Author: Anna Vainio

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The Decorated Cloverleaves in our cells

Transfer RNAs (tRNAs) are often described as humble, clover shaped molecular servants. They participate in protein synthesis by performing codon recognition on messenger RNAs (mRNAs) and by delivering amino acids necessary for translation. Despite technically being an accurate description, it underestimates the profound intricacies of the tRNAs. Humans have around 600 tRNA genes in the genome, and when expressed and processed, the beautiful clover leaf tRNA is reshaped to an L-shaped configuration and will acquire variable chemical modifications, increasing the diversity of tRNAs (Lant et al., 2019). Altogether, the modified L-shaped tRNAs comprise 15% of the total RNA found in the cell, whilst mRNA only comprises 1-5 % (Delaunay et al., 2024). Disruptions in tRNA expression, regulation and mutations have been linked with neurological and metabolic disorders and cancer (Lant et al., 2019). tRNAs are found in all forms of life (Delaunay et al., 2024), underscoring their fundamental role in biology. Understanding tRNAs is essential for understanding life: after all, would life even exist without the humble tRNA?

Modification of cellular macromolecules is crucial for accurate and efficient gene regulation. Many are familiar with DNA modifications, such as cytosine methylation, which may affect gene activity and chromatin structure (Liyanage et al., 2014). Cellular RNAs are also targets of post-transcriptional modifications (PTMs) with the N6-Methyladenosine (m6A) modification being one of the most widely studied (Lauman & Garcia, 2020). RNA modifications have essential regulatory implications: in mRNAs, certain modifications can affect transcript stability, localisation, splicing patterns, and translation (Delaunay et al., 2024). PTMs can be found in ribosomal RNA, long non-coding RNAs, and small non-coding RNAs (Delaunay et al., 2024). Unsurprisingly, PTMs are also seen in tRNAs. In fact, tRNAs are the most abundantly modified RNA species in the cell (Zhang et al., 2022). Our understanding of their effects is still limited but we know that the modifications can affect tRNA stability, tRNA-RNA interactions, tRNA-protein interactions, folding and mRNA decoding (Delaunay et al., 2024). Over 150 tRNA modifications have been identified (Delaunay et al., 2024) and as tRNAs are abundant in the cells, studying tRNA modifications becomes a very intriguing area of research.

Adenosine and N6-Methyladenosine. Created with BioRender.com.

During my time as a HiLIFE trainee I had the opportunity to delve into the science of tRNA modifications at the RNAcious laboratory, University of Helsinki. I participated in two projects, one where the aim was to produce hypomodified tRNAs and another where the aim was to determine the tRNA modification landscapes in different mouse tissues. You can find my first blog post here: https://blogs.helsinki.fi/hilife-trainees/2023/06/26/my-battle-against-rnases/

The making of plain cloverleaves: Hypomodified tRNAs

Modifications on tRNAs are so abundant that it would be difficult, or maybe even impossible, to extract hypomodified tRNAs from the cell: you see, in eukaryotes there are on average 13 modifications on each ~80nt long tRNA (Zhang et al., 2022). For methylations alone, there are around 40 proteins, known as modification writers (e.g. methyltransferases) and erasers (e.g. demethylases), which moderate tRNA modifications (Delaunay et al., 2024). One way to produce hypomodified tRNAs is through in vitro transcription (IVT), which essentially is a cell free transcription system. As IVT is cell free, it lacks the writer and eraser enzymes which modify the modification profile. My task was to develop a method to produce and isolate hypomodified tRNAs utilizing IVT, ribozyme- and MS2 based techniques.

Differentially decorated cloverleaves: tRNA modifications in different organs

We know that there are different tRNA modification landscapes in different organs (de Crécy-Lagard et al., 2019) but they have not yet been studied extensively. A tRNA modification landscape is the entire modification profile of the tRNAs of a specific organ. Uncovering the modification landscape would offer valuable insights into both the frequency and positional distribution of specific modifications within the tRNAs across various organs. Mass spectrometry is an effective tool to identify and study the location of modifications on single nucleotides (Lauman & Garcia, 2020), and certain reverse transcriptases can be used to study the location of modifications on the tRNA molecule (Zhang et al., 2022).

