Molecular Human Reproduction, Vol. 6, No. 12, 1131-1139,
December 2000
© 2000 European Society of Human Reproduction and Embryology
Pregnancy |
Uterine expression of alternatively spliced mRNAs of mouse splicing factor SC35 during early pregnancy
1 Prince Henry's Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia
Abstract
RNA differential display was applied to identify genes critical for the establishment of pregnancy in the mouse. One of the gene fragments identified was homologous to human SC35 splicing factor; the mouse counterpart had not then been cloned. To obtain the full cDNA sequence of the mouse gene, a cDNA library was screened and four positive clones were fully analysed. Sequencing analysis indicated that we had cloned alternatively spliced mRNA species of mouse SC35 splicing factor. A map of splicing structure for this gene's pre-mRNA was then proposed and region-specific mRNA species were tested on Northern blots. This analysis indicated that the overall expression level of SC35 mRNA was much higher in implantation sites than in inter-implantation sites in the mouse uterus during early pregnancy. The expression of alternatively spliced mRNAs for SC35 was differently regulated both during early pregnancy and by steroid hormones. Embryo-derived factors were also implicated in the up-regulation of SC35 mRNA at implantation sites. These results demonstrate, for the first time, that an essential splicing factor is regulated in a complex manner during implantation in the mouse uterus. Hence, its correct regulation could be important for the success of pregnancy.
implantation/mouse/pregnancy/RNA differential display/SC35 splicing factor
Introduction
Embryo implantation is the most relevant factor limiting successful pregnancy. It is a complex process involving active interactions between the blastocyst and the uterus. During this process, the uterus undergoes morphological and physiological changes in a transformation from a non-receptive to a receptive state. The main driving force for this transformation is the ovarian hormones which act through their intracellular receptors to regulate gene expression and hence influence cellular proliferation and differentiation. In the uterus, it is anticipated that a unique set of genes are expressed in a timely manner during the preparation for receptivity. Indeed, induction of specific genes including those encoding some growth factors and cytokines has been reported in the uterus during the peri-implantation period (Huet-Hudson et al., 1990
; Stewart et al., 1992; Robb et al., 1998; Zhu et al., 1998; Das et al., 1999). However, given the complexity of the process, the exact molecular events during the uterine transition from the non-receptive to receptive state are still not well understood, and it is clear that many other molecules critical for implantation are still unidentified.
In this study, we used the mouse as a model and searched for unrecognized molecules of importance for the very early stage of implantation. In the mouse on day 4.5 of pregnancy (vaginal plug = day 0), the uterus undergoes dramatic morphological changes in association with cell proliferation and differentiation, leading to the acquisition of a receptive state (Finn and McLaren, 1967; Abrahamsohn and Zorn, 1993). Uterine remodelling at this time is marked by an increase in vascular permeability at implantation sites (Psychoyos, 1973). We hypothesized that the proliferation and differentiation of endometrial cells at this time are associated with up- or down-regulation of a number of genes, many of which are still unknown (Nie et al., 1997). To identify those uterine genes which are potentially critical for uterine receptivity, we used the technique of RNA differential display polymerase chain reaction (DDPCR) (Liang and Pardee, 1992, 1993) and compared the mRNA expression pattern of implantation and inter-implantation sites on day 4.5 of pregnancy (Nie et al., 2000a,b).
One of the genes identified as being differently regulated between the two sites was splicing factor SC35, an essential component of the spliceosome. SC35 protein is required for the first step in the splicing of precursor mRNAs (Fu and Maniatis, 1992a). Gene and cDNA sequences for SC35 splicing factor in the human have been published (Fu and Maniatis, 1992b; Sureau and Perbal, 1994), but in the mouse, the full cDNA sequence had not been reported and only one short partial gene sequence was available (accession no. X98511) when our studies commenced. To the best of our knowledge, nothing is known about this splicing factor in the mouse uterus and in particular in the process of embryo implantation. In the present investigation, we have cloned alternatively spliced mRNA sequences of mouse SC35 splicing factor and examined their uterine expression during early pregnancy. We also show that this essential splicing factor itself may be alternatively spliced and regulated in the uterus at implantation.
