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CFIm25 links alternative polyadenylation to glioblastoma tumour suppression.

Masamha CP, Xia Z, Yang J, Albrecht TR, Li M, Shyu AB, Li W, Wagner EJ - Nature (2014)

Bottom Line: Applying a regression model on standard RNA-sequencing data for novel APA events, we identified at least 1,450 genes with shortened 3' UTRs after CFIm25 knockdown, representing 11% of significantly expressed mRNAs in human cells.Marked increases in the expression of several known oncogenes, including cyclin D1, are observed as a consequence of CFIm25 depletion.Downregulation of CFIm25 expression in glioblastoma cells enhances their tumorigenic properties and increases tumour size, whereas CFIm25 overexpression reduces these properties and inhibits tumour growth.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry and Molecular Biology, The University of Texas Medical School at Houston, Houston, Texas 77030, USA [2].

ABSTRACT
The global shortening of messenger RNAs through alternative polyadenylation (APA) that occurs during enhanced cellular proliferation represents an important, yet poorly understood mechanism of regulated gene expression. The 3' untranslated region (UTR) truncation of growth-promoting mRNA transcripts that relieves intrinsic microRNA- and AU-rich-element-mediated repression has been observed to correlate with cellular transformation; however, the importance to tumorigenicity of RNA 3'-end-processing factors that potentially govern APA is unknown. Here we identify CFIm25 as a broad repressor of proximal poly(A) site usage that, when depleted, increases cell proliferation. Applying a regression model on standard RNA-sequencing data for novel APA events, we identified at least 1,450 genes with shortened 3' UTRs after CFIm25 knockdown, representing 11% of significantly expressed mRNAs in human cells. Marked increases in the expression of several known oncogenes, including cyclin D1, are observed as a consequence of CFIm25 depletion. Importantly, we identified a subset of CFIm25-regulated APA genes with shortened 3' UTRs in glioblastoma tumours that have reduced CFIm25 expression. Downregulation of CFIm25 expression in glioblastoma cells enhances their tumorigenic properties and increases tumour size, whereas CFIm25 overexpression reduces these properties and inhibits tumour growth. These findings identify a pivotal role of CFIm25 in governing APA and reveal a previously unknown connection between CFIm25 and glioblastoma tumorigenicity.

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Design and optimization of the qRT-PCR assay to monitor APA of three test genes(a) Schematic denotes the relative location of the common and distal primer annealing sites in each test gene and the approximate locations of the annotated proximal and distal poly(A) sites depicted as pPAS and dPAS, respectively. The numbers demarcate where the 3′UTR starts and ends according to ENSEMBL. (b) Ethidium stained agarose gel of RT-PCR products of equal cycle number from the different amplicons using HeLa cell mRNA. (c) Both the common and distal Cyclin D1 amplicons were cloned into the same pcDNA3 plasmid in tandem. Three dilutions of each plasmid were made and amplified individually with each amplicon in triplicate. The 2 lines on the graph depict the amplification curve for the common and distal amplicons. The expectation is that identical Ct values should be attained for each, given that the PCR reactions were conducted using identical amounts of starting material. The average of 3 individual experiments is shown for each dilution and the average Ct deviation of either amplicon at all of the dilutions was calculated as a correction factor. (d) The experiment shown in panel c was repeated for Dicer-1 and Timp-2 to determine their respective correction factors, which was then applied to experiments shown in Figure 1.
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Figure 1: Design and optimization of the qRT-PCR assay to monitor APA of three test genes(a) Schematic denotes the relative location of the common and distal primer annealing sites in each test gene and the approximate locations of the annotated proximal and distal poly(A) sites depicted as pPAS and dPAS, respectively. The numbers demarcate where the 3′UTR starts and ends according to ENSEMBL. (b) Ethidium stained agarose gel of RT-PCR products of equal cycle number from the different amplicons using HeLa cell mRNA. (c) Both the common and distal Cyclin D1 amplicons were cloned into the same pcDNA3 plasmid in tandem. Three dilutions of each plasmid were made and amplified individually with each amplicon in triplicate. The 2 lines on the graph depict the amplification curve for the common and distal amplicons. The expectation is that identical Ct values should be attained for each, given that the PCR reactions were conducted using identical amounts of starting material. The average of 3 individual experiments is shown for each dilution and the average Ct deviation of either amplicon at all of the dilutions was calculated as a correction factor. (d) The experiment shown in panel c was repeated for Dicer-1 and Timp-2 to determine their respective correction factors, which was then applied to experiments shown in Figure 1.

