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 Also my data is unpublished and I am paranoid about getting scooped so at this time I am not sharing it widely.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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