Materials and methods
Animals and tissue preparation
Swiss O/B mice were housed and handled according to the Monash University animal ethics guidelines on the care and use of laboratory animals. All experimentation was approved by the Institutional Animal Ethics Committee at the Monash Medical Centre. Adult female mice (6–8 weeks old) were mated with fertile males of the same strain to produce normal pregnant animals or mated with vasectomized males to produce pseudopregnant mice. The morning of finding a vaginal plug was designated as day 0 of pregnancy. Uterine tissues were collected from non-pregnant mice, pregnant mice on days 3–11 and pseudopregnant mice on days 3–5. A selection of other mouse organs was also collected from non-pregnant mice. Tissues were snap-frozen in liquid nitrogen for Northern blot analysis. Each experiment was repeated in three to four animals.
For non-pregnant, pseudopregnant and 3-day pregnant mice, the entire uterus was collected. For 4.5-day pregnant mice, implantation sites were visualized by i.v. injections of a Chicago blue dye solution (1% in saline, 0.1 ml/mouse) into the tail vein 5 min before killing the animals. Implantation sites were separated from inter-implantation sites and frozen separately. For pregnant mice from day 5.5 onwards, implantation and inter-implantation sites were visualized without dye injection.
For non-pregnant mice, the uterus was also collected from different stages of the oestrous cycle: met-oestrus, dioestrus, pro-oestrus and oestrus. The stages of the cycle were determined by analysis of vaginal smears (Rugh, 1994). For ovarian hormone treatments, the animals were first ovariectomized under anaesthesia with avertin without regard to the stage of the oestrous cycle (Rugh, 1994). The animals were allowed to rest for 2 weeks, then treated with daily s.c. injections (0.1 ml per mouse) of steroid hormones for 3 days as follows: 17ß-oestradiol (100 ng), progesterone (1 mg), or a combination of both hormones. The steroids (Sigma Chemical Co, St Louis, MO, USA) were initially dissolved in minimal amounts of ethanol before dilution in peanut oil. Animals injected with oil alone served as controls. Mice were killed 24 h after the last injection.
RNA differential display PCR, re-amplification of cDNAs and sub-cloning
RNA differential display polymerase chain reaction (DDPCR) was performed as previously published (Nie et al., 2000b) and as described originally (Liang and Pardee, 1992, 1993). Uterine mRNA expression was compared between implantation and inter-implantation sites on day 4.5 of pregnancy in two separate experiments. To avoid embryonic contamination, the embryos were removed under the light microscope from the implantation sites. Total RNA (1 µg, DNA-free) from implantation and inter-implantation sites was used as the template for the first-strand cDNA synthesis in a 20 µl reaction mixture in the presence of 20 µmol/l dNTPs, 50 µmol/l oligo-dT anchored primers (one of T12 MG, T12MC, T12MA and T12MT, M can be A, G or C), 10 mmol/l dithiothreitol, 10 IU RNasin (Promega, Madison, WI, USA), 25 IU AMV reverse transcriptase (Boehringer Mannheim, Nanawading, Victoria, Australia) and cDNA synthesis buffer. The resultant cDNA was then amplified by PCR in 20 µl with the following components: 2 µl of cDNA, 1x PCR buffer (10 mmol/l Tris–HCl, 1.5 mmol/l MgCl2, 50 mmol/l KCl, pH 8.3), 10 µmol/l dNTPs, 10 pmol of one random decamer (Operon 10-mer kit A; Operon, Alameda, CA, USA), 50 pmol of one oligo-dT anchored primer (as used in cDNA synthesis), 2 
Ci of [33P]-dATP (Du Pont Australia Ltd, North Sydney, Australia) and 1 IU of Taq DNA polymerase (Boehringer Mannheim). The PCR was performed in a Hybaid OmniGene PCR system (Hybaid Lit, Middlesex, UK) with the following conditions: initial denaturation at 94°C for 5 min; then 40 cycles of denaturation at 94°C for 30 s, primer annealing at 39°C for 2 min and extension at 72°C for 30 s; and a final extension at 72°C for 10 min. The PCR products (4 µl) were run on a 6% high resolution polyacrylamide/urea gel and visualized by autoradiography. The differential display pattern was further confirmed by Northern blotting analysis using re-amplified PCR products as probes. Those bands giving differential expression patterns on the Northern blots were sub-cloned into pGEM-T vector (Promega) and sequenced.
