CFIm25 regulates glutaminase alternative terminal exon definition to modulate miR-23 function

Abstract

Alternative polyadenylation (APA) and alternative splicing (AS) provide mRNAs with the means to avoid microRNA repression through selective shortening or differential usage of 3 ′ UTRs. The two glutaminase (GLS) mRNA isoforms, termed KGA and GAC, contain distinct 3 ′ UTRs with the KGA isoform subject to repression by miR-23. We show that depletion of the APA regulator CFIm25 causes a strong shift to the usage of a proximal poly(A) site within the KGA 3 ′ UTR and also alters splicing to favor exclusion of the GAC 3 ′ UTR. Surprisingly, we observe that while miR-23 is capable of down-regulating the shortened KGA 3 ′ UTR, it has only minor impact on the full-length KGA 3 ′ UTR, demonstrating that additional potent negative regulation of GLS expression exists beyond this single microRNA targeting site. Finally, we show that the apoptosis induced upon downregulation of the GAC isoform can be alleviated through concurrent reduction in CFIm25 expression, revealing the sensitivity of glutaminase expression to the levels of RNA processing factors. These results exemplify the complex interplay between RNA processing and microRNA repression in controlling glutamine metabolism in cancer cells.

Molecular targeting of glutaminase sensitizes ovarian cancer cells to chemotherapy

“…Non‐small cell lung cancer lines predominantly express the shorter splice variant GAC, and this form is believed to be important for tumorigenesis in this cancer type . We were previously only able to detect the GAC isoform in HeLa cells under normal conditions . However, since KGA is also overexpressed in some tumors, it has also been suggested that the KGA isoform should also be targeted for cancer therapy .…”

“…In addition, we recently discovered a novel shorter form of the KGA transcript which shares the same identical exons with the longer KGA isoform but has a truncated 3′‐UTR . The GAC form has no known miRNA binding sites, whereas both the KGA long, and short forms contain a well‐known miR23 binding site making them subject to miRNA regulation …”

“…9 To add to the complexity, GLS1 undergoes alternative splicing, and alternative polyadenylation of the same GLS1 pre-mRNA, giving rise to several transcripts that share the same first 14 exons but have distinct C-termini. These GLS1 transcripts are the GAC isoform (genomic exons [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] and the traditionally recognized longer isoform of KGA (genomic exons 1-14 and exons [16][17][18][19] with intact 3′-UTR). 16,17 In addition, we recently discovered a novel shorter form of the KGA transcript which shares the same identical exons with the longer KGA isoform but has a truncated 3′-UTR.…”

Molecular Regulation of Alternative Polyadenylation (APA) within the Drosophila Nervous System

“…2 a), focusing on the analysis of CFI. Our choice of studying CFI in higher detail is based on several considerations: (i) CFI represents one of the hallmarks of metazoan CPA composition, (ii) it affects APA reactions in vitro [28] , [29] , (iii) its molecular structure is well understood in mammals [20] , and (iv) one of its members, CFI25, was shown to be involved in human pathologies including cancer and tumorigenesis [30] . Remarkably, expression patterns of both CFI25 and CFI68 showed a clear tissue-specific pattern with higher levels of expression of the two factors within the developing central nervous system (CNS) of the Drosophila embryo ( Fig.…”

Several Faces of Glutaminase Regulation in Cells

“…In addition, Masamha and colleagues described a more complex GLS regulation mechanism involving CFIm25 and ncRNA (6). Depletion of the alternative polyadenylation (APA) regulator CFIm25 was shown to cause a shift toward the usage of a proximal poly(A) site within the KGA 3 0 untranslated region (UTR), which altered splicing to favor the exclusion of the GAC 3 0 UTR.…”