Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins to silencer elements in the exon and that down-regulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This article demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.Alternative splicing of pre-mRNA leads to the synthesis of multiple protein isoforms from a single gene. It is an important mechanism for regulating gene expression and may be utilized by 40 -60% of human genes (1-4). Thus, the estimated 25,000 to 30,000 genes of the human genome can generate a much larger number of proteins. Regulation of alternative splicing occurs in both a tissue-and development-specific manner, resulting in alterations in the structure and function of critical proteins. Altered splicing regulation can also be of widespread importance in the etiology of human disease (5-7).The protein 4.1 gene family serves as an excellent model for investigating the regulation of alternative splicing. The four genes that comprise the family (4 .1R, 4.1G, 4.1B, and 4.1N) display a remarkable array of highly regulated, tissue-specific splicing events. These alternative splicing events facilitate expression of distinct isoforms of 4.1 protein in cells of erythroid, epithelial, neural, and muscle origin (8 -14); thus, they provide opportunities for understanding the mechanisms that regulate alternative splicing in several different cell types. To date, mechanistic studies have focused predominantly on erythroid cells, in which 4.1R protein is a structural component of the erythrocyte plasma membrane and is important for structural integrity and stability of the membrane skeleton. In differentiating erythroid progenitor cells, a dramatic switch in pre-mRNA splicing result...

A physiologically important alternative pre-mRNA splicing switch, involving activation of protein 4.1R exon 16 (E16) splicing, is required for the establishment of proper mechanical integrity of the erythrocyte membrane during erythropoiesis. Here we identify a conserved exonic splicing silencer element (CE 16 ) in E16 that interacts with hnRNP A/B proteins and plays a role in repression of E16 splicing during early erythropoiesis. Experiments with model premRNAs showed that CE 16 can repress splicing of upstream introns, and that mutagenesis or replacement of CE 16 can relieve this inhibition. An af®nity selection assay with biotinylated CE 16 RNA demonstrated speci®c binding of hnRNP A/B proteins. Depletion of hnRNP A/B proteins from nuclear extract signi®cantly increased E16 inclusion, while repletion with recombinant hnRNP A/B restored E16 silencing. Most importantly, differentiating mouse erythroblasts exhibited a stage-speci®c activation of the E16 splicing switch in concert with a dramatic and speci®c down-regulation of hnRNP A/B protein expression. These ®ndings demonstrate that natural developmental changes in hnRNP A/B proteins can effect physiologically important switches in premRNA splicing. Keywords: alternative splicing/exonic splicing silencer/ hnRNP A and B/protein 4.1R

B-cell lymphoma (BCL) is the most common hematologic malignancy. While sequencing studies gave insights into BCL genetics, identification of non-mutated cancer genes remains challenging. Here, we describe PiggyBac transposon tools and mouse models for recessive screening and show their application to study clonal B-cell lymphomagenesis. In a genome-wide screen, we discover BCL genes related to diverse molecular processes, including signaling, transcriptional regulation, chromatin regulation, or RNA metabolism. Cross-species analyses show the efficiency of the screen to pinpoint human cancer drivers altered by non-genetic mechanisms, including clinically relevant genes dysregulated epigenetically, transcriptionally, or post-transcriptionally in human BCL. We also describe a CRISPR/Cas9-based in vivo platform for BCL functional genomics, and validate discovered genes, such as Rfx7 , a transcription factor, and Phip , a chromatin regulator, which suppress lymphomagenesis in mice. Our study gives comprehensive insights into the molecular landscapes of BCL and underlines the power of genome-scale screening to inform biology.