After my time as a HiLIFE trainee, I’ve truly gained a deep appreciation for the complexities of the humble tRNA. While I metaphorically refer to tRNA modifications as “decorations”, in reality, these modifications play essential roles in biological processes. Learning about them has been truly fascinating.

To end this journey, I would like to express my gratitude to Docent Peter Sarin and his group of bright researchers, especially my supervisor Jenni Pedor, who always supported me during my time in the research laboratory. The pioneering and motivational environment provided an invaluable experience and an inspiration for my career moving forward. I would encourage anyone interested in expanding their understanding of tRNA modifications and tRNA biology to explore the research conducted at RNAcious laboratory.

 

References

de Crécy-Lagard, V., Boccaletto, P., Mangleburg, C. G., Sharma, P., Lowe, T. M., Leidel, S. A., & Bujnicki, J. M. (2019). Matching tRNA modifications in humans to their known and predicted enzymes. Nucleic acids research, 47(5), 2143–2159. https://doi.org/10.1093/nar/gkz011

Delaunay, S., Helm, M., & Frye, M. (2024). RNA modifications in physiology and disease: towards clinical applications. Nature reviews. Genetics, 25(2), 104–122. https://doi.org/10.1038/s41576-023-00645-2

Lant, J. T., Berg, M. D., Heinemann, I. U., Brandl, C. J., & O’Donoghue, P. (2019). Pathways to disease from natural variations in human cytoplasmic tRNAs. The Journal of biological chemistry, 294(14), 5294–5308. https://doi.org/10.1074/jbc.REV118.002982

Lauman, R., & Garcia, B. A. (2020). Unraveling the RNA modification code with mass spectrometry. Molecular omics, 16(4), 305–315. https://doi.org/10.1039/c8mo00247a

Liyanage, V. R., Jarmasz, J. S., Murugeshan, N., Del Bigio, M. R., Rastegar, M., & Davie, J. R. (2014). DNA modifications: function and applications in normal and disease States. Biology, 3(4), 670–723. https://doi.org/10.3390/biology3040670

Zhang, W., Foo, M., Eren, A. M., & Pan, T. (2022). tRNA modification dynamics from individual organisms to metaepitranscriptomics of microbiomes. Molecular cell, 82(5), 891–906. https://doi.org/10.1016/j.molcel.2021.12.007

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My battle against RNases

 

Hi! I’m Anna and I’m a Master’s student at University of Helsinki (UH) in the programme of Genetics and Molecular Biosciences. I am honored to have been chosen as one of the HiLIFE research trainees for the year 2023. 

For as long as I can remember I have been interested in life sciences. Nowadays, my main academic interests lie in functional genetics, RNA biology, translational biology, and biomedicine. I received my Bachelor’s degree at UH and wrote my thesis on the canonical and non-canonical functions of aminoacyl-tRNA synthetases. That was when my passion for RNA and translation really began. Learning about and understanding the complex systems that facilitate the basics for life is something that fascinates me deeply. In my spare time I am an active board member in the student organization Svenska Naturvetarklubben (SvNK), where I work as the vice chairman for the year 2023. A large portion of my spare time goes to the student organization, but I also love to draw and do some science related arts and crafts (such as making earrings out of the elegant tRNA secondary structure).  

RNA is part of the central dogma of molecular biology, but it has many other functions in the cell beyond that. There is a lot of ongoing research on a variety of different RNA types, but there is still a lot to learn about one of the first discovered, and no less fascinating, RNAs: tRNAs. For a long time, tRNAs had been thought to be mere vessels for amino acids, that their only job was to bring the amino acid to the ribosome for translation. tRNAs are indeed essential players in the basics of the translational system, but they also have many interesting regulatory functions. tRNAs have been shown to have a part in both transcriptional and translational regulation, as well as apoptotic pathways (Avcilar-Kucukgoze, I. & Kashina, A. 2020). Recently discovered tRNA derived RNA fragments have been shown to regulate both transcription and translation in miRNA-like ways and are seen as a part of the ncRNA family (Liu et al, 2022).   

