered yet. But I need to focus & get us to RNA processsing because there are some cool differences in that between mitochondria RNA & more "traditional" (for lack of better word) RNA .
Mitochondrial DNA (mtDNA) is transcribed into two long RNA strands. These strands contain the 37 genes: 13 messenger RNAs (mRNAs) which the mitoribosome translates to proteins, 2 ribosomal RNAs (rRNAs) & 22 transfer RNAs (tRNAs) which are the keys to decoding.
The mt-tRNAs have another interesting but passive role, they bookend the different mt-mRNAs and mt-rRNAs. This is called the tRNA punctuation model. This model explains how all these different RNAs are freed from the long strand to act as individual RNAs.
The mt-tRNAs are recognized by 2 enzymes, mt-RNAse P and mt-RNase Z, which cut on either side of the mt-tRNAs; thus, not only cutting out the tRNAs but also freeing the mRNAs and rRNAs that they flank. (Image adapted from: DOI: 10.1016/j.semcdb.2017.08.037)
This process is called endonucleolytic cleavage. Endo = internal, within. Nucleo = nucleic acid. Lytic = perform lysis. Endonucleolytic cleavage is an early mtRNA processing step.
And as you can imagine, if we cannot properly cut out these RNAs we will not be able to properly make the proteins needed to power our cells to survive. Thus, this process is a very important processing step.
mt-RNase P is built of 3 proteins (Mitochondrial RNAse P Protein 1-3 or MRPP1, MRPP2, MRPP3) and mt-RNase Z was thought to only be 1 (ELAC2). But, a recent study showed that 2 of the proteins in mt-RNase P (MRPP1 & MRPP2) are also needed for mt-RNase Z to cut RNAs in vitro.
MRPP1 & MRPP2 form a stable platform by binding each other. This study showed this platform is shared between mt-RNase Z & mt-RNase P. The thought is the MRPP1/2 platform holds the mt-tRNA and fold it into a shape that makes the cutting of it accessible. (doi:10.1093/nar/gkx902).
The tRNA punctuation model is unique to the mitochondria because the mt-RNA is transcribed in those 2 long strands. More "traditional" RNA is transcribed differently, where the individual RNAs are transcribed individually so there is no need to cut them apart from each other.
However, "traditional" RNA has introns (non-coding regions) & exons (coding regions) so it has pieces of itself, introns, cut out because they aren't need for translation & this process is called RNA splicing. mt-RNA does not have introns so they do not have RNA splicing.
Another difference in mt-mRNA vs "traditional" mRNA processing is mt-mRNA does not undergo 5'-capping aka mRNA capping. 5'-capping is the addition guanine attached in a unusual way on the 5' end. This cap is used for nuclear export, to prevent degradation & promote translation.
The last major difference I want to talk about is mt-mRNAs do not undergo traditional polyadenylation at the 3' end. Nuclear mRNAs have multiple adenines added to the 3' end, with the average between 50-100 nucleotides.
The product of polyadenylation, the poly(A) tail is important for nuclear export, translation & stability. mt-mRNAs have a much shorter tail 0-10 nucleotides so they are often referred to being oligoadenylated. But there are some exceptions. Oligo = few. Poly = many.
To sum up these major differences in mt-mRNA vs. "traditional" mRNA processing.
mt-mRNA has:
1. No introns, no RNA splicing
2. No 5'-end capping
3. Shorter or no poly(A) tails
mt-mRNA has:
1. No introns, no RNA splicing
2. No 5'-end capping
3. Shorter or no poly(A) tails
mt-tRNAs also are different with there being less (only 22) and having a more variable shape than the traditional cloverleaf.
mt-tRNAs undergo similar processing to mature as other tRNAs including -CCA addition & base modifications like pseudouridylation.
mt-RNA undergoes a lot of different processing but what I am interested in is WHERE this processing is occurring.
This FINALLY leads us to what I have been wanting to talk about all week: Mitochondrial RNA granules (MRGs). MRGs are a mitochondrial substructure where newly transcribed mt-RNA organizes.
We know that MRGs are membrane-less. And we know MRGs are platforms for many types of RNA processing and maturation. However, we do not know how they form, what keeps them together (remember they are membrane-less), and what determines protein and RNA composition.
Why care about this? Because incorrect processing and maturation of mtRNA are the cause of most human mitochondrial disease. And mitochondrial dysfunction is involved in aging and many common diseases such as Parkinson’s disease, Alzheimer’s disease, diabetes, and cancer.
A lot of RNA processing steps occur at the MRG which means a lot of different proteins hang out there (some of which are listed in this figure). I want to know if these various proteins always hang out or if not, what tells them to join vs leave? (doi.org/10.1016/j.tibs…)
Obviously, I can't answer that question about every protein that hangs out there since there are just too many. So I'm focused on determining if mt-tRNA cleavage by the formerly mentioned mt-RNase P & mt-RNase Z is confined to the MRG.
I'm also interested in understanding the biophysical properties of the MRG. One hypothesis in the field is that MRGs form due to phase separation aka condensates. So what is phase separation?
Liquid-liquid phase separation (LLPS) is like salad dressing. No seriously it is! LLPS in a vinaigrette occurs because oil molecules & water molecules repel each other.
LLPS inside cells or if it does occur in mitochondria happens due to the interactions of biological polymers. Polymers are large molecules made up of many repeating subunits (monomers) strung together in a chain. DNA & RNA are both polymers.
What I want to study is mtRNA & mtRNA-binding proteins & see if their interactions are capable of LLPS. We know that long RNAs with stable secondary structure can capture RNA-binding proteins out of solution, leading to phase-separated compartments. science.sciencemag.org/content/360/63…
I'm running out of time & I, myself, am also a novice to LLPS so I'm going to focus on sharing some articles I like. Here is a nice article to prime you on the concept of LLPS: What lava lamps and vinaigrette can teach us about cell biology nature.com/articles/d4158…
The structures LLPS creates are called condensates or membraneless organelles. This article can introduce you to the condensates we know a bit about: These Organelles Have No Membranes the-scientist.com/features/these…
But what about phase separation in mitochondria themselves? Well check out this recent preprint:
Mitochondrial RNA granules are fluid condensates, positioned by membrane dynamics
doi.org/10.1101/747055
Mitochondrial RNA granules are fluid condensates, positioned by membrane dynamics
doi.org/10.1101/747055
And here is another preprint but this one is focused on nucleoid (where mtDNA resides):
Self-assembly of multi-component mitochondrial nucleoids via phase separation
doi.org/10.1101/822858
Self-assembly of multi-component mitochondrial nucleoids via phase separation
doi.org/10.1101/822858
