that decreases surface tension at the alveolar air/liquid interface. SP-A has important ... Instruments, Westbury, NY, U.S.A.) controlled by a rheostat set at 30% of ...
Biochem. J. (1995) 307, 327-330 (Printed in Great Britain)
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RESEARCH COMMUNICATION
Translation in vivo of 5' untranslated-region splice variants of human surfactant protein-A Anne M. KARINCH and Joanna FLOROS* Department of Cellular and Molecular Physiology, The Pennsylvania State University, College of Medicine, Hershey, PA 17033, U.S.A.
Transcripts of human SP-A genes, SP-Al and SP-A2, undergo alternative splicing of 5' untranslated-region exons. We reversetranscribed and amplified free cytoplasmic and polysome-bound RNA and showed that (a) all splice variants of both genes are
translated in vivo, (b) the relative translatability of splice variants can differ among individuals, and (c) the relative levels of different SP-A splice variants differ among individuals.
INTRODUCTION
acetate, 25 Tris, pH 7.7, 5 ,-mercaptoethanol. Approx. 700 ,ul of supernatant was layered on top of each gradient, and the gradients were centrifuged at 175000g (ray 110.2mm) in a Beckman SW41Ti rotor at 4 °C for 3.5 h. The gradients were fractionated with an Isco Density Gradient Fractionator (Lincoln, NE, U.S.A.). The A254 of the gradient was monitored during fractionation, and polysomal and non-polysomal fractions of interest were pooled. To each pooled fraction, guanidine thiocyanate, sodium citrate and fl-mercaptoethanol (final concns. 4 M, 0.25 M and 4.1 % respectively)- were added. The samples were stored frozen at -70 'C.
Surfactant-association protein A (SP-A) is the major lung-specific protein present in pulmonary surfactant, the lipoprotein complex that decreases surface tension at the alveolar air/liquid interface. SP-A has important functions at a number of points in the 'life cycle' of surfactant [1-3], and also appears to play a role in local host defence in the lung [4]. In humans, SP-A is encoded by two functional genes, SP-A1 [5] and SP-A2 [6], both of which are expressed [7]. Both SP-A1 and SP-A2 exhibit a high degree of complexity at the level of the gene [8-12]. The complexity includes alternative splicing of 5' untranslated (5' UT) exons [8,12]. Studies in vitro showed that the major 5' UT splice variants of the mRNA of each SP-A gene, and some of the minor variants, can be translated into protein [8]. The goal of the work reported here was to determine whether all or some of the SP-A splice variants are translated into protein in the intact lung.
EXPERIMENTAL Tissue acquisition Human lung tissue used for total RNA extraction and polysome fractionation was discarded surgical tissue. These specimens were 'normal', as assessed by gross inspection by the pathologist.
Polysome fractionation The polysome fractionation procedure used was a modification of that described by Vary and colleagues [13]. Fresh lung tissue (0.5-1.0 g) was homogenized in 4 vol. of homogenization buffer, by using two or three brief bursts with a Polytron (Brinkman Instruments, Westbury, NY, U.S.A.) controlled by a rheostat set at 30 % of maximum speed [homogenization buffer was (in mM): 25 Hepes, pH 7.5, 250 KCI, 1 magnesium acetate, 1 dithiothreitol, 250 sucrose, 0.1 EDTA]. The homogenate was centrifuged in a Beckman JA-17 rotor at 630 g (r8v 90 mm) at 2°C for 2min. A volume of 10% Triton X-100/10% deoxycholate was added to the supernatant and mixed. The homogenate was then centrifuged at 10100 g (rav 90 mm) at 2 °C for 10 min. Linear 0.6-2.0 M sucrose density gradients were formed in buffer containing (in mM) 250 KCI, 2.5 magnesium I 9
Isolation of RNA and reverse-transcription (RT) PCR RNA was isolated from pooled gradient fractions essentially as described by Ausubel and colleagues for single-step RNA isolation from cultured cells or tissues [14]. Total RNA was isolated from frozen human lung tissue as previously described [15]. RT was carried out in a total volume of 25 ,1 with MMLV reverse transcriptase (Gibco/BRL, Gaithersburg, MD, U.S.A.). For RT of total lung RNA, 1 ,ug of RNA was incubated with 15 ng of primer at 70 'C for 10 min. The reaction mixture was incubated at room temperature for 15 min with buffer provided by the vendor of the reverse transcriptase, plus 1 mM of each dNTP, 10 mM dithiothreitol and 0.5 #1 of RNase block. MMLV reverse transcriptase (0.75 1l, 150 units) was added and the reaction mixture incubated at 46 'C for 1 h. The reaction was terminated by heating at 95 'C for 5 min. For RNA isolated from polysome gradients, 1 ,ug of RNA from the 'free' fraction and 2 ,ug of RNA from the 'bound' fraction (most of RNA in the bound fraction is RNA) were incubated with 25 ng of primer. PCR was carried out by using 1 c1 of RT reaction mixture as DNA template in a 50 ,u reaction volume. The reaction mixture contained buffer supplied by the polymerase vendor, 100 ,M of each dNTP, 1.5 mM MgCl2, 150 ng of each primer, 1 unit of AmpliTaq- polymerase (Perkin-Elmer Cetus, Norwalk, CT, U.S.A.). For radiolabelled PCR reactions, 0.8 pmol of 32P-5'end-labelled primer was added to the reaction mixture. Cycling conditions were: 94 'C, 30 s; 57 'C, 30 s; 72 'C, 30 s; for 40 cycles plus a 10 min extension at 72 'C. RT-PCR amplification products were separated on either non-denaturing 12 % polyacrylamide gels or 6% polyacrylamide/6 M urea sequencing
Abbreviations used: SP-A, pulmonary-surfactant-associated protein A; 5' UT, 5' untranslated * To whom correspondence should be addressed.
region; RT,
reverse
transcription.
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Research Communication
4- 9
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4- 292 4- 293
96
U AA' A" -
B-
B'-
C C'
11
DD'
Figure 1 Genomic structure of the 5' UT region of human SP-A White blocks (A, B, C, D) are 5' UT exons. Bars beneath the exons illustrate length variants of the exons. The 5' ends of exons A, B, C are exaggerated to illustrate the size differences. The black blocks represent the first and second coding exons (I and 11). The position and orientation of oligonucleotide primers used for RT and amplification in the studies described in the text are shown above the exons.
gels, and were revealed by staining with ethidium bromide or by autoradiography of the dried gel, respectively. SP-A splice variant standards were amplified by using the above conditions and cDNA clones as template. Sequences and positions of primers were (numbering of White et al. [5]): 96, 5'-TCCTTTGACACCATCTC-3', antisense (nucleotides 1185-1201); 292, 5'-CCATTATTCCCAGGAGGACATGGTG-3', antisense (1555-1579); 293, 5'-CCATCATTTCCAGGAGGACATGGCA-3', antisense (1555-1579); 32, 5'-CTGGAGGCTCTGTGTGTGGG-3', sense (181-200); 9, 5'-GGCACAGCCACATGGCTCTG-3', antisense (1039-1058).
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