MicroRNA (miRNA)-mediated Interaction between Leukemia/Lymphoma-related Factor (LRF) and Alternative Splicing Factor/Splicing Factor 2 (ASF/SF2) Affects Mouse Embryonic Fibroblast Senescence and Apoptosis

Abstract

Leukemia/lymphoma-related factor (LRF) is a transcriptional repressor, which by recruiting histone deacetylases specifically represses p19/ARF expression, thus behaving as an oncogene. Conversely, in mouse embryonic fibroblasts (MEF), LRF inhibition causes aberrant p19ARF up-regulation resulting in proliferative defects and premature senescence. We have recently shown that LRF is controlled by microRNAs. Here we show that LRF acts on MEF proliferation and senescence/apoptosis by repressing miR-28 and miR-505, revealing a regulatory circuit where microRNAs (miRNAs) work both upstream and downstream of LRF. By analyzing miRNA expression profiles of MEF transfected with LRF-specific short interfering RNAs, we found that miR-28 and miR-505 are modulated by LRF. Both miRNAs are predicted to target alternative splicing factor/splicing factor 2 (ASF/SF2), a serine/arginine protein essential for cell viability. In vertebrates, loss or inactivation of ASF/SF2 may result in genomic instability and induce G 2 cell cycle arrest and apoptosis. We showed that miR-28 and miR-505 modulate ASF/SF2 by directly binding ASF/ SF2 3-UTR. Decrease in LRF causes a decrease in ASF/SF2, which depends on up-regulation of miR-28 and miR-505. Alteration of each of the members of the LRF/miR-28/miR-505/ASF/ SF2 axis affects MEF proliferation and the number of senescent and apoptotic cells. Consistently, the axis is coordinately modulated as cell senescence increases with passages in MEF culture. In conclusion, we show that LRF-dependent miRNAs miR-28 and miR-505 control MEF proliferation and survival by targeting ASF/SF2 and suggest a central role of LRF-related miRNAs, in addition to the role of LRF-dependent p53 control, in cellular homeostasis. Leukemia/lymphoma-related factor (LRF)2 is a BTB/POZ domain Krüppel-like zinc finger transcription factor belonging to the POK protein family. POK proteins exert transcriptional repressive activity by recruiting histone deacetylases and subsequent remodeling of chromatin (1, 2). LRF can act as both transcriptional repressor and activator depending on the promoter context (3-6). LRF is known to have pleiotropic functions during development and differentiation, and it has recently been shown to act as an oncogene (7). Transgenic mouse cell lines overexpressing LRF developed aggressive tumors, and aberrant LRF overexpression was observed in a high percentage of human lymphomas. Conversely, LRF loss caused proliferative defects, premature senescence, and unresponsiveness to oncogenic stimuli. The main mode of action of LRF seems to be through the direct binding to p19ARF, a major tumor suppressor. Alterations of LRF therefore affect the p19ARF/MDM2/p53 pathway (7,8). However, LRF is also overexpressed in solid tumors, such as colon and bladder cancer, which have frequently lost the p14ARF/p53 pathway (8), and a positive relationship between LRF and p14ARF has been observed in several lung cancers (9). These data suggest possible additional target genes through which LRF might exert its activity.We have...

Alternative splicing factor or splicing factor‐2 plays a key role in intron retention of the endoglin gene during endothelial senescence

“…(A) Quantitative RT-PCR analysis of total endoglin, the intron responsible for S-endoglin and PAI-1, an endothelial senescence marker (Comi et al, 1995;Erusalimsky, 2009) demonstrated in endothelial cells (Figs 5 and 6), suggesting that the splicing factor ASF ⁄ SF2 is a new target of senescence. This is in line with the reported role of ASF ⁄ SF2 as modulator of the senescence induced by oncogene overexpression in mouse embryonic fibroblasts (Verduci et al, 2010) as well as with the upregulation of ASF ⁄ SF2 in various human tumors and with the oncoprotein function of ASF ⁄ SF2 itself (Karni et al, 2007). Moreover, a direct correlation was established between the preferential cytoplasmic localization of ASF ⁄ SF2 and S-endoglin expression at the senescent stage of endothelial cells.…”

Changes in splicing factor expression are associated with advancing age in man

“…The regulatory role of ATM in miRNA biogenesis is modulated by KH-type splicing regulatory protein (KSRP) (Zhang et al, 2011). Accordingly, it has subsequently been reported that SRSF1 is regulated by miR-505 and miR-28 in mouse embryonic fibroblasts (Verduci et al, 2010). These findings indicate that investigation of ATM , KSRP and microRNA regulation is potentially a fruitful future avenue of study.…”

miR-28-5p Promotes Chromosomal Instability in VHL-Associated Cancers by Inhibiting Mad2 Translation

“…The mechanism(s) mediating miR-28-5p induction is not known. However, the proto-oncogene zinc finger and BTB domain containing 7A (ZBTB7A, also known as LRF) has been shown to negatively regulate miR-28 expression (38) and to compete with the Sp1 transcription factor for target binding (39). Whether ZBTB7A/LRF acts downstream of pVHL to control of miR-28 expression remains to be investigated.…”