Translational control analysis by translationally active RNA capture/microarray analysis (TrIP-Chip)

Abstract

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.

Figure 1.

Schematic diagram of TrIP–Chip approach. Affinity beads with covalently attached anti-Hsp70 antibodies are used to immunoprecipitate a cross-linked complex composed of Hsp70, nascent peptides, polysomes and the actively translating mRNAs. The mRNAs are used to conduct qPCR or converted to a labeled cRNA in a two-step reaction, and hybridized to whole genome microarrays for analysis. If translation is not taking place, there are no nascent peptides with which Hsp70 can associate.

Figure 2.

Western immunoblot analysis of in vitro translated TS protein expression isolated using hsp70/hsc70 antibody affinity capture beads (lane 2); unrelated α-tubulin antibody beads were used as negative control (lane 1) (A). Real-time qRT-PCR analysis of in vitro transcribed TS mRNA expression (lane 1, control; lane 2, TS mRNA) (B).

Figure 3.

Real-time qRT-PCR analysis of TS and p53 mRNA expression from total RNA isolated from control and 5-FU-treated HCT116 cells (n = 3, P < 0.05) (A); Real-time qRT-PCR analysis of TS, p53 and α-tubulin mRNA expression from hsp70/hsc70 antibody affinity capture bead isolated RNAs from control and 5-FU-treated HCT116 cells (n = 3, P < 0.05) (B); Western immunoblot analysis of the expression of TS and p53 proteins isolated using hsp70/hsc70s antibody affinity capture beads (lane 1, control; lane 2, treated with 5-FU) (C). The lower panel shows the quantified protein expression levels for TS and p53.

Figure 4.

GO analysis was performed using the DAVID bioinformatics suite. The DAVID Gene Functional Classification algorithm allows us to condense our gene list into organized classes based on cellular functions. The genes are grouped and clustered based on their cellular and molecular functions, which helps to identify functionally related genes, presented as a fuzzy heat map graphic view. The annotation terms are ordered based on the enrichment scores associated with the groups. Green represents a positive association between the gene terms; conversely, black represents an unknown relationship. The scattered pattern indicates functional differences. More than 20 clusters were represented with unique functions such as protein synthesis, cell cycle control and RNA binding (A). A zoomed-in view of the upper left cluster representing genes involved in translational initiation (B). The distribution of each set of classified genes is presented as a pie chart (C).

Figure 5.

Western immunoblot analysis of PP2A protein expression isolated using hsp70/hsc70 antibody affinity capture beads from control and 5-FU-treated HCT116 colon cancer cells (lane 1, control; lane 2, plus 5-FU). Quantification of the protein levels for PP2A is provided in the lower panel (A). Real-time qRT-PCR analysis of total PP2A mRNA and polysome associated mRNA levels in control and 5-FU-treated HCT-116 colon cancer cells (B).