Thermostable group II intron reverse transcriptases and their applications in next generation RNA sequencing, diagnostics, and precision medicine
| dc.contributor.advisor | Lambowitz, Alan | |
| dc.contributor.committeeMember | Iyer, Vishwanath R | |
| dc.contributor.committeeMember | Krug, Robert M | |
| dc.contributor.committeeMember | Russell, Rick | |
| dc.contributor.committeeMember | Stevens, Scott W | |
| dc.contributor.committeeMember | Sullivan, Christopher S | |
| dc.creator | Qin, Yidan | |
| dc.date.accessioned | 2016-10-12T15:54:51Z | |
| dc.date.accessioned | 2018-01-22T22:30:44Z | |
| dc.date.available | 2016-10-12T15:54:51Z | |
| dc.date.available | 2018-01-22T22:30:44Z | |
| dc.date.issued | 2016-05 | |
| dc.date.submitted | May 2016 | |
| dc.date.updated | 2016-10-12T15:54:52Z | |
| dc.description.abstract | Thermostable group II intron reverse transcriptases (TGIRTs) from thermophilic bacteria are advantageous for biotechnological applications that require cDNA synthesis, such as RT-qPCR and RNA-seq. TGIRTs have higher thermostability, processivity and fidelity than conventional retroviral RTs, along with a novel end-to-end template-switching activity that attaches RNA-seq adapters to target RNAs without RNA ligation. First, I optimized the TGIRT template-switching method for RNA-seq analysis of small non-coding RNAs (ncRNAs). I showed that TGIRT-seq gives full-length reads of tRNAs, which are refractory to retroviral RTs, and enables identification of a variety of base modifications in tRNAs by distinctive patterns of misincorporated nucleotides. With collaborators, I developed an efficient and quantitative high-throughput tRNA sequencing method, identified RNAs bound by the human interferon-induced protein IFIT5, yielding new insights into its functions in tRNA quality control and innate immunity, and uncovered a novel mRNA-independent mechanism for elongation of nascent peptides. Second, I developed a new, streamlined TGIRT-seq method for comprehensive analysis of all RNA size classes in a single RNA-seq. This method enables RNA-seq library construction from <1 ng of fragmented RNAs in <5 h. By using the method, I showed that human plasma contains large numbers of protein-coding and long ncRNAs together with diverse classes of small ncRNAs, which are mostly present as full-length transcripts. With collaborators, I showed that TGIRT-seq analysis of circulating RNAs identified potential biomarkers at different stages of multiple myeloma and may provide a sensitive, non-invasive diagnostic tool for a variety of human diseases. Finally, I adapted TGIRTs for use in mapping of RNA structures and RNA-protein interaction sites, and identification of RNA targets of cellular RNA-binding proteins. My research led to a series of new biological insights, which would have been difficult or impossible to obtain by current methods, and established TGIRTs as a tool for a broad range of applications in RNA research and diagnostics. | |
| dc.description.department | Microbiology | |
| dc.format.mimetype | application/pdf | |
| dc.identifier | doi:10.15781/T2SN01588 | |
| dc.identifier.uri | http://hdl.handle.net/2152/41602 | |
| dc.language.iso | en | |
| dc.subject | RNA-seq | |
| dc.subject | Diagnostics | |
| dc.subject | Precision medicine | |
| dc.subject | Non-coding RNA | |
| dc.subject | Circulating RNA | |
| dc.title | Thermostable group II intron reverse transcriptases and their applications in next generation RNA sequencing, diagnostics, and precision medicine | |
| dc.type | Thesis | |
| dc.type.material | text |