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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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Altered expression of CFIm25 modulates GBM tumor growth(a) The global analysis of 3′UTR changes in GBM patient samples with either high or low levels of CFIm25. Scatterplot of PDUIs from both datasets using the same cutoffs as in Figure 2c. The shifting to proximal PAS in the low CFIm25 group is significant (P<2.2e-16; binomial test). (b) Representative UCSC Genome Browser images of RNA-seq data demonstrating 3′UTR shortening after CFIm25 knockdown in HeLa and in GBM patient samples having high (blue) or low CFIm25 expression (red). (c) Western blot analysis of lysates from two GBM cell lines. Note the overexpressed myc-CFIm25 also increases endogenous CFIm25 levels in U251 cells. (d) Growth comparison of U251 tumors overexpressing either GFP (control) or CFIm25 and data represents the average of 10 mice per group. Right panel is representative H/E and Ki67 staining of U251-GFP tumors (upper) or U251-CFIm25 tumors (lower). (e) Growth comparison of LN229 tumors derived from cells transduced with lentiviruses expressing a scrambled shRNA (control) or with lentiviruses expressing shRNA targeting CFIm25. The data represents the average of 10 mice per group. Right panel is representative H/E and Ki67 staining of LN229 tumors expressing shRNA targeting CFIm25 (upper) or LN229 tumors expressing scrambled shRNA (lower).
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Figure 13: Altered expression of CFIm25 modulates GBM tumor growth(a) The global analysis of 3′UTR changes in GBM patient samples with either high or low levels of CFIm25. Scatterplot of PDUIs from both datasets using the same cutoffs as in Figure 2c. The shifting to proximal PAS in the low CFIm25 group is significant (P<2.2e-16; binomial test). (b) Representative UCSC Genome Browser images of RNA-seq data demonstrating 3′UTR shortening after CFIm25 knockdown in HeLa and in GBM patient samples having high (blue) or low CFIm25 expression (red). (c) Western blot analysis of lysates from two GBM cell lines. Note the overexpressed myc-CFIm25 also increases endogenous CFIm25 levels in U251 cells. (d) Growth comparison of U251 tumors overexpressing either GFP (control) or CFIm25 and data represents the average of 10 mice per group. Right panel is representative H/E and Ki67 staining of U251-GFP tumors (upper) or U251-CFIm25 tumors (lower). (e) Growth comparison of LN229 tumors derived from cells transduced with lentiviruses expressing a scrambled shRNA (control) or with lentiviruses expressing shRNA targeting CFIm25. The data represents the average of 10 mice per group. Right panel is representative H/E and Ki67 staining of LN229 tumors expressing shRNA targeting CFIm25 (upper) or LN229 tumors expressing scrambled shRNA (lower).

Mentions: The collective observations that CFIm25 depletion leads to broad 3′UTR shortening, enhanced expression of growth promoting genes, and increased cell proliferation support the hypothesis that CFIm25 is a novel anti-proliferative gene whose levels may be reduced in human cancers. We focused our analysis on glioblastoma (GBM) as recent reports indicate that brain tissue possesses the longest 3′UTRs26,27. We reasoned that tumors derived from these cells might be more sensitive to changes in CFIm25 levels than other cancers. To test this prediction, we downloaded archived patient RNA-seq data from TCGA, stratified it according to CFIm25 expression, and analyzed it using DaPars. Indeed, following the same cutoffs in our HeLa RNA-seq 3′UTR analysis, we identified 60 genes with altered 3′UTRs and 59 of those experienced shortening in GBM (Fig. 4a and Table S2). Among those genes, a significant number of events (24 genes; P=2.2e-12 by hypergeometric testing) also shortened in CFIm25 knockdown HeLa cell line and this percentage of overlap increased dramatically to 86% as the ΔPDUI cutoff increased from 0.2 to 0.4 (Extended Data Fig. 6). Two representative examples of genes, Fos-related antigen 2 (Fra-2) and MecP2, with shortened 3′UTRs in low CFIm25 expressing GBM tumors is shown in Figure 4b demonstrating a compelling similarity between the patient samples and HeLa cells before and after CFIm25 knockdown. Overexpression of either of these genes has been shown to enhance cell proliferation18,28.


