Identification of a bidirectional splicing enhancer: differential involvement of SR proteins in 5' or 3' splice site activation.

The adenovirus E1A pre-mRNA undergoes alternative splicing whose modulation occurs during infection, through the use of three different 5' splice sites and of one major or one minor 3' splice site. Although this pre-mRNA has been extensively used as a model to compare the transactivation properties of SR proteins, no cis-acting element has been identified in the transcript sequence. Here we describe the identification and the characterization of a purine-rich splicing enhancer, located just upstream of the 12S 5' splice site, which is formed from two contiguous 9-nucleotide (nt) purine motifs (Pu1 and Pu2). We demonstrate that this sequence is a bidirectional splicing enhancer (BSE) in vivo and in vitro, because it activates both the downstream 12S 5' splice site through the Pu1 motif and the upstream 216-nt intervening sequence (IVS) 3' splice site through both motifs. UV cross-linking and immunoprecipitation experiments indicate that the BSE interacts with several SR proteins specifically, among them 9G8 and ASF/SF2, which bind preferentially to the Pu1 and Pu2 motifs, respectively. Interestingly, we show by in vitro complementation assays that SR proteins have distinct transactivatory properties. In particular, 9G8, but not ASF/SF2 or SC35, is able to strongly activate the recognition of the 12S 5' splice site in a BSE-dependent manner in wild-type E1A or in a heterologous context, whereas ASF/SF2 or SC35, but not 9G8, activates the upstream 216-nt IVS splicing. Thus, our results identify a novel exonic BSE and the SR proteins which are involved in its differential activity.

Characterization of SRp46, a novel human SR splicing factor encoded by a PR264/SC35 retropseudogene.

The highly conserved SR family contains a growing number of phosphoproteins acting as both essential and alternative splicing factors. In this study, we have cloned human genomic and cDNA sequences encoding a novel SR protein designated SRp46. Nucleotide sequence analyses have revealed that the SRp46 gene corresponds to an expressed PR264/SC35 retropseudogene. As a result of mutations and amplifications, the SRp46 protein significantly differs from the PR264/SC35 factor, mainly at the level of its RS domain. Northern and Western blot analyses have established that SRp46 sequences are expressed at different levels in several human cell lines and normal tissues, as well as in simian cells. In contrast, sequences homologous to SRp46 are not present in mice. In vitro splicing studies indicate that the human SRp46 recombinant protein functions as an essential splicing factor in complementing a HeLa cell S100 extract deficient in SR proteins. In addition, complementation analyses performed with beta-globin or adenovirus E1A transcripts and different splicing-deficient extracts have revealed that SRp46 does not display the same activity as PR264/SC35. These results demonstrate, for the first time, that an SR splicing factor, which represents a novel member of the SR family, is encoded by a functional retropseudogene.

Prochymosin polymorphism in calves of black-and-white cattle and their crosses with Simental bulls.

Polymorphism of prochymosin was observed in individual calf abomasa, using agarose gel electrophoresis followed by detection of proteolytic activity. Abomasum samples were randomly collected during slaughtering from 239 and 146 calves (3-5 weeks old) of Black-and-White cattle and their crosses with Simental bulls, respectively. Four distinct prochymosins were found and, according to their decreasing electrophoretic mobility in alkaline agarose gel, termed as prochymosin A, D, B and C which occurred singly and in pairs (then with equal proteolytic activities of both components). Prochymosin A, B and C (designation according to FOLTMANN, 1966) activated at pH 4.7 was transformed into electrophoretically distinct chymosin. When prochymosin D was activated at this pH, chymosin D showed similar mobility as chymosin B both at alkaline and acidic pHs. Prochymosin variants occurred at genetical equilibrium in nine and ten phenotypes in the first and second genetic group. The distribution of phenotypes in the two groups differed significantly (P < 0.05). The gene frequencies of prochymosin A, D, B and C were 0.35, 0.11, 0.52 and 0.02 in Black-and-White calves, and 0.39, 0.08, 0.47 and 0.06 in crosses, respectively. These prochymosins were controlled by four pairs of codominant alleles. A possible correlation of the results obtained by FOLTMANN (1966) with ours and those of ASATO and RAND (1972, 1977) was discussed.

The gene encoding human splicing factor 9G8. Structure, chromosomal localization, and expression of alternatively processed transcripts.

