Background: WTAP is a ubiquitously expressed nuclear protein that is required for mammalian early embryo development and cell cycle progression. Results: WTAP forms a complex with several splicing regulators. Conclusion: WTAP regulates both the cell cycle and alternative splicing by the formation of a protein complex. Significance: Characterization of this protein complex will help to elucidate the critically important function of WTAP in alternative splicing and cell proliferation.

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Wilms' tumor 1-associating protein (WTAP) has been reported to be a ubiquitously expressed nuclear protein. Although a relation to splicing factors has been postulated, its actual physiological function still remains to be elucidated. To investigate the role of WTAP, we generated WTAP-knockout mice and performed small interfering RNA (siRNA)-mediated knockdown analyses in primary cultured cells. In DNA microarrays using human umbilical vein endothelial cells, WTAP-targeted siRNA treatment resulted in markedly reduced expression of cell-cycle-related genes. siRNA-mediated WTAP knockdown down-regulated the stability of cyclin A2 mRNA through a nine-nucleotide essential sequence in cyclin A2 mRNA 3 UTR. WTAP knockdown induced G 2 accumulation, which is partially rescued by adenoviral overexpression of cyclin A2. Moreover, WTAP-null mice exhibited proliferative failure with death resulting at approximately embryonic day 6.5, an etiology almost identical to cyclin A2-null mice. Collectively, these findings establish WTAP as an essential factor for the stabilization of cyclin A2 mRNA, thereby regulating G 2͞M cell-cycle transition.mRNA stability ͉ cell-cycle regulation W ilms' tumor 1-associating protein (WTAP) was identified as a protein that specifically interacts with Wilms' tumor 1 (WT1) in both in vitro and in vivo assays (1). The Wilms tumor suppressor gene WT1 is essential for the normal development of the bipotential gonad, and the primordial kidney (2-4) and is associated with a common form of pediatric kidney cancer (5). WTAP and WT1 are present together throughout the nucleoplasm as well as in nuclear speckles and partially colocalize with splicing factors (1). WTAP was also identified as the mammalian homologue of the Drosophila female-lethal-2-D [fl(2)D]. fl(2)D is required for the female developmental pathway because of its activation of female-specific patterns of alternative splicing on SXL and transformer (tra) premRNA (6, 7). The presence of the lethal phenotype of the fl(2)D mutation in both sexes suggests an additional function for the gene (8). In vitro splicing assays demonstrated that depletion of WTAP from HeLa nuclear extracts affects tra but not AdML (a model adenovirus RNA) splicing, suggesting a biochemical function in female-specific splicing regulation (9). Furthermore, proteomic studies isolated WTAP as one of 145 spliceosomal proteins assembled on two distinct premRNAs, adenovirus major late and Fushi tarazu (10). While investigating GATA function in vascular endothelial cells, we serendipitously identified WTAP, in addition to GATAassociating proteins and hematopoietically expressed homeobox, from a yeast two-hybrid screen (11). In spite of these refined studies, the exact function of WTAP remains unknown.In the present study, we investigated the function of WTAP in vitro by using RNAi in primary cultured cells and in vivo by generating WTAP-knockout mice. Here, we demonstrate that WTAP is a factor essential for cyclin A2 mRNA stabilization and G 2 ͞M transition, the understanding of w...

Adipocyte differentiation is regulated by various mechanisms, of which mitotic clonal expansion (MCE) is a key step. Although this process is known to be regulated by cell cycle modulators, the precise mechanism remains unclear.

The U2AF35-like ZRSR1 has been implicated in the recognition of 3' splice site during spliceosome assembly, but ZRSR1 knockout mice do not show abnormal phenotypes. To analyze ZRSR1 function and its precise role in RNA splicing, we generated ZRSR1 mutant mice containing truncating mutations within its RNA-recognition motif. Homozygous mutant mice exhibited severe defects in erythrocytes, muscle stretch, and spermatogenesis, along with germ cell sloughing and apoptosis, ultimately leading to azoospermia and male sterility. Testis RNA sequencing (RNA-seq) analyses revealed increased intron retention of both U2- and U12-type introns, including U12-type intron events in genes with key functions in spermatogenesis and spermatid development. Affected U2 introns were commonly found flanking U12 introns, suggesting functional cross-talk between the two spliceosomes. The splicing and tissue defects observed in mutant mice attributed to ZRSR1 loss of function suggest a physiological role for this factor in U12 intron splicing.