We report here the cDNA and amino acid sequences of a human 58-kilodalton type II keratin, K5, which is coexpressed with a 50-kilodalton type I keratin partner, K14, in stratified squamous epithelia. Using a probe specific for the 3'-noncoding portion of this K5 cDNA, we demonstrated the existence of a single human gene encoding this sequence. Using Northern (RNA) blot analysis and in situ hybridization with cRNA probes for both KS 11 (10, 19, 25). Type I keratins are generally small (40 to 56.5 kDa) and relatively acidic (pKi, 4.5 to 5.5), while type II keratins are larger (53 to 67 kDa) and more basic (pKi, 6.5 to 7.5) (14,25,33,40,44). Type I and type II keratins are frequently expressed as specific pairs, and at least one pair of keratins is always expressed in any epithelial cell (8). Changes in differentiation (13,48,50) and development (7, 35) in epithelial cells often coincide with alterations in keratin synthesis, suggesting that the expression of keratins might be finely tailored to suit the particular and varied structural requirements of each epithelial cell.In the epidermis, keratin synthesis is especially abundant, leaving the fully differentiated squames with 85% of their protein as keratin. As judged with keratin extracts from epidermal sections cut parallel to the skin surface, the pattern of keratins undergoes change as an epidermal cell undergoes a commitment to terminally differentiate (13). In the basal layer, two keratins are expressed: a type I 50-kDa keratin, K14, and a type II partner 58-kDa keratin, K5 (36). As a differentiating epidermal cell migrates outward toward the skin surface, it synthesizes new keratins of both the type I class (K1O and Kll) and the type II class (Kl and K2) (13,24,35,44,50). Immunofluorescence studies have shown that the iniduction of the large keratins appears to take place in the first suprabasal layer (27,35,48). Analysis of keratins from tissue sectioning has indicated that the K5-K14 pair may also be present in the suprabasal layers (13, 50). However, owing to (i) the undulating nature of the epidermis and (ii) the masking of some critical antigenic determinants of the keratins in the suprabasal layers (50), it has been difficult to assess to what extent the expression of the basal keratins continues during terminal differentiation and wheth-* Corresponding author. er this expression is a consequence of a stable preexisting protein or new protein synthesis.To elucidate the molecular mechanisms underlying the differential expression of keratin pairs in the epidermis, we previously isolated and characterized a cDNA clone (18) and a gene (29,30) for K14 (50 kDa). We now report the isolation and characterization of a cDNA encoding its partner keratin, K5. We examine its complexity in the human genome and explore the coordinate expression of K5 and K14 in the epidermis and in cultured epidermal cells.Isolation and identification of a cDNA encoding human keratin KS. Previously, a pBR322 cDNA library was prepared from cultured human epidermal cell mRNAs, whic...

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Alternative splicing plays a major role in regulating tissue-specific expression of cytoskeletal protein 4.1R isoforms. In particular, expression of the protein's functionally critical spectrin-actin binding domain, essential for maintenance of red cell membrane mechanical properties, is governed by a developmentally regulated splicing switch involving alternative exon 16. Using a model 3-exon 4.1R pre–messenger RNA (pre-mRNA), we explored the sequence requirements for excision of the introns flanking exon 16. These studies revealed that splicing of this alternative exon occurs preferentially in an ordered fashion. The first step is excision of the downstream intron to join exons 16 and 17, followed by excision of the upstream intron. Constructs designed to test the converse pathway were spliced less efficiently and with less fidelity, in part due to activation of a cryptic 5′ splice site in exon 16. This downstream-first model for ordered splicing is consistent with the hypothesis that regulated alternative splicing requires cooperation between multiple exonic and/or intronic regulatory elements whose spatial organization is critical for recruitment of appropriate splicing factors. Our results predict that exon 16 splicing is regulated at the first step—excision of the downstream intron—and that cells unable to catalyze this step will exhibit exon 16 skipping. In cells that include exon 16, adherence to an ordered pathway is important for efficient and accurate production of mature 4.1R mRNA encoding an intact spectrin-actin binding domain.

In addition to controlling somatic sexual development in Drosophila melanogaster, the Sex-lethal (Sxl) gene is required for proper differentiation of female germ cells. To investigate its role in germ-line development, we have examined the expression of Sxl in wild-type ovaries and ovaries that are defective in early steps of germ cell differentiation. As in the soma, the basic mechanism for on/off regulation of Sxl relies on sex-specific processing of its transcripts in germ cells. One class of female-sterile mutations, which includes fs(1)1621 and the tumorous-ovary-producing allele of the ovarian tumor gene, otu1, is defective in the splicing process. These mutants have germ lines with high amounts of Sxl RNA spliced in the male mode and a severe reduction of protein levels in the germ cells. Another class of female-sterile mutations produces a phenotype similar to that seen in fs(1)1621 and otu1 but appears to express normal levels of Sxl protein in the germ cells. However, this second class does not show the changes in protein distribution normally observed in wild-type germ cells. In the wild-type germarium, the non-differentiated germ cells show a strong cytoplasmic accumulation of Sxl protein followed, as the germ cells differentiate, by a dramatic reduction and redistribution of the protein into nuclear foci. Interestingly, two female-sterile alleles of Sxl, Sxlf4 and Sxlf5 belong to the second class, which shows persistent cytoplasmic accumulation of Sxl protein. These Sxl female-sterile mutants encode an altered protein indicating that Sxl regulates processes that eventually lead to the changes in Sxl protein distribution. Lastly, we demonstrate that during the final stages of oogenesis several mechanisms must operate to prevent the progeny from inheriting Sxl protein. Conceivably, this regulation safeguards the inadvertent activation of the Sxl autoregulatory feedback loop in the male zygote.

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