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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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Shortened transcripts have more “UGUA” CFIm25 binding motifs than unaltered transcripts(a) CFIm25 is known to bind to the UGUA motif. The number of UGUA motifs within the 3′UTRs of genes with 3′UTR shortening after CFIm25 knockdown relative to genes with unaltered 3′UTRs was calculated and compared. Here we selected the genes without 3′UTR change according to /ΔPDUI/≤0.05. (b) iCLIP tags from Martin et al (GEO accession number GSE37398) was superimposed onto data derived from PDUI analysis of CFIm25 knockdown cells. The box plot demonstrates the enrichment of CFIm25 binding within 3′UTRs that are altered after CFIm25 knockdown (P = 6.1e-107, t-test).
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Figure 3: Shortened transcripts have more “UGUA” CFIm25 binding motifs than unaltered transcripts(a) CFIm25 is known to bind to the UGUA motif. The number of UGUA motifs within the 3′UTRs of genes with 3′UTR shortening after CFIm25 knockdown relative to genes with unaltered 3′UTRs was calculated and compared. Here we selected the genes without 3′UTR change according to /ΔPDUI/≤0.05. (b) iCLIP tags from Martin et al (GEO accession number GSE37398) was superimposed onto data derived from PDUI analysis of CFIm25 knockdown cells. The box plot demonstrates the enrichment of CFIm25 binding within 3′UTRs that are altered after CFIm25 knockdown (P = 6.1e-107, t-test).

Mentions: Based on this promising observation, we applied a novel bioinformatics algorithm termed “Dynamic Analysis of Alternative Polyadenylation from RNA-Seq” (DaPars; see Supplemental Methods) for the de novo identification of all instances of 3′UTR alterations between control and CFIm25 knockdown cells, regardless of a pre-annotated pPAS within each Refseq transcript. DaPars uses a linear regression model to identify the exact location of this novel proximal 3′UTR as the optimal fitting point (red point in Fig. 2b) as well as the abundance of both novel and annotated UTRs. The degree of difference of 3′UTR usage between the samples was then quantified as a change in percentage dPAS usage index (ΔPDUI), which is capable of identifying lengthening (positive index) or shortening (negative index) within the 3′UTR. When applied to the 12,273 RefSeq transcripts whose average terminal exon sequence coverage is more than 30-fold, DaPars identified 1,453 transcripts possessing a significant, reproducible shift in 3′UTR usage in response to CFIm25 depletion (Fig. 2c and Extended Data Fig. 2c/d). Strikingly, among this group of transcripts, 1,450 are shifted to proximal PAS usage in CFIm25 knockdown. We found a significant enrichment of the CFIm25 UGUA binding motif and previously reported CFIm25 iCLIP sequence tags14 within 3′UTRs that shortened after CFIm25 knockdown relative to transcripts exhibiting no length change (Extended Data Fig. 3). Moreover, we determined that 70% of transcripts whose 3′UTR is shortened after CFIm25 knockdown, utilize a pPAS within the first one-third of their 3′UTR. In contrast, only 29% of multi-PAS transcripts that did not alter 3′UTR length in response to CFIm25 have an annotated pPAS in the first one-third of their 3′UTR. This demonstrates that CFIm25 APA targets are enriched with pPASs positioned close to the stop codon to maximize their degree of 3′UTR shortening. Collectively, these results clearly indicate that the function of CFIm25 is to broadly repress proximal poly(A) site choice, and consequently, the shortening of 3′UTR length is dramatic for the majority of CFIm25 regulated transcripts upon its depletion.


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)

Shortened transcripts have more “UGUA” CFIm25 binding motifs than unaltered transcripts(a) CFIm25 is known to bind to the UGUA motif. The number of UGUA motifs within the 3′UTRs of genes with 3′UTR shortening after CFIm25 knockdown relative to genes with unaltered 3′UTRs was calculated and compared. Here we selected the genes without 3′UTR change according to /ΔPDUI/≤0.05. (b) iCLIP tags from Martin et al (GEO accession number GSE37398) was superimposed onto data derived from PDUI analysis of CFIm25 knockdown cells. The box plot demonstrates the enrichment of CFIm25 binding within 3′UTRs that are altered after CFIm25 knockdown (P = 6.1e-107, t-test).
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Figure 3: Shortened transcripts have more “UGUA” CFIm25 binding motifs than unaltered transcripts(a) CFIm25 is known to bind to the UGUA motif. The number of UGUA motifs within the 3′UTRs of genes with 3′UTR shortening after CFIm25 knockdown relative to genes with unaltered 3′UTRs was calculated and compared. Here we selected the genes without 3′UTR change according to /ΔPDUI/≤0.05. (b) iCLIP tags from Martin et al (GEO accession number GSE37398) was superimposed onto data derived from PDUI analysis of CFIm25 knockdown cells. The box plot demonstrates the enrichment of CFIm25 binding within 3′UTRs that are altered after CFIm25 knockdown (P = 6.1e-107, t-test).
Mentions: Based on this promising observation, we applied a novel bioinformatics algorithm termed “Dynamic Analysis of Alternative Polyadenylation from RNA-Seq” (DaPars; see Supplemental Methods) for the de novo identification of all instances of 3′UTR alterations between control and CFIm25 knockdown cells, regardless of a pre-annotated pPAS within each Refseq transcript. DaPars uses a linear regression model to identify the exact location of this novel proximal 3′UTR as the optimal fitting point (red point in Fig. 2b) as well as the abundance of both novel and annotated UTRs. The degree of difference of 3′UTR usage between the samples was then quantified as a change in percentage dPAS usage index (ΔPDUI), which is capable of identifying lengthening (positive index) or shortening (negative index) within the 3′UTR. When applied to the 12,273 RefSeq transcripts whose average terminal exon sequence coverage is more than 30-fold, DaPars identified 1,453 transcripts possessing a significant, reproducible shift in 3′UTR usage in response to CFIm25 depletion (Fig. 2c and Extended Data Fig. 2c/d). Strikingly, among this group of transcripts, 1,450 are shifted to proximal PAS usage in CFIm25 knockdown. We found a significant enrichment of the CFIm25 UGUA binding motif and previously reported CFIm25 iCLIP sequence tags14 within 3′UTRs that shortened after CFIm25 knockdown relative to transcripts exhibiting no length change (Extended Data Fig. 3). Moreover, we determined that 70% of transcripts whose 3′UTR is shortened after CFIm25 knockdown, utilize a pPAS within the first one-third of their 3′UTR. In contrast, only 29% of multi-PAS transcripts that did not alter 3′UTR length in response to CFIm25 have an annotated pPAS in the first one-third of their 3′UTR. This demonstrates that CFIm25 APA targets are enriched with pPASs positioned close to the stop codon to maximize their degree of 3′UTR shortening. Collectively, these results clearly indicate that the function of CFIm25 is to broadly repress proximal poly(A) site choice, and consequently, the shortening of 3′UTR length is dramatic for the majority of CFIm25 regulated transcripts upon its depletion.

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