6426

SRSF1

serine/arginine-rich splicing factor 1;SRSF1;serine/arginine-rich splicing factor 1

SR splicing factor 1|alternative-splicing factor 1|pre-mRNA-splicing factor SF2, P33 subunit|splicing factor 2|splicing factor, arginine/serine-rich, 30-KD, A

17q22

protein-coding

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.

Alternative splicing involving intron retention plays a key role in the regulation of gene expression. We previously reported that the alternatively spliced short isoform of endoglin (S-endoglin) is induced during the aging or senescence of endothelial cells by a mechanism of intron retention. In this work, we demonstrate that the alternative splicing factor or splicing factor-2 (ASF/SF2) is involved in the synthesis of endoglin. Overexpression of ASF/SF2 in endothelial cells switched the balance between the two endoglin isoforms, favoring the synthesis of S-endoglin. Using a minigene reporter vector and RNA immunoprecipitation experiments, it was shown that ASF/SF2 interacts with the nucleotide sequence of the endoglin minigene, suggesting the direct involvement of ASF/SF2. Accordingly, the sequence recognized by ASF/SF2 in the endoglin gene was identified inside the retained intron near the consensus branch point. Finally, the ASF/SF2 subcellular localization during endothelial senescence showed a preferential scattered distribution throughout the cytoplasm, where it interferes with the activity of the minor spliceosome, leading to an increased expression of S-endoglin mRNA. In summary, we report for the first time the molecular mechanisms by which ASF/SF2 regulates the alternative splicing of endoglin in senescent endothelial cells, as well as the involvement of ASF/SF2 in the minor spliceosome.