5482 Nucleic Acids Research, Vol. 20, No. 20
k. 1992 Oxford University Press
ASF alternative transcripts are highly conserved between mouse and man Roland Tacke, Annie Boned and Christo Goridis Centre d'Immunologie, INSERM-CNRS de Marseille-Luminy, case 906, F-13288 Marseille Cedex 9, France Submitted September 3, 1992 The human altemative splicing factor ASF/SF2 contains an RNA binding domain (RBD) characteristic for a family of RNA processing factors (1, 2). Recent cloning data indicated e existence of hee alternative transcripts in man ially givig rise to three different ASF isoforms (ASPI, ASF2, and ASP3) that share the RNA binding domain but differ in most of the remaining sequences (1). However, whereas ASFl (248 amino acids) accounts for all of the known functions of ASF/SP2, the existence of ASF2 and ASF3 in vivo has yet to be documented. The ASF2 and ASF3 transcripts, respectively, contain part (the 3' most 17 nt) or all (199 nt) of an intron inserted between ASFI codons 184 and 185. The 17 nt insertion introduces a shift in the ASF2 reading frame (292 codons) which terminates 119 nt 3' of the ASFl stop codon. In ASF3, which would consist of 201 amino acid residues, the C-terminal region of ASFI is replaced by 17 amino acid residues encoded by the 5'-part of the 199 nt intron. Using a combination of cDNA library screening and reverse transcription/PCR (RT/PCR) we isolated five independent clones of mouse ASF. As an inititial probe we generated a 95 bpfragment by RT/PCR using total RNA from N2a neuroblastoma cells and a pair of degenerated primers derived from the amino acid sequence of the human ASF RBD. Clone asf.5' was ilated from an N2a cell cDNA library, conins 80bp of5' uanslaed region (5'UTR) and the 5' part of the coding region up to an internal Nodl-site 273 bp downstream from the translaion start. Clone asf.IVS, isolated from a mouse brain cDNA library, is about 2 kb long, starts at position 550 and contains the complete altemative ASF intron. Three different PCR clones were amplified from cDNA reverse transcribed from N2a total RNA with a primer complementary to the sequence at position 1041-1064, 18 nt downstream from the ASF1 tamlatioh stop. Due to the 5' primer used for amplification, all te PCR clones start with the translation start codon. Clone asfl terminates with the ASFI stop codon and contains the complete ASFI rading frame. The two remaining clones tmin 4 nt downstream fron the ASF intron of which they contain only the 3' most 17 nt (asf2) or the entire sequence (asf3). Thus asf3 contains the complete open reading frame for ASF3 whereas asf2 represents a partial ASF2 cDNA clone. Apart from the 5' UTR the alternative transcripts of human (1, 2) and mouse ASF are highly homologous. The position of the intron with respect to the ASFl reading frame and the splice sites are conserved. The murine version of the intron is 3 nt shorter than its human counterpart; the sequence identity is 86%. Notably, the 3'44 nt of the intron are identical in human and
EMBL accession no. X66091 mouse. This sequence cares the two alternative 3' splice sites used for the generation of ASFI and ASF2 mRNAs, r cy. Similarly, the 5' splice site of the intron is absolutely conserved. 100% sequene identity at the nucleotide level is found for the part of ASF RNA that is used in two different reading frames. The amino acid sequences of the human and mouse ASFI reading frames are identical. The nucleotide sequence coding for ASF2 downstream from the ASFI stop codon is conserved except for two tansitions and an insertion. The latter itroduces a stop codon into the mouse sequence at position 1099. As a result, mouse ASF2 would be 12 amino acids shorter dtn its human homologue. Mouse and human ASF3 differ in tree amino acid residues owing to sequence divergencies in the ASF intron. We have sequenced the 5' 565 nt of the mouse 3'UTR (with respect to the ASFI stop codon). Surprisingly, we find 95% identity with the corresponding sequence of human SF2 (2). The similarity with the corresponding sequence of human ASF (1) is lower due to strong divergence 3' of position 1511. The sriking homology is illustrated by a stretch of 227 nt (position 1344-1571 in the mouse sequence) which is absolutly conserved between man and mouse. Any of the six possible reading fra-es includes one to several stop codons, and no obvious splice sites are present in the sequence on either strand. Hence it is unlikely that this sequence possesses any coding potential. Recently, absolute conservation between human and rabbit has been shown for the 5' 60 nt of the 3' UTR of the elongation factor 1 alpha mRNA, which appear to participate in a stable secondary structure (3). We are unable to provide arable evidence for the ASF 3' UTR, but this may simply be because the homology could comprise a more extended region than has been sequenced. Together, our data confirm the existence of altemative ASF transcripts previously predicted by human cDNA clones. ASF2 and ASF3 share the RBD with ASF1 but differ from it in that they lack an arginine/serine (RS) domain. The RS domain of U2AF appears to be an important effector domain of this factor (4) and the RS domain of ASFI may be of similar importance for ASF/SF2-specific function. ASF2 and ASF3 can thus be expected to have RNA-binding activities similar to ASF1 but altered or deficient effector activities.
REFERENCES 1. 2. 3. 4.
Ge,H., Zuo,P. and Manley,J.L. (1991) Cell 66, 373-382. Krai,A.R., Mayeda,A., Kozak,D. and Bims,G. (1991) Cel 66, 383-394. Cavallius,J. and Merrick,W.C. (1992) Nucleic Acids Re. 20, 1422. Zamore,P.D., Patton,J.G. and Green,M.R. (1992) Nature 355, 609-614.