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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

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).
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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).

Mentions: To determine the feasibility of isolating the hsp70-associated translational complex, we utilized an in vitro rabbit reticulocyte lysate coupled transcription/translation system with an expression construct pGEM-pcHTS-1 providing expression of full-length TS mRNA (15). Our results clearly indicate that we can immunoprecipitate the TS translation complex with hsp70 antibody affinity capture beads as shown in Figure 2. The absence of TS protein with control beads coupled to an anti-tubulin antibody (lane 1) indicates the specificity of our hsp70 antibody affinity capture beads (Figure 2A). We can detect the presence of newly synthesized TS protein in the isolated complex using western immunoblot analysis (lane 2) (Figure 2A). The associated TS mRNA template was also detected using real-time qRT-PCR analysis (lane 2, Figure 2B).Figure 2.


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)

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

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

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).
Mentions: To determine the feasibility of isolating the hsp70-associated translational complex, we utilized an in vitro rabbit reticulocyte lysate coupled transcription/translation system with an expression construct pGEM-pcHTS-1 providing expression of full-length TS mRNA (15). Our results clearly indicate that we can immunoprecipitate the TS translation complex with hsp70 antibody affinity capture beads as shown in Figure 2. The absence of TS protein with control beads coupled to an anti-tubulin antibody (lane 1) indicates the specificity of our hsp70 antibody affinity capture beads (Figure 2A). We can detect the presence of newly synthesized TS protein in the isolated complex using western immunoblot analysis (lane 2) (Figure 2A). The associated TS mRNA template was also detected using real-time qRT-PCR analysis (lane 2, Figure 2B).Figure 2.

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