cDNA Library Screening
To isolate full-length cDNA sequences, a mouse uterus 
gt11 cDNA library (Clontech, Palo Alto, CA, USA) was screened with the radio-labelled cloned PCR products as probes using standard methods (Sambrook et al., 1989). In the primary screen ~ 600 000 plaques were screened, and the resultant four positive clones were plated for the second and tertiary screens to get 100% positivity. The positive phage DNA was isolated by the Lambda mini kit (Qiagen Pty Ltd, Clifton Vic, Australia) and digested with EcoR1 restriction enzyme to liberate the cDNA insert from the phage; the insert was then sub-cloned into modified pGEM-T easy vector and sequenced as described previously (Nie et al., 2000b).
Northern blot and RT–PCR analyses
For Northern blot analysis, the basic method was similar to that previously published (Nie et al., 2000a). No attempt was made to separate the embryos from the decidua before day 8 of pregnancy, but for 8- and 11-day pregnant mice, embryos were separated from the uterine tissue. Total RNA (10–15 µg) was denatured at 50°C for 60 min in 50% dimethylsulphoxide (DMSO) and 1 mol/l glyoxal, the denatured RNA was fractionated by electrophoresis through a 1.2% agarose gel in 10 mmol/l sodium phosphate buffer (pH 7.0) and transferred to positively charged nylon membranes (Hybond-N+, Amersham, Australia) by overnight capillary blotting in 5x SSPE (1x SSPE = 150 mmol/l NaCl, 10 mmol/l NaH2PO4, 1 mmol/l EDTA, pH 7.4). Probes were generated by random primer labelling of 25 ng cDNA with [32P]-deoxy-CTP (50 µCi/reaction). Unincorporated nucleotides were removed with a MicroSpin S-200 HR column (AMRAD Pharmacia Biotech, Melbourne, Australia). To correct lane to lane loading variation, each blot was also probed with a mouse cDNA probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
To validate the proposed splicing structure of mouse SC35 splicing factor primary RNA presented in Figure 4 and Table I, region-specific probes were generated by either restriction enzyme digestion or reverse transcription (RT)–PCR and tested on Northern blots. The region 1-specific probe was a 345 bp fragment between nucleotides (nt) 115–459 of MS-SC35. It was generated by RT–PCR of mouse uterine RNA with a upper primer (5'CTG TCC GGG GCG TTA GGG TCT C3') and a lower primer (5'GCG GCT GTG GTG CGA GTC3'). The region 2-specific probe was a 451 bp fragment between nt 366–816 of clone 8–5, the sequence between HindIII and ApaI sites. It was therefore obtained by digestion of clone 8–5 sequence with these two enzymes. The region 4-specific probe was a 361 bp sequence between nt 1–361 of clone 8–15. The region 5-specific probe was the sequence between nt 362–642 of clone 8–15. The latter two probes were generated by Alw 441 (SnoI) digestion (cut between nt 361–362) of clone 8–15.
