Limits...
Comparing proteomics and RISC immunoprecipitations to identify targets of Epstein-Barr viral miRNAs.

Kuzembayeva M, Chiu YF, Sugden B - PLoS ONE (2012)

Bottom Line: Epstein-Barr virus is a gamma-herpes virus that is causally associated with several lymphomas and carcinomas.Currently, the function of these viral miRNAs and the contribution they provide to EBV's life-cycle remain largely unknown, due to difficulties in identifying cellular and viral genes regulated by these miRNAs.We have compared and contrasted two methods to identify targets of viral miRNAs in order to identify the advantages and limitations of each method to aid in uncovering the functions of EBV's miRNAs.

View Article: PubMed Central - PubMed

Affiliation: McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin, United States of America.

ABSTRACT
Epstein-Barr virus is a gamma-herpes virus that is causally associated with several lymphomas and carcinomas. This virus encodes at least 25 pre-miRNAs, which are expressed in infected cells to yield more than 50 detected mature miRNAs. miRNAs are small, non-coding RNAs that inhibit gene expression by promoting the inhibition of translation or of degradation of mRNAs. Currently, the function of these viral miRNAs and the contribution they provide to EBV's life-cycle remain largely unknown, due to difficulties in identifying cellular and viral genes regulated by these miRNAs. We have compared and contrasted two methods to identify targets of viral miRNAs in order to identify the advantages and limitations of each method to aid in uncovering the functions of EBV's miRNAs.

Show MeSH

Related in: MedlinePlus

Validating the candidate genes obtained via 2D DIGE and RISC IPs.A. Measuring levels of candidate transcripts obtained by RISC IPs in Ago2-immunoprecipitated RNA samples of BJAB BARTs and BJAB Empty cell lines by RT-PCR revealed an enrichment for IPO7 transcripts in immunoprecipitates of BJAB BARTs cells (*indicates p-value <0.0005, Student’s t-test). B. Reporter constructs containing the 3′UTRs of candidate genes were transfected into BJAB BARTs and BJAB Empty cells. The normalized luciferase activity in BJAB Empty cells was arbitrarily set to 100% (*indicates p-value <0.05, Student’s t-test). The luciferase activity of IPO7 3′UTR-pmiRGLO construct in BJAB BARTs cells decreased by 33%. C. Expression levels of CEBPA, PUMA, CNBP1 and IPO7 in BJAB Empty and BJAB BARTs cells were analyzed by immunoblotting, quantified by Image J software and normalized to levels of alpha-tubulin. The levels of expression of all proteins are indicated under the respective bands. The levels of IPO7 protein were found to decrease by 33% in the presence of BART miRNAs in three biological replicates while the levels of the other three proteins were unaffected by them. D. Nine candidates identified either by 2D DIGE or RISC IPs were examined further either by RT-PCR to measure their levels in RISCs, in reporter assays, or by immunoblotting where appropriate antibodies were available. IPO7 was validated by all three methods.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3472983&req=5

pone-0047409-g001: Validating the candidate genes obtained via 2D DIGE and RISC IPs.A. Measuring levels of candidate transcripts obtained by RISC IPs in Ago2-immunoprecipitated RNA samples of BJAB BARTs and BJAB Empty cell lines by RT-PCR revealed an enrichment for IPO7 transcripts in immunoprecipitates of BJAB BARTs cells (*indicates p-value <0.0005, Student’s t-test). B. Reporter constructs containing the 3′UTRs of candidate genes were transfected into BJAB BARTs and BJAB Empty cells. The normalized luciferase activity in BJAB Empty cells was arbitrarily set to 100% (*indicates p-value <0.05, Student’s t-test). The luciferase activity of IPO7 3′UTR-pmiRGLO construct in BJAB BARTs cells decreased by 33%. C. Expression levels of CEBPA, PUMA, CNBP1 and IPO7 in BJAB Empty and BJAB BARTs cells were analyzed by immunoblotting, quantified by Image J software and normalized to levels of alpha-tubulin. The levels of expression of all proteins are indicated under the respective bands. The levels of IPO7 protein were found to decrease by 33% in the presence of BART miRNAs in three biological replicates while the levels of the other three proteins were unaffected by them. D. Nine candidates identified either by 2D DIGE or RISC IPs were examined further either by RT-PCR to measure their levels in RISCs, in reporter assays, or by immunoblotting where appropriate antibodies were available. IPO7 was validated by all three methods.

