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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

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).
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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).

Mentions: It should be noted that although technically changes in mRNA levels are being measured in these hybridization assays, increased mRNA abundance in this polysome fraction would be expected to indicate increased levels of translation. However, some of which will also reflect in the levels of increased transcription or mRNA turn over. Changes in transcription would only be revealed when total steady-state mRNA abundance is measured if there is no translation control. We performed GO analysis using the DAVID Bioinformatics Resource to discover important gene pathways involved in post-transcriptional regulation (http://david.abcc.ncifcrf.gov/) (31) (Figure 4). 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 help us to identify functional related genes presented as a fuzzy heat map graphic view (Figure 4A). A zoomed-in view of cluster one is shown in Figure 4B. Most of the genes in cluster one are associated with protein translational initiation process. The fuzziness capability is a unique feature to maximally preserve biological patterns and to discover fine differences for a given gene. More than 20 clusters were represented with unique functions, such as protein synthesis, cell cycle control and RNA binding. The distribution of major classified gene clusters was presented as a pie chart (Figure 4C).Figure 4.


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)

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

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

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).
Mentions: It should be noted that although technically changes in mRNA levels are being measured in these hybridization assays, increased mRNA abundance in this polysome fraction would be expected to indicate increased levels of translation. However, some of which will also reflect in the levels of increased transcription or mRNA turn over. Changes in transcription would only be revealed when total steady-state mRNA abundance is measured if there is no translation control. We performed GO analysis using the DAVID Bioinformatics Resource to discover important gene pathways involved in post-transcriptional regulation (http://david.abcc.ncifcrf.gov/) (31) (Figure 4). 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 help us to identify functional related genes presented as a fuzzy heat map graphic view (Figure 4A). A zoomed-in view of cluster one is shown in Figure 4B. Most of the genes in cluster one are associated with protein translational initiation process. The fuzziness capability is a unique feature to maximally preserve biological patterns and to discover fine differences for a given gene. More than 20 clusters were represented with unique functions, such as protein synthesis, cell cycle control and RNA binding. The distribution of major classified gene clusters was presented as a pie chart (Figure 4C).Figure 4.

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