The alpha V/41 polymorphism of erythroid alpha-spectrin has been characterized initially by an increased susceptibility to proteolysis of the alpha IV-alpha V domain junction (Alloisio N., L. Morlé, J. Maréchal, A.-F. Roux, M.-T. Ducluzeau, D. Guetarni, B. Pothier, F. Baklouti, A. Ghanem, R. Kastally, et al. 1991. J. Clin. Invest. 87:2169-2177). Until now, it has been found associated invariably with a low expression level of the corresponding alpha chain. Among 61 chromosomes investigated in French and North African individuals or kindreds, we observed 19 chromosomes with the alpha V/41 polymorphism. With no single exception, the latter displayed a point mutation in exon 40 (Leu-->Val; CTA-->GTA) at position alpha 1857. According to the triple helical model of spectrin structure, this change accounts for the peptide maps' abnormalities. Sequencing the entire alpha V domain cDNA disclosed, in addition, a partial skipping of exon 46. At the gene level, a substitution (C-->T) was evidenced at nucleotide -12 of intron 45. This mutation appeared linked to the exon 40 mutation in 17 chromosomes, again with no single exception, among 53 examined chromosomes. We hypothesized that the lack of exon 46 would hamper the nucleation process and eventually account for the low expression feature. The present doubly mutated allele was renamed allele alpha LELY (low expression, Lyon).

Protein 4.1 is a globular 80-kDa component of the erythrocyte membrane skeleton that enhances spectrin-actin interaction via its internal 10-kDa domain. Previous studies have shown that protein 4.1 mRNA is expressed as multiple alternatively spliced isoforms, resulting from the inclusion or exclusion of small cassette sequences called motifs. By tissue screening for protein 4.1 isoforms, we have observed new features of an already complex pattern of alternative splicing within the spectrin/actin binding domain. In particular, we found a new 51-nt exon that is present almost exclusively in muscle tissue. In addition, we have isolated multiple genomic clones spanning over 200 kb, containing the entire erythroid and nonerythroid coding sequence of the human locus. The exon/intron structure has now been characterized; with the exception of a 17-nt motif, all of the alternatively spliced motifs correspond to individual exons. The 3'-untranslated region (UTR) has also been completely sequenced using various PCR and genomic-sequencing methods. The 3' UTR, over 3 kb, accounts for one-half of the mature mRNA.

Spectrin alpha-chain mutants associated with hereditary elliptocytosis are highly variable in their level of expression. It has been assumed that the degree of elliptocytosis can be increased when the spectrin alpha chain, encoded by the alpha gene in trans to the variant, is expressed at a low level. We now provide strong evidence for the existence of low-level expression of spectrin alpha chains. This condition is referred to as the alpha V/41 polymorphism. It has been observed in 15 different families or individuals of French, North African, and African ancestry in which seven distinct elliptocytogenic alpha-spectrin variants were co-inherited. Whenever the alpha V/41 polymorphism was present, the severity of the biochemical, morphological, and, sometimes, the clinical phenotype of elliptocytosis was increased. The alpha V/41 polymorphism was also frequently encountered among 36 unrelated control subjects in the heterozygous or homozygous states, and was entirely asymptomatic in both cases. The main biochemical feature was an increased susceptibility to proteolysis of the alpha IV-alpha V domain junction. Alteration of the facing beta IV domain of spectrin was demonstrated by in vitro spectrin dimer reconstitution experiments. It appears that the alpha V/41 polymorphism is often required for alpha-spectrin elliptocytogenic variants to become manifest in the heterozygous state. Thus, alpha-spectrin-related elliptocytosis may be viewed as a bifactorial condition.

The inclusion of exon 16 in the mature protein 4.1R messenger RNA (mRNA) is a critical event in red blood cell membrane biogenesis. It occurs during late erythroid development and results in inclusion of the 10-kd domain needed for stabilization of the spectrin/actin lattice. In this study, an experimental model was established in murine erythroleukemia cells that reproduces the endogenous exon 16 splicing patterns from a transfected minigene. Exon 16 was excluded in predifferentiated and predominantly included after induction. This suggests that the minigene contained exon and abutting intronic sequences sufficient for splicing regulation. A systematic analysis of the cis-acting regulatory sequences that reside within the exon and flanking introns was performed. Results showed that (1) the upstream intron of 4.1R pre-mRNA is required for exon recognition and it displays 2 enhancer elements, a distal element acting in differentiating cells and a proximal constitutive enhancer that resides within the 25 nucleotides preceding the acceptor site; (2) the exon itself contains a strong constitutive splicing silencer; (3) the exon has a weak 5′ splice site; and (4) the downstream intron contains at least 2 splicing enhancer elements acting in differentiating cells, a proximal element at the vicinity of the 5′ splice site, and a distal element containing 3 copies of the UGCAUG motif. These results suggest that the interplay between negative and positive elements may determine the inclusion or exclusion of exon 16. The activation of the enhancer elements in late erythroid differentiation may play an important role in the retention of exon 16.

SR proteins exhibit diverse functions ranging from their role in constitutive and alternative splicing, to virtually all aspects of mRNA metabolism. These findings have attracted growing interest in deciphering the regulatory mechanisms that control the tissue-specific expression of these SR proteins. In this study, we show that SRSF5 protein decreases drastically during erythroid cell differentiation, contrasting with a concomitant upregulation of SRSF5 mRNA level. Proteasome chemical inhibition provided strong evidence that endogenous SRSF5 protein, as well as protein deriving from stably transfected SRSF5 cDNA, are both targeted to proteolysis as the cells undergo terminal differentiation. Consistently, functional experiments show that overexpression of SRSF5 enhances a specific endogenous pre-mRNA splicing event in proliferating cells, but not in differentiating cells, due to proteasome-mediated targeting of both endogenous and transfection-derived SRSF5. Further investigation of the relationship between SRSF5 structure and its post-translation regulation and function, suggested that the RNA recognition motifs of SRSF5 are sufficient to activate pre-mRNA splicing, whereas proteasome-mediated proteolysis of SRSF5 requires the presence of the C-terminal RS domain of the protein. Phosphorylation of SR proteins is a key post-translation regulation that promotes their activity and subcellular availability. We here show that inhibition of the CDC2-like kinase (CLK) family and mutation of the AKT phosphorylation site Ser86 on SRSF5, have no effect on SRSF5 stability. We reasoned that at least AKT and CLK signaling pathways are not involved in proteasome-induced turnover of SRSF5 during late erythroid development.