My adventure in tRNA biology, so far.

When the RNA journal started in March 1995, I was a second year Assistant Professor still trying to establish my research program. My postdoctoral work with Olke Uhlenbeck at the University of Colorado at Boulder was on finding new ribozymes through in vitro selection. I was very excited to continue the research in RNA catalysis and RNA folding in my own lab. Our first paper in RNA, published in June 1996, described our finding of the folding domains in bacterial RNase P RNA. This work has been cited 189 times as of December 2014 and led to the eventual success of the first high resolution RNase P RNA structure in 2003. After I received tenure in 2000, I had a period of “tenure blues,” when I tried to figure out whether to stay the course of what I had done or venture into new directions. The decisive moment for me to change my research into biology was the publication of the draft human genome sequence in February 2001. I remember reading the Nature article where Table 20 showed the entire known classes of human RNA at the time: this table covered less than one half of a page! I thought that there must still be ample opportunities to study RNA biology. Indeed, the field of micro-RNA and RNA interference leapt into prominence shortly thereafter, and RNA biology rapidly expanded into all areas of biological inquiries. With no prior experience and training in biological work, I decided to stay with at least something I knew: transfer RNA. I decided early on that my entry to success in tRNA biology would have to come from developing new techniques that are specifically tailored for tRNA. I was very fortunate to recruit a very talented graduate student, Kimberly Dittmar, to initiate this work in my lab. In 2001, all ‘omics research on nucleic acids were done using microarrays. Although commercial microarrays were very powerful, they were very expensive, costing ∼$1000 per chip. Further, because of the abundance of post-transcriptional modifications that interfere with quantitative cDNA synthesis and extensive secondary structure that interfere with hybridization, tRNA expression was difficult to quantify. Standard methods at the time required cDNA synthesis for fluorescent labeling, and the commercial microarrays typically used short antisense oligonucleotides that were not efficient for tRNA hybridization on the chip. We came up with an idea to selectively label only tRNA in the total RNA without cDNA synthesis (based on the universal 3′CCA in all tRNA), and obtained custom-made microarrays with probes covering the entire length of the tRNA. These developments not only made the quantitative assessment of tRNA in biological samples possible, it also reduced the cost to ∼$100 per array. The drawback of our method was a reduction of resolution from single nucleotide level for mRNA chips to ∼8 nucleotides for tRNA. This was not a problem for bacterial and yeast work where almost all individual tRNA isoacceptors can be distinguished at this resolution. However, as I will describe below, this resolution was not useful in addressing certain questions on mammalian tRNA biology. Nevertheless, we successfully applied our microarray method for many aspects of tRNA biology in bacteria, yeast and mammals, including the identification of new tRNA species in HIV virions and the demonstration of initiator tRNA overexpression leading to global reprogramming of tRNA expression and increased proliferation, published in RNA in 2010 and 2013, respectively.