Downregulation of splicing factor SRSF3 induces p53ß, an alternatively spliced isoform of p53 that promotes cellular senescence.

Most human pre-mRNA transcripts are alternatively spliced, but the significance and fine-tuning of alternative splicing in different biological processes is only starting to be understood. SRSF3 (SRp20) is a member of a highly conserved family of splicing factors that have critical roles in key biological processes, including tumor progression. Here, we show that SRSF3 regulates cellular senescence, a p53-mediated process to suppress tumorigenesis, through TP53 alternative splicing. Downregulation of SRSF3 was observed in normal human fibroblasts undergoing replicative senescence, and was associated with the upregulation of p53ß, an alternatively spliced isoform of p53 that promotes p53-mediated senescence. Knockdown of SRSF3 by short interfering RNA (siRNA) in early-passage fibroblasts induced senescence, which was associated with elevated expression of p53ß at mRNA and protein levels. Knockdown of p53 partially rescued SRSF3-knockdown-induced senescence, suggesting that SRSF3 acts on p53-mediated cellular senescence. RNA pulldown assays demonstrated that SRSF3 binds to an alternatively spliced exon uniquely included in p53ß mRNA through the consensus SRSF3-binding sequences. RNA crosslinking and immunoprecipitation assays (CLIP) also showed that SRSF3 in vivo binds to endogenous p53 pre-mRNA at the region containing the p53ß-unique exon. Splicing assays using a transfected TP53 minigene in combination with siRNA knockdown of SRSF3 showed that SRSF3 functions to inhibit the inclusion of the p53ß-unique exon in splicing of p53 pre-mRNA. These data suggest that downregulation of SRSF3 represents an endogenous mechanism for cellular senescence that directly regulates the TP53 alternative splicing to generate p53ß. This study uncovers the role for general splicing machinery in tumorigenesis, and suggests that SRSF3 is a direct regulator of p53.

Structure and promoter analysis of the human unc-33-like phosphoprotein gene. E-box required for maximal expression in neuroblastoma and myoblasts.

The human unc-33-like phosphoprotein (hUlip/CRMP-4) is a member of a family of developmentally regulated genes that are highly expressed in the nervous system. Mutations in the C. elegans unc-33 gene lead to worms with abnormal movements. The hUlip gene encodes a 570-amino acid protein with 98% homology to its murine (Ulip) (Byk, T., Dobransky, T., Cifuentes-Diaz, C., and Sobel, A. (1996) J. Neurosci. 16, 688-701) and rat (CRMP-4) (Wang, L. H., and Strittmatter, S. M. (1996) J. Neurosci. 16, 6197-6207) counterparts (Gaetano, C., Matsuo, T., and Thiele, C. J. (1997) J. Biol. Chem. 272, 12195-12201). The hUlip gene was isolated from a human genomic library. It contains 15 exons, including an exon defined by an anaplastic oligodendroglioma expressed sequence tag, and spans at least 61.7 kilobases. hUlip lacks sequences corresponding to the first six exons found in unc-33. unc-33 exons correspond to homologous hUlip exons as follows: VII to 1 and 2, VIII to 3-9, IX to 10-12, and X to 13 and 14. Using the hUlip clone 1 phage, fluorescence in situ hybridization analysis indicates that the hybridization signal localizes to human chromosome 5q32. Deletion analysis of 5'-flanking sequences delineated the sequences sufficient to express a reporter gene in both neuroblastoma cells and myoblasts. A consensus MyoD/myogenin binding site is located in a region of the downstream promoter that is nearly identical to its mouse homologue. Mutagenesis shows that this conserved MyoD/myogenin site is necessary for full promoter activity in both myoblasts and neuroblastoma cells.

Autonomous activity of the alternate aldolase A muscle promoter is maintained by a sequestering mechanism.

The mouse aldolase A gene contains two closely-spaced alternate promoter/first exons. The more distal of the two, the M promoter, is muscle-specific while the 3' promoter, the H promoter, is expressed constitutively. Various segments from these promoter regions were linked to a reporter gene and used to transfect the myogenic cell line C2C12 and the hepatoma cell line BWTG3. A muscle-specific enhancer, MEN1, responsible for 80% of promoter M activity and containing 4 consensus MyoD binding sites was localized between -2578 to -2723 of the M promoter. Another muscle-specific enhancer and a restrictive element, MEN2/MSE, were found in the interval -1100 to -350. The MSE restrictive element was found to prohibit inappropriate up-regulation of the M promoter by selectively sequestering it from H promoter elements in both myoblasts and myotubes. Among the H promoter elements was found an enhancer, HEN, situated between -533 and -200 which did not function in myotubes. These studies also show that H promoter elements can act synergistically with a non-specific element, MAE, located between -350 and -130 of the M cap site greatly stimulating M promoter transcription in all cell types when the MSE restrictive element was absent. Through the analysis of interactions between these elements and the aldolase A and HSV-TK promoters we showed that neither the enhancers nor the promoter proximal sequences by themselves contain adequate information to reproduce the native pattern of aldolase A promoter modulation. Rather, the sequestering of the M promoter by the MSE restrictive element and the relative positioning and context of promoters M and H appear critical to the regulated expression of aldolase A.

Unique use of alternative polyadenylation signals in the mouse aldolase B gene.

We isolated and sequenced two mouse aldolase B cDNAs. They differ only in the length of the 3' untranslated region. This is consistent with Northern blot analysis of liver RNA which shows two transcripts differing by 400 nucleotides. We also isolated and sequenced the corresponding 3' genomic region and found four polyadenylation signals in the final exon. RNase protection studies demonstrate that all four of these signals are utilized, but not equally. This is unique to the mouse aldolase B gene.

Nonconservative utilization of aldolase A alternative promoters.

Recently, analysis of the sequence and expression of the human aldolase A gene revealed the unique arrangement of three tandem promoters and exons preceding a common coding sequence. A muscle-specific promoter (M) and two flanking widely used promoters (N and H) produce mRNA species which, in their mature forms, differ only in the sequence of their 5'-untranslated regions. We have isolated and investigated the expression of a mouse aldolase A gene. This mouse gene represents a functional gene by sequence analysis, recombinational screening, and by transfection into C2C12 cells. Although there is a high degree of sequence similarity between the mouse and the human gene in the region of the alternative first exons, we have been unable to detect a functional utilization of the 5'-most promoter (N) in the mouse. Steady state mRNAs isolated from a variety of adult tissues and cultured cells were analyzed by RNase protection and primer extension to identify first exon utilization. Consistent with previous reports, exon M is found only in skeletal muscle and exon H, the "housekeeping" exon, is utilized in every tissue where aldolase A is expressed. Under identical conditions we fail to see any evidence of the N exon. Therefore, although sequence homology exists between rodents and primates in the N region, the absence of selective pressure to preserve its primate pattern of expression may have resulted in functional promoter extinction.