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

Kudo K, Xi Y, Wang Y, Song B, Chu E, Ju J, Russo JJ, Ju J - Nucleic Acids Res. (2010)

Bottom Line: 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.In contrast, the protein expression and polysome-associated mRNA levels of both genes were increased.

View Article: PubMed Central - PubMed

Affiliation: Mitchell Cancer Institute, Mobile, AL 36688, USA.

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.

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Related in: MedlinePlus

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.
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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.

Mentions: The new approach described here stemmed from previous studies indicating that mRNAs being actively translated are associated with multiple units of ribosomes (polyribosomes or polysomes) and the newly synthesized polypeptides are closely associated with molecular chaperones, including members of the hsp70 and hsc70 families (9–11). Chaperones can assist in the efficient folding of newly translated proteins as they are being synthesized on the ribosome and can maintain pre-existing proteins in a stable conformation to avoid premature folding and modifications (12,13). We therefore reasoned that such chaperones can provide a molecular anchor for separating polysome-loaded mRNAs from free mRNAs. In this study, we apply this principle to develop and optimize antibody affinity capture beads for capturing actively translated mRNAs associated with hsp70 family chaperones. The translationally active mRNAs in the polysome complex are pelleted by an antibody recognizing the hsp70/hsc70 chaperone proteins associated with nascent polypeptides of the complex (Figure 1). Bound RNAs are then purified and used for high-throughput expression profiling analysis.Figure 1.


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

Kudo K, Xi Y, Wang Y, Song B, Chu E, Ju J, Russo JJ, Ju J - Nucleic Acids Res. (2010)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875024&req=5

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.
Mentions: The new approach described here stemmed from previous studies indicating that mRNAs being actively translated are associated with multiple units of ribosomes (polyribosomes or polysomes) and the newly synthesized polypeptides are closely associated with molecular chaperones, including members of the hsp70 and hsc70 families (9–11). Chaperones can assist in the efficient folding of newly translated proteins as they are being synthesized on the ribosome and can maintain pre-existing proteins in a stable conformation to avoid premature folding and modifications (12,13). We therefore reasoned that such chaperones can provide a molecular anchor for separating polysome-loaded mRNAs from free mRNAs. In this study, we apply this principle to develop and optimize antibody affinity capture beads for capturing actively translated mRNAs associated with hsp70 family chaperones. The translationally active mRNAs in the polysome complex are pelleted by an antibody recognizing the hsp70/hsc70 chaperone proteins associated with nascent polypeptides of the complex (Figure 1). Bound RNAs are then purified and used for high-throughput expression profiling analysis.Figure 1.

Bottom Line: 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.In contrast, the protein expression and polysome-associated mRNA levels of both genes were increased.

View Article: PubMed Central - PubMed

Affiliation: Mitchell Cancer Institute, Mobile, AL 36688, USA.

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.

Show MeSH
Related in: MedlinePlus