The 9G8 factor is a 30-kDa member of the SR splicing factor family. We report here the isolation and characterization of the human 9G8 gene. This gene spans 7745 nucleotides and consists of 8 exons and 7 introns within the coding sequence, thus contrasting with the organization of the SC35/PR264 or RBP1 SR genes. We have located the human 9G8 gene in the p22-21 region of chromosome 2. The 5'-flanking region is GC-rich and contains basal promoter sequences and potential regulatory elements. Transfection experiments show that the 400-base pair flanking sequence has a promoter activity. Northern blot analysis of poly(A)+ RNA isolated from human fetal tissues has allowed us to identify five different species, generated by alternative splicing of intron 3, which may be retained or excised as a shorter version, as well as the use of two polyadenylation sites. We also show that the different isoforms are differentially expressed in the fetal tissues. The persistence of sequences between exon 3 and 4 results in the synthesis of a 9G8 protein lacking the SR domain which is expected to be inactive in constitutive splicing. Thus, our results raise the possibility that alternative splicing of intron 3 provides a mechanism for modulation of the 9G8 function.

Characterization and cloning of the human splicing factor 9G8: a novel 35 kDa factor of the serine/arginine protein family.

By adopting a monoclonal antibody approach, we have identified a novel splicing factor of 35 kDa which we have termed 9G8. The isolation and characterization of cDNA clones indicate that 9G8 is a novel member of the serine/arginine (SR) splicing factor family because it includes an N-terminal RNA binding domain (RBD) and a C-terminal SR domain. The RNA binding domain of 9G8 is highly homologous to those of the SRp20 and RBP1 factors (79-71% identity), but the homology is less pronounced in the cases of SF2/ASF and SC35/PR264 (45-37% identity). Compared with the other SR splicing factors, 9G8 presents some specific sequence features because it contains an RRSRSXSX consensus sequence repeated six times in the SR domain, and a CCHC motif in its median region, similar to the zinc knuckle found in the SLU7 splicing factor in yeast. Complete immunodepletion of 9G8 from a nuclear extract, which is accompanied by a substantial depletion of other SR factors, results in a loss of splicing activity. We show that a recombinant 9G8 protein, expressed using a baculovirus vector and excluding other SR factors, rescues the splicing activity of a 9G8-depleted nuclear extract and an S100 cytoplasmic fraction. This indicates that 9G8 plays a crucial role in splicing, similar to that of the other SR splicing factors. This similarity was confirmed by the fact that purified human SC35 also rescues the 9G8-depleted extract. The identification of the 9G8 factor enlarges the essential family of SR splicing factors, whose members have also been proposed to play key roles in alternative splicing.

Occurrence of prochymosin variants in the abomasum of bovine foetuses and calves.

Prochymosin variants were analysed in single abomasa from 67 foetuses (3-9 months of gestation) and 33 calves (about 6 weeks old) of Black-and-White cattle collected in a slaughter house. The method of agarose gel electrophoresis followed by detection of proteolytic activity was used. Three distinct prochymosins A, B and C that occurred singly or in pairs (with equal proteolytic activities of both components) were found. Chymosin A, B and C obtained after conversion of corresponding prochymosins, demonstrated similar electrophoretical mobilities like the three chymosin fractions contained in commercial rennin (Sigma, USA). Our chymosin B showed identical mobility as the amidated form of recombined chymosin B contained in Chymogen (Chr. Hansen's Lab., Denmark A/S). Prochymosin A, B and C in the examined animals were precursors of corresponding chymosins and were controlled by three separate codominant alleles. The following prochymosin phenotypes were found: AA (30), AB (32), AC (4), BB (29), BC (4) and CC (1). Chi-square analysis demonstrated significant differences between the observed and expected numbers of phenotypes. The gene frequencies of prochymosin A, B and C were 0.48, 0.47 and 0.05, respectively.

Contrasted cis-acting effects of downstream 5' splice sites on the splicing of a retained intron: the adenoviral E1A pre-mRNA model.

The adenoviral E1A pre-mRNA contains an upstream intron (the 216 nucleotide intron) which is spliced only weakly both in vivo and in vitro. We have chosen the E1A transcript as a model to analyse, in vitro, the role of downstream cis-elements involved in the alternative splicing of this retained intron. By using a series of constructs containing specific deletions, mutations and/or truncations, we show that the 13S 5' splice site, positioned 259 nucleotides downstream of the 216 nucleotide intron, is the main cis-element which activates the splicing of this intron. Our results establish the importance of a downstream 5' splice site for the activation of the 3' splice site, which is known to be suboptimal within this retained intron. Unexpectedly, the 12S 5' splice site, although positioned at an ideal distance (121 nucleotides) from the upstream intron, does not exhibit such a cis-acting effect. In contrast, its improvement to a consensus sequence may even result in a slight negative cis-acting effect in the presence of the 13S 5' splice site, which is the first observation of such a feature. We have shown that this unexpected behaviour is due, at least partly, to the unusual characteristics of the wild-type upstream intron, which requires a hairpin structure between the branch sites and the 3' splice site to reduce the operational distance between these two sites. Possible mechanisms involved in the contrasted cis-acting effects of the 13S and 12S 5' splice sites are discussed.