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To RT–PCR amplify the sequence across regions 2 and 4, an upper primer was designed on region 2 and a lower primer on region 4 (Figure 4
). The upper primer (5'AAC ATC CTC AAC TGC CAA C3') was located at nt 834–853 of clone 8–5, and the lower primer (5'GGC CAC AAA TTA GGT TAC CA3') was at nt 595–614 of clone 8–17. Results
DDPCR results and confirmation by Northern analysis
To identify genes whose expression changes in mouse uterus during the initial implantation process, the gene expression pattern of implantation and inter-implantation sites on day 4.5 of pregnancy was compared using the DDPCR technique. Several bands were detected which showed differential expression between implantation and inter-implantation sites. One of these bands, designated as band 8, was found to be up-regulated in the implantation sites. This DDPCR result was verified by Northern blot analysis using cDNA extracted from band 8 as a probe (Figure 1). On this initial blot, multiple mRNAs were detected with the main transcript being ~2.0 kb.
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The extracted cDNA of band 8 from the DDPCR gel was re-amplified and cloned into pGEM-T vector. Northern blot analysis was repeated using the cloned inserts as probes and clone 8, which contained a cDNA of 271 bp, was confirmed to contain the cDNA representing the original expression pattern of band 8 (data not shown).
To systematically determine the expression pattern of this gene in the uterus in relation to the time of implantation and early pregnancy, total RNA from the uterus of non-pregnant mice (at oestrus) and pregnant mice at the initial stage of implantation (day 4.5 of pregnancy) through to fully established placentation (day 10.5 of pregnancy) was analysed by Northern analysis using the 271 bp cDNA of clone 8 as a probe (Figure 2). When the whole uteri were examined before and after pregnancy, it appeared that the expression level was much higher in the pregnant animals. However, it was evident that at around the time of initial embryo attachment and during the actual implantation period (day 4.5–6.5 of pregnancy), the mRNA level in the implantation sites was much higher than that in the inter-implantation sites which displayed expression levels equivalent to that in the non-pregnant uterus. After day 8.5, high expression was also seen in the embryo or/and extra-embryonic tissues. These results indicated that this gene was increased during early pregnancy and the expression level was much higher in implantation sites compared with inter-implantation sites. The observed difference between the two sites on this blot was similar to the result shown in Figure 1 when the original DDPCR-derived cDNA fragment was used as probe.
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Cloning the full cDNA sequence and prediction of alternatively spliced mRNAs
The nucleotide sequence contained in clone 8 (271 bp) was determined and is shown in Figure 3
. When compared with the GenBank database, the sequences showing the highest homology (>98% over 269 nucleotides) with clone 8 were some mouse ESTs (e.g. entry codes: MMAA20888, MMAA77395). However, the most meaningful cDNAs with known function which showed high homology with clone 8 were the human splicing factor SC35 mRNA (82% over 276 nts, accession no. M90104) and its gene (82% identity in 262 nts, accession no. X75755) and mouse putative myelin regulatory factor (92% over 141 nts, accession no. U14648). Because myelin regulatory factor is a protein closely related to SC35 splicing factor (Haque et al., 1994), it was therefore very likely that clone 8 contained the partial cDNA sequence of mouse splicing factor SC35 which had not been reported as being cloned at that time.
