Translational control analysis by translationally active RNA capture/microarray analysis (TrIP-Chip).
We have developed a new approach to systematically study post-transcriptional regulation in a small number of cells. Actively translating mRNAs are associated with polysomes and the newly synthesized peptide chains are closely associated with molecular chaperones such as hsp70s, which assist in the proper folding of nascent polypeptides into higher ordered structures. These chaperones provide an anchor with which to separate actively translating mRNAs associated with polysomes from free mRNAs. Affinity capture beads were developed to capture hsp70 chaperones associated with the polysome complexes. The isolated actively translating mRNAs were used for high-throughput expression profiling analysis. Feasibility was demonstrated using an in vitro translation system with known translationally regulated mRNA transcript thymidylate synthase (TS). We further developed the approach using HCT-116 colon cancer cells with both TS and p53 as positive controls. The steady-state levels of TS and p53 mRNAs were unaltered after 5-fluorouracil treatment as assessed by real-time qRT-PCR analysis. In contrast, the protein expression and polysome-associated mRNA levels of both genes were increased. These differences in translational rate were revealed with our new approach from 500 cells. This technology has the potential to make investigation of translational control feasible with limited quantities of clinical specimens.
Kudo
Kenji
K
Mitchell Cancer Institute, Mobile, AL 36688, USA.
Xi
Yaguang
Y
Wang
Yuan
Y
Song
Bo
B
Chu
Edward
E
Ju
Jingyue
J
Russo
James J
JJ
Ju
Jingfang
J
D000906:Antibodies; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D045744:Cell Line, Tumor; D005472:Fluorouracil / Q000494:pharmacology; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D018840:HSP70 Heat-Shock Proteins / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D047468:Immunoprecipitation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000302:isolation & purification*
D000906:Antibodies; D000964:Antimetabolites, Antineoplastic; D018840:HSP70 Heat-Shock Proteins; D012333:RNA, Messenger; D005472:Fluorouracil