Motifs within the CA-repeat-rich region of Surfactant Protein B (SFTPB) intron 4 differentially affect mRNA splicing

Wenjun Yang; Lan Ni; Patricia Silveyra; Guirong Wang; Georgios T. Noutsios; Anamika Singh; Susan L. DiAngelo; Olabisi Sanusi; Manmeet Raval; Joanna Floros

Motifs within the CA-repeat-rich region of Surfactant Protein B (SFTPB) intron 4 differentially affect mRNA splicing

Wenjun Yang, Lan Ni, Patricia Silveyra, Guirong Wang, Georgios T. Noutsios, Anamika Singh, Susan L. DiAngelo, Olabisi Sanusi, Manmeet Raval, Joanna Floros

Abstract

The first half of the surfactant protein B (SP-B) gene intron 4 is a CA-repeat-rich region that contains 11 motifs. To study the role of this region on SP-B mRNA splicing, minigenes were generated by systematic removal of motifs from either the 5’ or 3’ end. These were transfected in CHO cells to study their splicing efficiency. The latter was determined as the ratio of completely to incompletely spliced SP-B RNA. Our results indicate that SP-B intron 4 motifs differentially affect splicing. Motifs 8 and 9 significantly enhanced and reduced splicing of intron 4, respectively. RNA mobility shift assays performed with a Motif 8 sequence that contains a CAUC cis-element and cell extracts resulted in a RNA:protein shift that was lost upon mutation of the element. Furthermore, in silico analysis of mRNA secondary structure stability for minigenes with and without motif 8 indicated a correlation between mRNA stability and splicing ratio. We conclude that differential loss of specific intron 4 motifs results in one or more of the following: a) altered splicing, b) differences in RNA stability and c)changes in secondary structure. These, in turn, may affect SP-B content in lung health or disease.

Keywords

RNA; SP-B; secondary structure; SRp20; SRSF3; mRNA splicing

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