Identification of miR-215 mediated targets/pathways via translational immunoprecipitation expression analysis (TrIP-chip).
Steady state mRNA expression profiling can identify the majority of miRNA targets. However, some translationally repressed miRNA targets are missed and thus not considered for functional validation. Therefore, analysis of mRNA translation can enhance miRNA target identification for functional studies. We have applied a unique approach to identify miRNA targets in a small number of cells. Actively translating mRNAs are associated with polyribosomes and newly synthesized peptide chains are associated with molecular chaperones such as HSP70s. Affinity capture beads were used to capture HSP70 chaperones associated with polyribosome complexes. The isolated actively translating mRNAs were used for high throughput expression profiling analysis. miR-215 is an important miRNA in colorectal cancer and loss of miR-215 is significantly associated with prognosis of this disease. miR-215 suppresses the expression of several key targets. We utilized the affinity capture approach to isolate miR-215 mediated mRNA target transcripts. This approach provides a unique way to identify targets regulated by non-coding RNAs and RNA binding proteins from a small number of cells.
Department of Pathology, Stony Brook University, School of Medicine, Stony Brook, NY, USA.
Andrew
A
Fesler
Icahn School of Medicine at Mount Sinai, New York, NY, USA.
Xiao
X
Xu
The Third Affiliated Hospital, Soochow University, China.
Xiao
X
Zheng
The Third Affiliated Hospital, Soochow University, China.
Xiaodong
X
Li
The Third Affiliated Hospital, Soochow University, China.
Jingting
J
Jiang
Center for Genome Technology and Biomolecular Engineering, Department of Chemical Engineering, Columbia University, New York, NY, USA.
James J
JJ
Russo
Department of Pathology, Stony Brook University, School of Medicine, Stony Brook, NY, USA.
Jingfang
J
Ju
D000465:Algorithms; D015179:Colorectal Neoplasms / Q000378:metabolism*; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D045325:HCT116 Cells; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D035683:MicroRNAs / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000378:metabolism
D018840:HSP70 Heat-Shock Proteins; C553667:MIRN215 microRNA, human; D035683:MicroRNAs; D018832:Molecular Chaperones; D012333:RNA, Messenger
HSP70; miR-215; protein chaperone; translation