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To obtain the full identity of this 271 cDNA fragment and clone the full cDNA sequence of this gene, a
gt11 cDNA library derived from mouse uterus was screened using the 271 bp cDNA as a probe. Four positive phages were selected from initial screening and subjected to secondary and tertiary screening to gain 100% positivity. The cDNAs were then sub-cloned into pGEM-T easy vector and designated as clone 8–5 (1835 bp, accession no. AF250135), 8–14 (1162 bp, accession no. AF250132), 8–15 (642 bp, accession no. AF250134) and 8–17 (1196 bp, accession no. AF250133) respectively. Sequencing analysis revealed differences among these four clones, although all had a similar 3' nucleotide sequence which was homologous (>99%) to the 271 bp cDNA probe (clone 8). During our efforts to clone the full length cDNA of this gene, a cDNA sequence (1900 bp) of mouse splicing factor SC35 became available in GenBank (accession no. AF077858). We designated this published sequence as MS-SC35. Comparison of our sequences with MS-SC35 revealed an interesting but complex alignment map (Figure 4A). The shortest (DDPCR-derived) clone 8 (271 bp) aligned (>99% identity) to the 3' ends of all of the longer sequences including the published sequence MS-SC35. Among the four longer clones resulting from the library screening, clone 8–14 (1162 bp, accession no. AF250132) was essentially the same as MS-SC35 (>99.5% identity), but it lacked the 715 bp of the 5' end of MS-SC35 and therefore contained only part of the coding sequence (CDS) (Figure 4A). For clone 8–17 (1196 bp, accession no. AF250133), from nucleotide 278 onwards its sequence was 100% identical to that of clone 8–14 (from its nt 111 onwards), but the initial 277 bp fragment of clone 8–17 was not found in clone 8–14 or MS-SC35. Clone 8–15 (642 bp, accession no. AF250134) contained a shorter sequence of 642 bp and it was identical to the region close to the 3' end of MS-SC35 (between nt 1066–1708), clone 8–14 (between nt 351–992) and clone 8–17 (between nt 518–1159). Clone 8–5 (1835 bp, accession no. AF250135) was the longest clone obtained from library screening and was the most unusual. The initial 198 nucleotides (nt 1–198 bp) of clone 8–5 aligned perfectly to nt 628–825 of MS-SC35, but the subsequent 1052 bp nucleotides between and including nt 199-1250 were not found in MS-SC35. After this long unique sequence which was absent from MS-SC35 (at its nt 825–826), the immediate following 105 nucleotides (nt between and including 1251–1355) of clone 8–5 then perfectly aligned to nt 826–930 of MS-SC35. Interestingly, the remaining sequence from nt 1356 onwards of clone 8–5 aligned not to the sequence from nt 931, but further towards the 3' end from nt 1433 onwards of MS-SC35 (Figure 4A). Therefore a section of 502 bp sequence between and including nt 931-1432 of MS-SC35 was missing in clone 8–5. In addition, clone 8–5 had a longer 3' end compared with the rest of the clones. Furthermore, the initial 277 nucleotides of clone 8–17 which were not found in MS-SC35 or clone 8–14, aligned perfectly to clone 8–5 (between nt 974–1250).
Previous Northern blot analysis detected multiple mRNA species (Figures 1 and 2). These bands may represent the products of alternatively spliced primary RNA of this gene. The above sequence comparison and alignment suggested that we had actually cloned alternatively spliced mRNA forms of mouse SC35 splicing factor. To prove this, we used the sequence comparison information presented in Figure 4A and tentatively proposed a splicing structure for mouse SC35 splicing factor primary RNA (Figure 4B and Table I). We then experimentally validated this hypothesis by Northern blot analysis.
As a proposal for the basic splicing structure for the primary RNA of this gene (Figure 4B), we arbitrarily assigned the sequence before nt 825 of MS-SC35 which includes the coding sequence (CDS) as one region (region 1) and the rest of the nucleotides which are 3' untranslated sequence (UTR) into four regions (region 2–5). The sequence before nt 825 of MS-SC35 was proposed as a single region because, firstly, none of the clones obtained from library screening showed a splicing site within that section. Secondly, only one mRNA species was RT–PCR amplified at that region (data not shown). However, analysis of the only available partial gene sequence of mouse SC35 splicing factor (1620 bp, accession no. X98511) indicated that the coding region could be thought to consist of two exons. In conjunction with our data, this implied that although the coding region may consist of two exons, both exons are always present together to form the complete coding region in the final mRNA. Therefore this section of the sequence can be regarded as one unit in terms of the final mRNA format. Thus we decided to assign regions rather than exons to avoid confusion and simplify the splicing structure. Figure 4B is presented in proportion to Figure 4A so that the length and position of each region in Figure 4B can be easily referenced to the actual sequences presented in Figure 4A.
