Transfer RNA (tRNA) Fragments Are Connected to Diseases

  • Specifically Formed tRNA Fragments (tRFs) can Repress Expression by RNAi 
  • Specific tRFs are Associated with Cancer and Other Diseases
  • Chemical Modifications in tRFs Pose a Challenge for Sequencing 

Researching new, trending topics for Zone in with Zon rewards me in several ways, including learning about important subject matter that I only vaguely knew about, or had been completely unaware of. The present blog is about tRNA fragments (tRFs), which was totally new subject matter for me that I found to be very interesting and worth sharing here.

But before getting to biological formation and functions of tRFs, I want to mention what led me to this intriguing class of RNA molecules. In a nutshell, TriLink’s R&D team decided to “brainstorm” on how its expertise in chemically modified RNA might be leveraged into new product offerings beyond its current lines of modified oligo RNA and modified messenger RNA (mRNA). Since tRNAs were long known to have numerous types of chemical modifications, as detailed elsewhere, TriLink’s R&D started to think about tRFs for reasons outlined below.

Biogenesis of tRFs

Formation of tRNA is a complex process. Initially, tRNA is transcribed in the form of a precursor (pre-tRNA) containing 50-nt leader and 30-nt trailer sequences, and in some cases introns in the anticodon loop. Pre-tRNAs then undergo various types of RNA processing steps to ultimately form mature tRNAs. During tRNA maturation, the 50-nt trailer is processed by RNase P, the 30-nt trailer is removed by RNase Z, and following 30-trailer removal, the 30-nt end of all human tRNAs is modified by enzymatic addition of the universal CCA triplet, as depicted here.

Pre-tRNA (left) and mature tRNA (right); adapted from Anderson & Ivanov FEBS Lett (2014)

Also depicted here are specific types of enzymatic cleavage reactions of mature tRNA by ribonucleases Dicer and angiogenin (ANG) that lead to formation of 5’-tRFs and 3’-CCA tRFs, as well as 5’-halves and 3’-halves. These tRFs derived from mature tRNAs, as well as tRFs from pre-tRNAs that will not be discussed here, have now been extensively characterized by high-throughput short RNA sequencing methods. Among new advances in this sequencing methodology, TriLink’s recent PLOS One publication of its innovative CleanTag™ sample prep procedure has already been viewed an impressive ~6,000 times since appearing online only ~14 months ago as of this writing.

Mature tRNA (adapted from Anderson & Ivanov)

It should be noted that tRFs are not restricted to humans but have been shown to exist in multiple organisms. Two online tools are available for those wishing to learn more about tRFs: the framework for the interactive exploration of mitochondrial and nuclear tRNA fragments (MINTbase) and the relational database of Transfer RNA related Fragments(tRFdb). MINTbase also provides a scheme for the naming of tRFs called tRF-license plates that is genome independent. A recent publication by Kim et al. is a good lead reference for various functions of tRFs, some of which include the following.

Possible Roles of tRFs in Human Diseases

In a review of this subject, Anderson & Ivanov emphasize that, while production of tRFs have been observed in several types of human diseases, it remains to be determined whether these tRFs contribute to disease pathogenesis. Landmark findings regarding functions of tRFs were published by a team, including Andrew Fire—2006 Nobel Laureate for  RNA interference (RNAi)—titled Human tRNA-derived small RNAs in the global regulation of RNA silencing that provided compelling evidence demonstrating that human tRFs can enter RNAi pathways. These findings by Fire & coworkers are now recognized as a previously unknown nexus of RNAi translational repression pathways involving tRFs and microRNAs (miRNAs) depicted here.

Schematic representation of the biogenesis of miRNAs and tRFs associated with Argonaute (AGO) proteins. Taken from Shigematsu & Kirino Gene Regul Syst Bio (2015)

tRFs and Cancer: In 2009, Lee et al. reported that a specific tRF, designated as tRF-1001, is highly expressed in a wide range of cancer cell lines but much less in tissues, and its expression in cell lines was tightly correlated with cell proliferation. Furthermore, siRNA-mediated knockdown of tRF-1001 impaired cell proliferation. Since that discovery, various research groups have similarly found specific tRFs associated with different types of cancer, as recently detailed by Croce & coworkers, who concluded the following:

“We found that tRNA-derived small RNAs (tsRNAs) [i.e. tRFs in this blog] are dysregulated in many cancers and that their expression is modulated during cancer development and staging. Indeed, activation of oncogenes and inactivation of tumor suppressors lead to a dysregulation of specific tRFs, and tRFs-knock out cells display a specific change in gene-expression profile. Thus, tRFs could be key effectors in cancer-related pathways. These results indicate active crosstalk between tRFs and oncogenes and suggest that tRFs could be useful [bio]markers for diagnosis or targets for therapy. Additionally, [overexpression of two specific tRFs] affect cell growth in lung cancer cell lines, further confirming the involvement of tRFs in cancer pathogenesis.”

