A cytosine-rich splice regulatory determinant enforces functional processing of the human α-globin gene transcript

Abstract

The establishment of efficient and stable splicing patterns in terminally differentiated cells is critical to maintenance of specific functions throughout the lifespan of an organism. The human α-globin (hα-globin) gene contains 3 exons separated by 2 short introns. Naturally occurring α-thalassemia mutations that trigger aberrant splicing have revealed the presence of cryptic splice sites within the hα-globin gene transcript. How cognate (functional) splice sites are selectively used in lieu of these cryptic sites has remained unexplored. Here we demonstrate that the preferential selection of a cognate splice donor essential to functional splicing of the hα-globin transcript is dependent on the actions of an intronic cytosine (C)-rich splice regulatory determinant and its interacting polyC-binding proteins. Inactivation of this determinant by mutation of the C-rich element or by depletion of polyC-binding proteins triggers a dramatic shift in splice donor activity to an upstream, out-of-frame, cryptic donor. The essential role of the C-rich element in hα-globin gene expression is supported by its coevolution with the cryptic donor site in primate species. These data lead us to conclude that an intronic C-rich determinant enforces functional splicing of the hα-globin transcript, thus acting as an obligate determinant of hα-globin gene expression.

Sequence and Evolutionary Features for the Alternatively Spliced Exons of Eukaryotic Genes

“…Based on the fluorescence-based splicing reporter, the experimental approach was developed to systematically identify SREs [41]. The genome-wide discovery of SREs have been significantly advanced by combining the high-throughput sequencing with immunoprecipitation, such as the crosslinking immunoprecipitation sequencing (CLIP-seq) and RNA immunoprecipitation sequencing (RIP-seq) [42,43,44]. On the whole, hundreds of SREs have been computationally or experimentally discovered and most of them are mainly involved in regulating tissue-specific alternative splicing of pre-mRNAs [45].…”

α-Globin pre-mRNA splicing, revisited

“…In this issue of Blood, Ji et al have identified a novel regulatory mechanism that ensures proper splicing of human a-globin pre-messenger RNA (pre-mRNA). 1 One could be forgiven for thinking that splicing of this well-studied gene is fully understood, but Ji et al have described an essential new element: a C-rich sequence located a few nucleotides downstream of the intron 1 splice donor site that favors proper splicing while suppressing use of a competing cryptic splice site in the exon (see figure). Moreover, they report that polyC-binding proteins must interact with this intronic element to enhance proper splicing.…”

“…10 In this issue of Blood, Pemmaraju et al explored the potential correlation between therapy with JAK inhibitors and the risk of a subsequent lymphoma in patients with myeloproliferative neoplasms (MPNs). 1 MPNs are clonal disorders complicated by vascular events, myelofibrosis, transformation to leukemia and, more rarely, the development of secondary malignancies. 2 Lymphoproliferative neoplasms (LPNs) in patients with MPNs are rare but occur at a higher frequency than that found in the general population.…”