From our data, most splicing events appear to occur between regions 1 and 5 (Figure 4). Even though it is difficult to predict the exact splicing possibilities without the gene sequence, based on our data we can assume that regions 1 (coding region) and 5 (region with poly (A) tail) are always in the final mRNA. Thus four possible mRNA forms of alternatively spliced mouse SC35 splicing factor primary RNA could be predicted (Table I). The first three types have already been confirmed by the cloning and sequencing experiments (Table I and Figure 4). The first possibility was represented by clone 8–17, the second by clone 8–5 and the third by clone 8–14. Our cloning experiments did not unveil the existence of the fourth form where only region 3 is spliced out (Table I). To validate the status of this possible alternatively spliced form, RT–PCR was performed on total RNA isolated from implantation sites of day 4.5 pregnant uterus with primers across regions 2 and 4 (see Materials and methods section). This approach amplified only one type of transcript, all of which included region 3 (data not shown). Therefore, it seems that the proposed fourth possible splicing event does not occur in the mouse uterus.
Northern blot analysis to confirm the alternatively spliced mRNAs
Northern blots, prepared from long RNA gels which were run very slowly to separate the multiple bands, were probed with cDNAs specific to each region hypothesized in Figure 4B. When a probe specific to region 1 (coding region) which included nucleotides between 115 and 459 of MS-SC35 was used, three clear bands at ~2.0, 3.2 and 3.5 kb were detected (Figure 5A). An identical band pattern was obtained with a probe specific to region 5 [region with poly(A) tail; data not shown]. This finding was in close agreement with the prediction presented in Table I where regions 1 and 5 are present in all three confirmed possible mRNA forms. However, each of the bands on the gel were larger than predicted. It may be that the actual mRNAs in the cell had longer poly A tails than seen by cloning. Furthermore, the region 1 specific probe used here was not from the library derived clones, but was an RT–PCR fragment close to the 5' end of MS-SC35. That both this probe and the region 5-specific probe showed identical band patterns on the Northern blot, unambiguously confirmed that, although our library-derived clones did not contain the full length sequence, they were alternatively spliced mRNAs of mouse SC35 splicing factor.
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As anticipated (Table I
), when a probe specific to region 2 was used on Northern blots, only the two larger bands were detected (Figure 5B). When a probe specific to region 4 was used, only the largest and smallest bands were detected (Figure 5C), and this is also in close agreement with the prediction (Table I). These findings clearly supported our hypothesis and prediction, and proved that mouse SC35 splicing factor primary RNA was alternatively spliced, resulting in multiple bands on Northern blots. In addition, the splicing events seem to be the same in both the uterus and the kidney (Figure 5).
In addition, the results presented in Figure 5 confirmed previous findings that the total amount of transcript of SC35 splicing factor was much higher in implantation than inter-implantation sites on both day 4.5 and 5.5 of pregnancy. However, it is evident that of the three bands, only the 2.0 kb transcript, which was the most abundant form, showed a clear and consistent pattern of regulation between implantation and inter-implantation from more than three separate experiments.
Surprisingly, this experiment also detected very different levels of transcripts in the two non-pregnant samples. This indicated that the expression of this gene might be regulated by ovarian hormones across the oestrous cycle in the mouse uterus.
Level of SC35 splicing factor mRNAs during the oestrous cycle and the effects of progesterone and oestradiol on its expression in ovariectomized mice
To determine the influence of the oestrous cycle on the expression of SC35 splicing factor mRNA in the non-pregnant uterus, total RNA from mice at different stages of the cycle (met-oestrus, dioestrus, pro-oestrus and oestrus) was examined by Northern blotting with the region 5-specific probe (Figure 6). All three bands were detected in all samples, but the expression level, particularly of the 2.0 kb band, changed across the cycle. Overall, expression was low at pro-oestrus and oestrus, increased during met-oestrus, and started to decline towards di-oestrus (Figure 6), indicating a likely influence of oestrogen and progesterone on the expression of this gene in the uterus.
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To verify that the ovarian steroids can regulate the expression of SC35 splicing factor in the uterus, oestradiol and/or progesterone were administered to ovariectomized mice and the expression level was determined by Northern analysis using the probe specific to region 5 (Figure 7
). The ovariectomized mice treated with vehicle (oil) showed very low expression of SC35 splicing factor mRNA. Animals treated with oestradiol alone had the same level of expression as the controls while those treated with progesterone had much higher expression compared with the control or oestradiol-treated animals. Animals treated with both steroids showed less response. Both the 2.0 kb and 3.5 kb bands showed changes, but only the 2.0 kb band was found to show consistent and reproducible changes.