The research in Docent Peter Sarin’s RNAcious laboratory focuses on molecular modifications on tRNA nucleotides. All natural tRNAs have molecular modifications on some nucleotides, such as methylation, acetylation and pseudouridylation. These modifications are important for the stability of the tRNA molecule, stress response in the cell, and regulation of translation (Koh & Sarin, 2018). For instance, hypomodification of the anticodon loop of tRNA molecules can disrupt anticodon recognition and may cause issues in protein homeostasis (Koh & Sarin, 2018). Diseases that have been associated with issues in the tRNA modificome, sometimes called tRNA modopathies, are e.g., microcephaly, intellectual disabilities, and multiple cancers (Chujo &Tomizawa, 2021). Modopathies can be caused by mutations or dysregulation of certain enzymes, such as methyltransferases and pseudouridine synthases, that regulate the modification profile of tRNAs (Chujo & Tomizawa, 2021).    

 We still have a lot to learn about the role of tRNA modifications in health and in disease. As Chujo & Tomizawa (2021) mention, we do not have a complete understanding of the general cytoplasmic tRNA modification profile in healthy humans, which in turn makes it difficult to study modopathies since we have no healthy reference. A big obstacle in the research of tRNA modifications has been the loss of molecular modifications during sample handling as well as lack of methods to identify all modifications and their exact location. The RNAcious laboratory does e.g., research on method development to identify different types of modifications and research on the role of tRNAs and tRNA modifications in viral infection.    

 

The research in RNAcious. From RNAcious laboratory website, 21.6.2023, https://www.helsinki.fi/en/researchgroups/rnacious-laboratory/research-topics-0

 

My HiLIFE traineeship at RNAcious started in the beginning of April, and it has been a great pleasure to work in a prestigious, diverse, and international research group. So far, I am involved in a project where the aim is to characterize tRNA modifications in different mouse organs. In addition, I am working on a project where the goal is to plan, produce and isolate hypomodified tRNAs. My work has e.g., comprised of protocol optimization, RNA and tRNA isolation, mass spectrometry sample preparation, In vitro transcription (IVT), IVT template design and protein production and isolation. It has been a privilege to work with pioneers in the field and share and ponder different ideas with them.     

 

 

One thing that I have concluded thus far is that all RNA researchers have one common enemy, RNases. Ribonucleases: the enzymes that seek to destroy what is dearest to us, and they reveal their destruction through smeary bands in polyacrylamide gels. That is why I comprised a short list below which includes a few different ways you can inhibit RNase activity.  

 RNA work 101:    

  • Use nuclease free eppendorfs.    
  • Use 3% Hydrogen peroxide to clean counter tops and gloves (anything that may be in contact with the sample).    
  • Always use gloves when working with RNA to not contaminate your sample with RNases from your skin.    
  • RNase inhibitors are your best friend.  
  • Always keep your sample on ice.  
  • Heating samples at 80°C for 5 min should inhibit RNase activity.  
  • Store your RNA in -80°C to inhibit degradation.   

I am excited to continue this RNAcious journey and fight my own battle against RNases!           

  

References:  

 Avcilar-Kucukgoze, I. & Kashina, A. (2020). Hijacking tRNAs From Translation: Regulatory Functions of tRNAs in Mammalian Cell Physiology. Front Mol Biosci 7, 610617.

Chujo, T., & Tomizawa, K. (2021). Human transfer RNA modopathies: diseases caused by aberrations in transfer RNA modifications. The FEBS journal, 288(24), 7096–7122. 

Koh, C. S., & Sarin, L. P. (2018). Transfer RNA modification and infection – Implications for pathogenicity and host responses. Biochimica et biophysica acta. Gene regulatory mechanisms, 1861(4), 419–432.

Liu, B., Cao, J., Wang, X. Guo, C.,Liu, Y., Wang, T (2022). Deciphering the tRNA-derived small RNAs: origin, development, and future. Cell Death Dis 13, 24.