Mentions: To measure relative changes in endogenous APA events, we devised a quantitative RT-PCR (qRT-PCR) assay to monitor the transcript-specific use of the distal PAS (dPAS) while normalizing for total mRNA levels for three test transcripts, Cyclin D1, Dicer1, and Timp2 known to undergo APA3,12. Using this approach, we readily detected appreciable usage of dPASs for all three genes in HeLa cells (Extended Data Fig. 1). This was somewhat surprising given their highly transformed state, but is consistent with previous reports that not all transformed cells tested exhibit appreciable 3′UTR shortening1,3. Previous studies implicate multiple members of the cleavage and polyadenylation (CPA) machinery as potentially regulating poly(A) site selection12-15. To test the relative contribution of these factors to the APA of the three test genes, we utilized systematic RNAi (Fig. 1a-c). We observed only small changes in the relative use of the dPAS after knockdown of members of the CPSF/CstF/CFIIm complexes (Fig. 1d-e). In contrast, we detected significant reduction in dPAS usage after knockdown of the members of the CFIm complex. These results are consistent with a recent report that CFIm68 depletion decreases 3′UTR length14; however, the most significant PAS switching was found to occur after knockdown of CFIm25. We therefore focused all further analyses on CFIm25.


CFIm25 links alternative polyadenylation to glioblastoma tumour suppression.

Masamha CP, Xia Z, Yang J, Albrecht TR, Li M, Shyu AB, Li W, Wagner EJ - Nature (2014)

Design and optimization of the qRT-PCR assay to monitor APA of three test genes(a) Schematic denotes the relative location of the common and distal primer annealing sites in each test gene and the approximate locations of the annotated proximal and distal poly(A) sites depicted as pPAS and dPAS, respectively. The numbers demarcate where the 3′UTR starts and ends according to ENSEMBL. (b) Ethidium stained agarose gel of RT-PCR products of equal cycle number from the different amplicons using HeLa cell mRNA. (c) Both the common and distal Cyclin D1 amplicons were cloned into the same pcDNA3 plasmid in tandem. Three dilutions of each plasmid were made and amplified individually with each amplicon in triplicate. The 2 lines on the graph depict the amplification curve for the common and distal amplicons. The expectation is that identical Ct values should be attained for each, given that the PCR reactions were conducted using identical amounts of starting material. The average of 3 individual experiments is shown for each dilution and the average Ct deviation of either amplicon at all of the dilutions was calculated as a correction factor. (d) The experiment shown in panel c was repeated for Dicer-1 and Timp-2 to determine their respective correction factors, which was then applied to experiments shown in Figure 1.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4128630&req=5

