Multiple cis elements regulate an alternative splicing event at 4.1R pre-mRNA during erythroid differentiation

Abstract

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 IntroductionProtein 4.1R is a critical 80-kd cytoskeletal protein found in circulating red blood cells (RBCs). It mediates the formation and maintenance of spectrin/actin complex and anchors the cytoskeleton to the overlying lipid bilayer. 1 Human 4.1R is encoded by a single genomic locus over 200 kb in length, 2 and is expressed as multiple isoforms resulting from complex alternative premessenger RNA (pre-mRNA) splicing pathways. Previous studies have shown that inclusion of a 21-amino acid sequence motif at the N-terminus of the 10-kd spectrin/actin-binding (SAB) domain is required to promote cytoskeletal junctional complex stability. 3,4 Genomic cloning of both the mouse and human genes confirmed that the 63-nucleotide (nt) motif is encoded by an individual exon, exon 16. 2,5 The splicing of this exon is therefore regulated in a cassette fashion. Exon 16 is omitted from much of the 4.1R mRNA of pre-erythroid cells but is included in most of the mRNA produced in late erythroid cells (Figure 1). 6,7 Splicing of exon 16 is thus highly regulated in a differentiation stage-specific manner, and this regulated event is critical for production of 4.1R isoforms that sustain the function of 4.1R in circulating RBCs.Alternative splicing of pre-mRNA is a fundamental mechanism for regulating eukaryotic gene expression. 8 In many cases, alternative RNA splicing contributes to developmentally regulated and cell type-specific patterns of gene expression. Although a great deal of information is available concerning the genera...

Subtle distinct regulations of late erythroid molecular events by PI3K/AKT-mediated activation of Spi-1/PU.1 oncogene autoregulation loop

“…This regulated splicing event underlies the production of a functional 4.1R; inclusion of the 21 amino acid peptide encoded by exon 16, at the N-terminus of the 10 kDa spectrin/actinbinding domain of 4.1R is indeed essential to promote high mechanical stability and high deformability of the mature red cell membrane. We have shown that Epononresponsive MEL cells reproduce exon 16-regulated splicing on DMSO induction to erythroid differentiation from either the endogenous pre-mRNA or transfected minigene transcript (Deguillien et al, 2001). More recently, we have observed that Eporesponsive erythroleukemia cells also efficiently regulate this splicing event when induced in culture with either DMSO or Epo (The´oleyre et al, 2004).…”

“…Inclusion of exon 16 in mature 4.1R mRNA was assessed by semiquantitative RT-PCR, as previously described (Deguillien et al, 2001;The´oleyre et al, 2004).…”

Spi-1/PU.1 but not Fli-1 inhibits erythroid-specific alternative splicing of 4.1R pre-mRNA in murine erythroleukemia cells

“…More recently, we have developed in MEL cells a stable transfection system, which reproduces this regulated splicing pattern, using minigene constructs bearing exon 16. We showed that multiple cis-acting elements, within the exonic and close vicinity intronic sequences, contribute coordinately to exon skipping in predifferentiated cells, and to promote exon inclusion as the cells differentiate (Deguillien et al, 2001).…”

Downregulation of the Spi-1/PU.1 oncogene induces the expression of TRIM10/HERF1, a key factor required for terminal erythroid cell differentiation and survival

“…To evaluate exon 16 inclusion, semi-quantitative RT-PCR experiments were performed as detailed previously [33].…”