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


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.


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


Ankö ML, Müller-McNicoll M, Brandl H, Curk T, Gorup C, Henry I, Ule J & Neugebauer KM 2012 The RNA-binding landscapes of two SR proteins reveal unique functions and binding to diverse RNA classes. Genome Biol 13 R17.

Beers MF, Atochina EN, Preston AM & Beck JM 1999 Inhibition of lung surfactant protein B expression during Pneumocystis carinii pneumonia in mice. J Lab Clin Med 133 423-433.

Buratti E & Baralle FE 2004 Influence of RNA secondary structure on the pre-mRNA splicing process. Mol Cell Biol 24 10505-10514.

Desmet FO, Hamroun D, Lalande M, Collod-Béroud G, Claustres M & Béroud C 2009 Human Splicing Finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res 37 e67.

Ewis AA, Kondo K, Dang F, Nakahori Y, Shinohara Y, Ishikawa M & Baba Y 2006 Surfactant protein B gene variations and susceptibility to lung cancer in chromate workers. Am J Ind Med 49 367-373.

Floros J & Fan R 2001 Surfactant protein A and B genetic variants and respiratory distress syndrome: allele interactions. Biol Neonate 80 Suppl 1 22-25.

Floros J, Fan R, Diangelo S, Guo X, Wert J & Luo J 2001 Surfactant protein (SP) B associations and interactions with SP-A in white and black subjects with respiratory distress syndrome. Pediatrics international : official journal of the Japan Pediatric Society 43 567-576.

Floros J, Veletza SV, Kotikalapudi P, Krizkova L, Karinch AM, Friedman C, Buchter S & Marks K 1995 Dinucleotide repeats in the human surfactant protein-B gene and respiratory-distress syndrome. Biochem J 305 ( Pt 2) 583-590.

Gooding C, Clark F, Wollerton MC, Grellscheid SN, Groom H & Smith CW 2006 A class of human exons with predicted distant branch points revealed by analysis of AG dinucleotide exclusion zones. Genome Biol 7 R1.

Gower WA & Nogee LM 2011 Surfactant dysfunction. Paediatr Respir Rev 12 223-229.

Guttentag SH, Beers MF, Bieler BM & Ballard PL 1998 Surfactant protein B processing in human fetal lung. Am J Physiol 275 L559-566.

Hallman M, Haataja R & Marttila R 2002 Surfactant proteins and genetic predisposition to respiratory distress syndrome. Semin Perinatol 26 450-460.

Hamvas A, Wegner DJ, Trusgnich MA, Madden K, Heins H, Liu Y, Rice T, An P, Watkins-Torry J & Cole FS 2005 Genetic variant characterization in intron 4 of the surfactant protein B gene. Hum Mutat 26 494-495.

Hargous Y, Hautbergue GM, Tintaru AM, Skrisovska L, Golovanov AP, Stevenin J, Lian LY, Wilson SA & Allain FH 2006 Molecular basis of RNA recognition and TAP binding by the SR proteins SRp20 and 9G8. EMBO J 25 5126-5137.

Hui J 2009 Regulation of mammalian pre-mRNA splicing. Sci China C Life Sci 52 253-260.

Hui J, Hung LH, Heiner M, Schreiner S, Neumuller N, Reither G, Haas SA & Bindereif A 2005 Intronic CA-repeat and CA-rich elements: a new class of regulators of mammalian alternative splicing. EMBO J 24 1988-1998.

Hung LH, Heiner M, Hui J, Schreiner S, Benes V & Bindereif A 2008 Diverse roles of hnRNP L in mammalian mRNA processing: a combined microarray and RNAi analysis. RNA 14 284-296.

Kala P, Ten Have T, Nielsen H, Dunn M & Floros J 1998 Association of pulmonary surfactant protein A (SP-A) gene and respiratory distress syndrome: interaction with SP-B. Pediatr Res 43 169-177.

Kerr MH & Paton JY 1999 Surfactant protein levels in severe respiratory syncytial virus infection. Am J Respir Crit Care Med 159 1115-1118.

Korbie DJ & Mattick JS 2008 Touchdown PCR for increased specificity and sensitivity in PCR amplification. Nat Protoc 3 1452-1456.

Lin Z, Thomas NJ, Wang Y, Guo X, Seifart C, Shakoor H & Floros J 2005 Deletions within a CA-repeat-rich region of intron 4 of the human SP-B gene affect mRNA splicing. Biochem J 389 403-412.

Mathews DH, Sabina J, Zuker M & Turner DH 1999 Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J Mol Biol 288 911-940.

Melo KF, Martin RM, Costa EM, Carvalho FM, Jorge AA, Arnhold IJ & Mendonca BB 2002 An unusual phenotype of Frasier syndrome due to IVS9 +4C>T mutation in the WT1 gene: predominantly male ambiguous genitalia and absence of gonadal dysgenesis. J Clin Endocrinol Metab 87 2500-2505.

