Development Of A New Method For Detecting Stress-induced 5’ TRNA Halves And Preliminary Screening Of 5’ TRNA Halves Binding Proteins

Objective:The cellular tRNA halves may be generated during different stress conditions in a varity of cells. tRNA halves have important biological function. This study is to develop a simple and rapid method for detection of stress-induced 5’tRNA halves and to screen 5’tRNA halves binding proteins by RNA Pulldown and liquid chromatography-tandem mass spectrometry.Methods:1) The Poly(A) polymerase was used to polyadenylate total RNA prepared from stress induced cells, we provided the degenerate DNA probes designed to hybridize to 3’tRNA-halves of intact tRNAs. RNase H specifically degraded the 31 tRNA-halves strands in tRNA-DNA probes hybrids. The cDNA was synthesized with oligo (dT)n-anchored primer and the 5’tRNA halves were amplified with the primer of 5’tRNA halves and anchored-primer by PCR.2) The cells induced with PBS were lysised, the cell lysate was incubated with 51 tRNA halves-biotin, streptavidin beads were added to the respective samples in order to obtain 5’tRNA halves-biotin binding proteins. Then the eluted complexes can be electrophoresed in denaturing PAGE and the unique protein bands were cut for liquid chromatography-tandem mass spectrometry analyses. The expression of candidate RNA and protein levels of 51 tRNA half binding proteins in the cells induced with PBS, DIS3L and GRSF1, were then assessed by RT-PCR and western blot respectively.Results:The method of Poly(A)-tailed-RNase H digestion-RT-PCR can be used to detect stress-induced 5’tRNA halves successfully.5’Val-tRNA halves and 5’Gly-tRNA halves binding protein profiling were obtained by RNA Pulldown and liquid chromatography-tandem mass spectrometry. The expression of candidate RNA and protein levels, DIS3L and GRSF1, were disturbed in the cells induced with PBS.Conclusion:A simple and rapid method for detection of 51 tRNA halves has been established and the novel method is a convenient tool for 5’tRNA halves detection and function research. The technology of RNA Pulldown and liquid chromatography-tandem mass spectrometry can be used to screen 5’tRNA halves binding proteins. The candidate proteins, DIS3L and GRSF1, may be the participants in process of 5’tRNA halves biological function and stability.