RNA-binding protein exerts important biological function by specifically recognizing RNA motif. SELEX (Systematic evolution of ligands by exponential enrichment), an in vitro selection method, can obtain consensus motif with high-affinity and specificity for many target molecules from DNA or RNA libraries. Here, we combined SELEX with next-generation sequencing to study the protein-RNA interaction in vitro. A pool of RNAs with 20 bp random sequences were transcribed by T7 promoter, and target protein was inserted into plasmid containing SBP-tag, which can be captured by streptavidin beads. Through only one cycle, the specific RNA motif can be obtained, which dramatically improved the selection efficiency. Using this method, we found that human hnRNP A1 RRMs domain (UP1 domain) bound RNA motifs containing AGG and AG sequences. The EMSA experiment indicated that hnRNP A1 RRMs could bind the obtained RNA motif. Taken together, this method provides a rapid and effective method to study the RNA binding specificity of proteins.
Aptamers, Nucleotide ; Gene Library ; Heterogeneous Nuclear Ribonucleoprotein A1 ; genetics ; High-Throughput Nucleotide Sequencing ; Humans ; RNA ; SELEX Aptamer Technique

OBJECTIVETo identify and compare the expression profiles of differential proteins between chronic phase and blast crisis in chronic myeloid leukemia (CML) by proteomic analysis, and screen the proteins related to blast crisis.

METHODSThe total cellular proteins from the bone marrow cells at chronic phase (CP) and blast crisis (BC) in CML were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE) and analyzed with ImageMaster 5.0 software to screen the differential protein spots. Differential protein spots were identified by mass spectrometry for peptide mass fingerprint in combination with database searching from SWISS-PROT. Then 3 protein spots were selected to verify at protein and mRNA levels by Western blot and semi-quantitative RT-PCR, separately.

RESULTSComparing gel pages from CML-CP and CML-BC, the expression of 13 protein spots decreased and 25 protein spots increased significantly in CML-BC. Twenty differential protein spots were identified by mass spectrometry and 15 were successfully determined. The results of Western blotting were similar to those of 2-DE and showed a high expression of hnRNPK, annexin A1 and RhoA. Semi-quantitative RT-PCR analysis showed that there was no correlation between the protein expression changes and mRNA levels of hnRNPK, annexin A1 and RhoA.

CONCLUSIONA group of proteins associated with blast crisis are obtained and the results may provide clues for further research to elucidate the role of these proteins in CML-BC carcinogenesis and to develop potential associated biomarkers.

Adult ; Aged ; Annexin A1 ; genetics ; metabolism ; Blast Crisis ; genetics ; metabolism ; Female ; Gene Expression Profiling ; Heterogeneous-Nuclear Ribonucleoprotein K ; Humans ; Leukemia, Myeloid, Chronic-Phase ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Proteomics ; methods ; RNA, Messenger ; metabolism ; Ribonucleoproteins ; genetics ; metabolism ; Young Adult ; rhoA GTP-Binding Protein ; genetics ; metabolism