Biomarkers in blood, which I’ve blogged about previously, are a “hot topic” in disease diagnostics because they offer a more general, less invasive and safer means of patient sample access compared to traditional tumor biopsies.

tRFs and Pathological Stress Injuries: Stress-related cellular damage is central to disease pathogenesis that can be induced by hypoxia, nutrient deprivation, oxidative conditions and metabolic imbalance. Dhahbi et al. sequenced short RNAs from mouse serum and identified abundant 5′-halves derived from a small subset of tRNAs, implying that these tRFs are produced by tRNA type-specific biogenesis. A survey of somatic tissues revealed that these tRFs are concentrated within blood cells and hematopoietic tissues, with very little in other tissues, suggesting that they may be produced by blood cells. Serum levels of specific subtypes of these 5′ tRNA halves change markedly with age, either up or down, and these changes were prevented by calorie restriction.

Taken from Mishima et al. J Am Soc Nephrol (2014)

In a study by Mishima et al., it was shown in vivo that oxidative stress leads to conformational changes in tRNA that thus allows ANG-mediated productin of tRFs. This stress-induced conformational change allows 1-methyladenosine nucleoside (m1A), a modification important for stabilizing the L-shaped structure of tRNA, to be recognized by an m1A-specific antibody, as depicted here. This antibody was used to show that renal injury and cisplatin-mediated nephrotoxicity (which both induce tissue damage via oxidative stress) generate tRFs. Similar results were obtained using m1A-based immunohistochemistry to directly visualize damaged areas of kidneys, brain and liver. Mishima et al. further demonstrated that these tRFS avoid degradation in the blood because they are associated with circulating exosomes, which are extracellular vesicles packed with proteins and nucleic acids.

tRFS and Neurodegenerative Diseases: As detailed in the above mentioned review by Anderson & Ivanov, ANG mutants possessing reduced ribonuclease activity were reported in 2006 to be implicated in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS; aka Lou Gehrig disease), which is a fatal neurodegenerative disease that I have blogged about. In 2012, a subset of ALS-associated ANG mutants was also found in Parkinson’s Disease (PD) patients. Recombinant ANG is neuroprotective for cultured motor neurons, and administration of ANG to a standard mouse model for ALS significantly promotes both life-span and motor function.

Concluding Comments on Analysis of tRFs

Although I started this blog by refering to the fact that mature tRNAs are extensively modified by a wide variety of nucleobase and ribose chemical modifications, these modifcations were not further mentioned. That is because sample prep for short RNA sequencing uses reverse transcription to form cDNA that is then PCR amplified before sequencing, and it is widely acknowledged (e.g. Cozen et al.) that certain chemical modifications in RNA can interfere with reverse transcription. Thus, aside from reported use of demethylases to first remove interfering methyl groups from m1A, N1-methylguanosine, N3-methylcytosine, and N2,N2-dimethylguanosine, sequenced tRFs exclude many tRFs having chemical modifications that prevent reverse transcription.

Recognizing the need for alternative methods of determining structures of chemically modified tRFS, Limbach & Paulines have recently proposed the possibility of developing mass spectrometric (aka mass spec) approaches in a publication provocatively titled Going global: the new era of mapping modifications in RNA. I think this is a great idea, and hope that the mass spec community will soon address this challenge.

As usual, your comments are welcomed.

ADDENDUM

After writing this blog, Eng et al., who investigated the mosquito Aedes aegypti—the primary vector of human arboviral diseases caused by dengue, chikungunya and Zika viruses—reported the following:

Aedes aegypti mosquito. Taken from wcvb.com

“[A]single tRF derived from the precursor sequences of a tRNA-Gly was differentially expressed between males and females, developmental transitions and also upon blood feeding by females of two laboratory strains that vary in midgut susceptibility to dengue virus infection. The multifaceted functional implications of this specific tRF suggest that biogenesis of small regulatory molecules from a tRNA can have wide ranging effects on key aspects of Ae. aegypti vector biology.”

Click here to read my past blogs about Zika virus.

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