Mentions: 2D DIGE and subsequent protein identification of differentially expressed spots was carried out by Applied Biomics, Inc. (Hayward, CA) (Table 1, Figure S2). This approach separated 2351 spots on the gels (Figure S2), which is consistent with the resolution achieved by other groups that have used this technique. Out of the total number of spots separated, 35 were decreased in intensity by 1.3-fold (30%) or more when compared to the BJAB Empty control which does not express EBV’s BART miRNAs (Table 1). The application of MALDI TOF/TOF mass spectrometry identified 28 unique and characterized proteins out of the 35 selected spots. This list of candidates was narrowed for further validation in as unbiased a manner as practical. Specifically, genes were selected for validation if their 3′UTRs were predicted to have sites complementary to the 3′ ends of BART miRNAs (Table 2). We used the Probability of Interaction by Target Accessibility (PITA) prediction algorithm to identify potential miRNA binding sites because this algorithm takes into account hybridization energy and binding site accessibility based on the secondary structure of the transcript, and does not rely on conservation of the miRNAs, a commonly used parameter not applicable to viral miRNAs [26]. We also evaluated the number of sites present in a given 3′UTR of a candidate mRNA, and whether the miRNAs predicted to target this gene are expressed efficiently or not (Figure S1). Four candidates were selected for further evaluation and examined by RT-PCR and/or immunoblotting (Table 2, Figure 1A, 1C).


Comparing proteomics and RISC immunoprecipitations to identify targets of Epstein-Barr viral miRNAs.

Kuzembayeva M, Chiu YF, Sugden B - PLoS ONE (2012)

Validating the candidate genes obtained via 2D DIGE and RISC IPs.A. Measuring levels of candidate transcripts obtained by RISC IPs in Ago2-immunoprecipitated RNA samples of BJAB BARTs and BJAB Empty cell lines by RT-PCR revealed an enrichment for IPO7 transcripts in immunoprecipitates of BJAB BARTs cells (*indicates p-value <0.0005, Student’s t-test). B. Reporter constructs containing the 3′UTRs of candidate genes were transfected into BJAB BARTs and BJAB Empty cells. The normalized luciferase activity in BJAB Empty cells was arbitrarily set to 100% (*indicates p-value <0.05, Student’s t-test). The luciferase activity of IPO7 3′UTR-pmiRGLO construct in BJAB BARTs cells decreased by 33%. C. Expression levels of CEBPA, PUMA, CNBP1 and IPO7 in BJAB Empty and BJAB BARTs cells were analyzed by immunoblotting, quantified by Image J software and normalized to levels of alpha-tubulin. The levels of expression of all proteins are indicated under the respective bands. The levels of IPO7 protein were found to decrease by 33% in the presence of BART miRNAs in three biological replicates while the levels of the other three proteins were unaffected by them. D. Nine candidates identified either by 2D DIGE or RISC IPs were examined further either by RT-PCR to measure their levels in RISCs, in reporter assays, or by immunoblotting where appropriate antibodies were available. IPO7 was validated by all three methods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3472983&req=5

