Editing BioMicroCenter:Research (section)

===Sequencing of tRNAs - DEDON/NILES LABS - [http://web.mit.edu/be BE] and [http://web.mit.edu/cehs CEHS] ===
Emerging evidence points to selective translation of critical proteins as a major facet of the cellular stress response.  The Dedon lab has shown that while one contribution to this translational control of cell response involves changes in the relative quantities of dozens of modified ribonucleosides in tRNA, there is also evidence for control of the number of copies of individual tRNA molecules.  This dual control problem makes it difficult to distinguish changes observed in the level of tRNA modifications caused by altered activity of ribonucleoside-modifying enzymes from changes in the number of tRNA copies.  To solve this problem, the Dedon lab has undertaken the development of a high-throughput deep-sequencing method to identify and quantify individual species of tRNA in a bulk population of tRNAs.  This approach exploits the paucity of ribonucleoside modifications at the 3’-end of tRNA molecules, modifications that can interfere with RNA sequencing reactions. Following ligation of a custom primer to the 3’-ends of tRNA molecules, reverse transcription is performed to create cDNA that is subsequently subjected to linear amplification off of the custom primer.  The amplified products are then identified and quantified by Illumina sequencing. This method allows for the simultaneous quantification of individual tRNA species in total tRNA isolated from cells subjected to different stresses, with application to study controlled degradation of tRNA during cell responses and to differentiate between changes in tRNA copy numbers from changes in RNA modifications due to enzymatic activity. <BR><BR>
The BioMicro Center has been very instrumental in providing technical expertise towards optimization of experimental design for the conversion of tRNA to cDNA for sequencing, as well as assisting with bioinformatics analysis of the output data from the Illumina HiSeq 2000. The Dedon lab was able to barcode six different experimental samples so that multiple sequencing libraries could be run in a single lane of an Illumina flow cell. Using the Illumina HiSeq 2000, we were able to identify every tRNA species found in S.cerevisiae and are in the process of optimizing the sequencing method in order to reliably quantify relative changes in the levels of specific tRNAs isoacceptors from different populations of tRNAs. <BR><BR>
The Niles lab has partnered with the Dedon Lab and the BioMicro Center in their efforts to develop a protocol for sequencing bulk tRNA.  While the Dedon lab is investigating the effect of different stresses on tRNA expression, the Niles lab is interested in the basic endogenous biology of the malaria parasite.  Currently, very little is known in the field about tRNA dynamics across the complicated life cycle of Plasmodium falciparum.  While many published studies have tracked RNA expression using microarrays and deep sequencing, these protocols enrich for protein coding RNAs, excluding an important component of host biology.  Additionally, while the mitochondria does not have any genes to produce tRNA, the apicoplast organelle has a full complement of tRNA genes.  As the apicoplast organelle is required for successful parasite infection, having a better understanding of the expression of tRNA from the reduced but necessary apicoplast plastid will assist in the downstream search for both vaccines and pharmacological treatments.<BR><BR>
Using the method described above, all tRNAs found in both the genome and apicoplast were detected from extracts of Plasmodium falciparum.  However, there were several species of tRNA that jackpotted, appearing at concentrations several orders of magnitude above the remaining species.  We are continuing to work with the BioMicro Center and the Dedon Lab to troubleshoot the protocol to determine a more quantitative picture of tRNA dynamics in the parasite.<BR><BR>