Figure 1: Design and optimization of the qRT-PCR assay to monitor APA of three test genes(a) Schematic denotes the relative location of the common and distal primer annealing sites in each test gene and the approximate locations of the annotated proximal and distal poly(A) sites depicted as pPAS and dPAS, respectively. The numbers demarcate where the 3′UTR starts and ends according to ENSEMBL. (b) Ethidium stained agarose gel of RT-PCR products of equal cycle number from the different amplicons using HeLa cell mRNA. (c) Both the common and distal Cyclin D1 amplicons were cloned into the same pcDNA3 plasmid in tandem. Three dilutions of each plasmid were made and amplified individually with each amplicon in triplicate. The 2 lines on the graph depict the amplification curve for the common and distal amplicons. The expectation is that identical Ct values should be attained for each, given that the PCR reactions were conducted using identical amounts of starting material. The average of 3 individual experiments is shown for each dilution and the average Ct deviation of either amplicon at all of the dilutions was calculated as a correction factor. (d) The experiment shown in panel c was repeated for Dicer-1 and Timp-2 to determine their respective correction factors, which was then applied to experiments shown in Figure 1.
Mentions: To measure relative changes in endogenous APA events, we devised a quantitative RT-PCR (qRT-PCR) assay to monitor the transcript-specific use of the distal PAS (dPAS) while normalizing for total mRNA levels for three test transcripts, Cyclin D1, Dicer1, and Timp2 known to undergo APA3,12. Using this approach, we readily detected appreciable usage of dPASs for all three genes in HeLa cells (Extended Data Fig. 1). This was somewhat surprising given their highly transformed state, but is consistent with previous reports that not all transformed cells tested exhibit appreciable 3′UTR shortening1,3. Previous studies implicate multiple members of the cleavage and polyadenylation (CPA) machinery as potentially regulating poly(A) site selection12-15. To test the relative contribution of these factors to the APA of the three test genes, we utilized systematic RNAi (Fig. 1a-c). We observed only small changes in the relative use of the dPAS after knockdown of members of the CPSF/CstF/CFIIm complexes (Fig. 1d-e). In contrast, we detected significant reduction in dPAS usage after knockdown of the members of the CFIm complex. These results are consistent with a recent report that CFIm68 depletion decreases 3′UTR length14; however, the most significant PAS switching was found to occur after knockdown of CFIm25. We therefore focused all further analyses on CFIm25.

Bottom Line: Applying a regression model on standard RNA-sequencing data for novel APA events, we identified at least 1,450 genes with shortened 3' UTRs after CFIm25 knockdown, representing 11% of significantly expressed mRNAs in human cells.Marked increases in the expression of several known oncogenes, including cyclin D1, are observed as a consequence of CFIm25 depletion.Downregulation of CFIm25 expression in glioblastoma cells enhances their tumorigenic properties and increases tumour size, whereas CFIm25 overexpression reduces these properties and inhibits tumour growth.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry and Molecular Biology, The University of Texas Medical School at Houston, Houston, Texas 77030, USA [2].

ABSTRACT
The global shortening of messenger RNAs through alternative polyadenylation (APA) that occurs during enhanced cellular proliferation represents an important, yet poorly understood mechanism of regulated gene expression. The 3' untranslated region (UTR) truncation of growth-promoting mRNA transcripts that relieves intrinsic microRNA- and AU-rich-element-mediated repression has been observed to correlate with cellular transformation; however, the importance to tumorigenicity of RNA 3'-end-processing factors that potentially govern APA is unknown. Here we identify CFIm25 as a broad repressor of proximal poly(A) site usage that, when depleted, increases cell proliferation. Applying a regression model on standard RNA-sequencing data for novel APA events, we identified at least 1,450 genes with shortened 3' UTRs after CFIm25 knockdown, representing 11% of significantly expressed mRNAs in human cells. Marked increases in the expression of several known oncogenes, including cyclin D1, are observed as a consequence of CFIm25 depletion. Importantly, we identified a subset of CFIm25-regulated APA genes with shortened 3' UTRs in glioblastoma tumours that have reduced CFIm25 expression. Downregulation of CFIm25 expression in glioblastoma cells enhances their tumorigenic properties and increases tumour size, whereas CFIm25 overexpression reduces these properties and inhibits tumour growth. These findings identify a pivotal role of CFIm25 in governing APA and reveal a previously unknown connection between CFIm25 and glioblastoma tumorigenicity.

Show MeSH
Related in: MedlinePlus