pone-0047409-g001: Validating the candidate genes obtained via 2D DIGE and RISC IPs.A. Measuring levels of candidate transcripts obtained by RISC IPs in Ago2-immunoprecipitated RNA samples of BJAB BARTs and BJAB Empty cell lines by RT-PCR revealed an enrichment for IPO7 transcripts in immunoprecipitates of BJAB BARTs cells (*indicates p-value <0.0005, Student’s t-test). B. Reporter constructs containing the 3′UTRs of candidate genes were transfected into BJAB BARTs and BJAB Empty cells. The normalized luciferase activity in BJAB Empty cells was arbitrarily set to 100% (*indicates p-value <0.05, Student’s t-test). The luciferase activity of IPO7 3′UTR-pmiRGLO construct in BJAB BARTs cells decreased by 33%. C. Expression levels of CEBPA, PUMA, CNBP1 and IPO7 in BJAB Empty and BJAB BARTs cells were analyzed by immunoblotting, quantified by Image J software and normalized to levels of alpha-tubulin. The levels of expression of all proteins are indicated under the respective bands. The levels of IPO7 protein were found to decrease by 33% in the presence of BART miRNAs in three biological replicates while the levels of the other three proteins were unaffected by them. D. Nine candidates identified either by 2D DIGE or RISC IPs were examined further either by RT-PCR to measure their levels in RISCs, in reporter assays, or by immunoblotting where appropriate antibodies were available. IPO7 was validated by all three methods.
Mentions: 2D DIGE and subsequent protein identification of differentially expressed spots was carried out by Applied Biomics, Inc. (Hayward, CA) (Table 1, Figure S2). This approach separated 2351 spots on the gels (Figure S2), which is consistent with the resolution achieved by other groups that have used this technique. Out of the total number of spots separated, 35 were decreased in intensity by 1.3-fold (30%) or more when compared to the BJAB Empty control which does not express EBV’s BART miRNAs (Table 1). The application of MALDI TOF/TOF mass spectrometry identified 28 unique and characterized proteins out of the 35 selected spots. This list of candidates was narrowed for further validation in as unbiased a manner as practical. Specifically, genes were selected for validation if their 3′UTRs were predicted to have sites complementary to the 3′ ends of BART miRNAs (Table 2). We used the Probability of Interaction by Target Accessibility (PITA) prediction algorithm to identify potential miRNA binding sites because this algorithm takes into account hybridization energy and binding site accessibility based on the secondary structure of the transcript, and does not rely on conservation of the miRNAs, a commonly used parameter not applicable to viral miRNAs [26]. We also evaluated the number of sites present in a given 3′UTR of a candidate mRNA, and whether the miRNAs predicted to target this gene are expressed efficiently or not (Figure S1). Four candidates were selected for further evaluation and examined by RT-PCR and/or immunoblotting (Table 2, Figure 1A, 1C).

Bottom Line: Epstein-Barr virus is a gamma-herpes virus that is causally associated with several lymphomas and carcinomas.Currently, the function of these viral miRNAs and the contribution they provide to EBV's life-cycle remain largely unknown, due to difficulties in identifying cellular and viral genes regulated by these miRNAs.We have compared and contrasted two methods to identify targets of viral miRNAs in order to identify the advantages and limitations of each method to aid in uncovering the functions of EBV's miRNAs.

View Article: PubMed Central - PubMed

Affiliation: McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin, United States of America.

ABSTRACT
Epstein-Barr virus is a gamma-herpes virus that is causally associated with several lymphomas and carcinomas. This virus encodes at least 25 pre-miRNAs, which are expressed in infected cells to yield more than 50 detected mature miRNAs. miRNAs are small, non-coding RNAs that inhibit gene expression by promoting the inhibition of translation or of degradation of mRNAs. Currently, the function of these viral miRNAs and the contribution they provide to EBV's life-cycle remain largely unknown, due to difficulties in identifying cellular and viral genes regulated by these miRNAs. We have compared and contrasted two methods to identify targets of viral miRNAs in order to identify the advantages and limitations of each method to aid in uncovering the functions of EBV's miRNAs.

Show MeSH
Related in: MedlinePlus