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)

Altered expression of CFIm25 modulates GBM tumor growth(a) The global analysis of 3′UTR changes in GBM patient samples with either high or low levels of CFIm25. Scatterplot of PDUIs from both datasets using the same cutoffs as in Figure 2c. The shifting to proximal PAS in the low CFIm25 group is significant (P<2.2e-16; binomial test). (b) Representative UCSC Genome Browser images of RNA-seq data demonstrating 3′UTR shortening after CFIm25 knockdown in HeLa and in GBM patient samples having high (blue) or low CFIm25 expression (red). (c) Western blot analysis of lysates from two GBM cell lines. Note the overexpressed myc-CFIm25 also increases endogenous CFIm25 levels in U251 cells. (d) Growth comparison of U251 tumors overexpressing either GFP (control) or CFIm25 and data represents the average of 10 mice per group. Right panel is representative H/E and Ki67 staining of U251-GFP tumors (upper) or U251-CFIm25 tumors (lower). (e) Growth comparison of LN229 tumors derived from cells transduced with lentiviruses expressing a scrambled shRNA (control) or with lentiviruses expressing shRNA targeting CFIm25. The data represents the average of 10 mice per group. Right panel is representative H/E and Ki67 staining of LN229 tumors expressing shRNA targeting CFIm25 (upper) or LN229 tumors expressing scrambled shRNA (lower).
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Figure 13: Altered expression of CFIm25 modulates GBM tumor growth(a) The global analysis of 3′UTR changes in GBM patient samples with either high or low levels of CFIm25. Scatterplot of PDUIs from both datasets using the same cutoffs as in Figure 2c. The shifting to proximal PAS in the low CFIm25 group is significant (P<2.2e-16; binomial test). (b) Representative UCSC Genome Browser images of RNA-seq data demonstrating 3′UTR shortening after CFIm25 knockdown in HeLa and in GBM patient samples having high (blue) or low CFIm25 expression (red). (c) Western blot analysis of lysates from two GBM cell lines. Note the overexpressed myc-CFIm25 also increases endogenous CFIm25 levels in U251 cells. (d) Growth comparison of U251 tumors overexpressing either GFP (control) or CFIm25 and data represents the average of 10 mice per group. Right panel is representative H/E and Ki67 staining of U251-GFP tumors (upper) or U251-CFIm25 tumors (lower). (e) Growth comparison of LN229 tumors derived from cells transduced with lentiviruses expressing a scrambled shRNA (control) or with lentiviruses expressing shRNA targeting CFIm25. The data represents the average of 10 mice per group. Right panel is representative H/E and Ki67 staining of LN229 tumors expressing shRNA targeting CFIm25 (upper) or LN229 tumors expressing scrambled shRNA (lower).
Mentions: The collective observations that CFIm25 depletion leads to broad 3′UTR shortening, enhanced expression of growth promoting genes, and increased cell proliferation support the hypothesis that CFIm25 is a novel anti-proliferative gene whose levels may be reduced in human cancers. We focused our analysis on glioblastoma (GBM) as recent reports indicate that brain tissue possesses the longest 3′UTRs26,27. We reasoned that tumors derived from these cells might be more sensitive to changes in CFIm25 levels than other cancers. To test this prediction, we downloaded archived patient RNA-seq data from TCGA, stratified it according to CFIm25 expression, and analyzed it using DaPars. Indeed, following the same cutoffs in our HeLa RNA-seq 3′UTR analysis, we identified 60 genes with altered 3′UTRs and 59 of those experienced shortening in GBM (Fig. 4a and Table S2). Among those genes, a significant number of events (24 genes; P=2.2e-12 by hypergeometric testing) also shortened in CFIm25 knockdown HeLa cell line and this percentage of overlap increased dramatically to 86% as the ΔPDUI cutoff increased from 0.2 to 0.4 (Extended Data Fig. 6). Two representative examples of genes, Fos-related antigen 2 (Fra-2) and MecP2, with shortened 3′UTRs in low CFIm25 expressing GBM tumors is shown in Figure 4b demonstrating a compelling similarity between the patient samples and HeLa cells before and after CFIm25 knockdown. Overexpression of either of these genes has been shown to enhance cell proliferation18,28.

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