Neklason DW, Solomon CH, Dalton AL, Kuwada SK & Burt RW 2004 Intron 4 mutation in APC gene results in splice defect and attenuated FAP phenotype. Fam Cancer 3 35-40.

Nesslein LL, Melton KR, Ikegami M, Na CL, Wert SE, Rice WR, Whitsett JA & Weaver TE 2005 Partial SP-B deficiency perturbs lung function and causes air space abnormalities. Am J Physiol Lung Cell Mol Physiol 288 L1154-1161.

O'Reilly MA, Weaver TE, Pilot-Matias TJ, Sarin VK, Gazdar AF & Whitsett JA 1989 In vitro translation, post-translational processing and secretion of pulmonary surfactant protein B precursors. Biochim Biophys Acta 1011 140-148.

Pastor T, Talotti G, Lewandowska MA & Pagani F 2009 An Alu-derived intronic splicing enhancer facilitates intronic processing and modulates aberrant splicing in ATM. Nucleic Acids Res 37 7258-7267.

Pilot-Matias TJ, Kister SE, Fox JL, Kropp K, Glasser SW & Whitsett JA 1989 Structure and organization of the gene encoding human pulmonary surfactant proteolipid SP-B. DNA 8 75-86.

Raponi M, Upadhyaya M & Baralle D 2006 Functional splicing assay shows a pathogenic intronic mutation in neurofibromatosis type 1 (NF1) due to intronic sequence exonization. Hum Mutat 27 294-295.

Rova M, Haataja R, Marttila R, Ollikainen V, Tammela O & Hallman M 2004 Data mining and multiparameter analysis of lung surfactant protein genes in bronchopulmonary dysplasia. Hum Mol Genet 13 1095-1104.

Seifart C, Plagens A, Brödje D, Müller B, von Wichert P & Floros J 2002a Surfactant protein B intron 4 variation in German patients with COPD and acute respiratory failure. Dis Markers 18 129-136.

Seifart C, Seifart U, Plagens A, Wolf M & von Wichert P 2002b Surfactant protein B gene variations enhance susceptibility to squamous cell carcinoma of the lung in German patients. Br J Cancer 87 212-217.

Serrano AG, Ryan M, Weaver TE & Pérez-Gil J 2006 Critical structure-function determinants within the N-terminal region of pulmonary surfactant protein SP-B. Biophys J 90 238-249.

Slaugenhaupt SA, Blumenfeld A, Gill SP, Leyne M, Mull J, Cuajungco MP, Liebert CB, Chadwick B, Idelson M, Reznik L, Robbins C, Makalowska I, Brownstein M, Krappmann D, Scheidereit C, Maayan C, Axelrod FB & Gusella JF 2001 Tissue-specific expression of a splicing mutation in the IKBKAP gene causes familial dysautonomia. Am J Hum Genet 68 598-605.

Tokieda K, Iwamoto HS, Bachurski C, Wert SE, Hull WM, Ikeda K & Whitsett JA 1999 Surfactant protein-B-deficient mice are susceptible to hyperoxic lung injury. Am J Respir Cell Mol Biol 21 463-472.

Tokieda K, Whitsett JA, Clark JC, Weaver TE, Ikeda K, McConnell KB, Jobe AH, Ikegami M & Iwamoto HS 1997 Pulmonary dysfunction in neonatal SP-B-deficient mice. Am J Physiol 273 L875-882.

Tuohy TM, Done MW, Lewandowski MS, Shires PM, Saraiya DS, Huang SC, Neklason DW & Burt RW 2010 Large intron 14 rearrangement in APC results in splice defect and attenuated FAP. Hum Genet 127 359-369.

Varani L, Hasegawa M, Spillantini MG, Smith MJ, Murrell JR, Ghetti B, Klug A, Goedert M & Varani G 1999 Structure of tau exon 10 splicing regulatory element RNA and destabilization by mutations of frontotemporal dementia and parkinsonism linked to chromosome 17. Proc Natl Acad Sci U S A 96 8229-8234.

Wang G, Christensen ND, Wigdahl B, Guttentag SH & Floros J 2003 Differences in N-linked glycosylation between human surfactant protein-B variants of the C or T allele at the single-nucleotide polymorphism at position 1580: implications for disease. Biochem J 369 179-184.

Ward AJ & Cooper TA 2010 The pathobiology of splicing. J Pathol 220 152-163.

Weaver TE 1998 Synthesis, processing and secretion of surfactant proteins B and C. Biochim Biophys Acta 1408 173-179.

Weaver TE, Na CL & Stahlman M 2002 Biogenesis of lamellar bodies, lysosome-related organelles involved in storage and secretion of pulmonary surfactant. Semin Cell Dev Biol 13 263-270.

Wert SE, Whitsett JA & Nogee LM 2009 Genetic disorders of surfactant dysfunction. Pediatr Dev Pathol 12 253-274.

Zuker M & Stiegler P 1981 Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res 9 133-148.

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