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Effects of the embryo on uterine expression of SC35 splicing factor
To determine whether the presence of embryos in the uterus was essential for the observed changes in SC35 splicing factor mRNA during early pregnancy, total RNA was isolated from mice on day 3.5 and 4.5 of pseudopregnancy and the expression pattern compared with that in pregnant animals by Northern analysis (Figure 8
). As seen previously (Figure 5), a high level of expression of the 2.0 kb transcript was detected on both day 3.5 and 4.5 of normal pregnancy. Furthermore, on day 4.5 this high expression was seen only in the implantation sites while the expression in the inter-implantation sites was very low. Under pseudopregnant conditions, on both day 3.5 and 4.5, the uterus showed very low expression of SC35 splicing factor mRNA, with a value equivalent to that of inter-implantation sites of actual pregnancy (Figure 8). Thus the observed high expression of SC35 splicing factor mRNA during early pregnancy appears to be embryo-dependent.
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Tissue distribution of SC35 splicing factor mRNA
Multi-tissue Northern analysis was performed to investigate whether the expression of SC35 splicing factor mRNA was specific to uterus (Figure 9
). The mRNA was detected using region 5 specific probe in all of the samples examined, indicating that it is widely expressed. The 2.0 kb band was predominant in all tissues examined. The expression level was higher in the testis, ovary and uterus compared with lung or brain, while the lowest level was observed in the muscle.
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Discussion
The DDPCR technique has been extensively used to identify differentially regulated genes in a number of biological systems (Everett et al., 1997; Kothapalli et al., 1997; Das et al., 1999). We applied this technique to search for genes which are differentially expressed between implantation and inter-implantation sites in the mouse uterus on day 4.5 of pregnancy when the uterus shows the first morphological changes associated with pregnancy. We reasoned that up- or down-regulation of these genes would be potentially important for conversion of the uterus from the non-receptive to the receptive state. We detected several bands exhibiting different expression between the two sites, one of which we identified as mouse SC35 splicing factor.
The mRNA expression of this splicing factor was much higher in implantation sites than in inter-implantation sites during early pregnancy. In the pseudopregnant uterus, the level was low and equivalent to that in the inter-implantation sites of pregnant mice. This suggests that SC35 splicing factor mRNA is tightly regulated during early pregnancy, and that its pregnancy-related expression is dependent upon the presence of blastocysts in the uterus. The observed unique expression pattern of this splicing factor suggests that it plays an important role in the implantation process.
The removal of introns from pre-mRNA precursors by splicing is a fundamental process controlling gene expression during differentiation and development, and is an essential and often regulated step in the expression of eukaryotic genes (Rio, 1992). Nuclear pre-mRNA splicing takes place in a multi-component structure termed the spliceosome, a high molecular weight complex containing small nuclear ribonucleoprotein (snRNP) particles and several non-snRNP protein components (Rio, 1992). These non-snRNP proteins are designated as SR proteins because they have one or two copies of the RNA recognition motif (RRM) and contain regions rich in arginine and serine residues (RS domains). SC35 splicing factor is one of these non-snRNP proteins and a member of the SR protein family; it has also been termed PR264 (Vellard et al., 1992). SC35 splicing factor is an important component for constitutive splicing of precursor mRNAs (Fu et al., 1992; Fu and Maniatis, 1992a). Among the SR family members, ASF/SF2 and SC35 exhibit different substrate specificities suggesting they are non-redundant components of the splicing machinery (Gallego et al., 1997). Furthermore, it has been suggested that the relative abundance of each SR protein and the molar ratio of each SR protein to snRNP (e.g. the A1 type) may determine the patterns of alternative splicing of many genes expressed in a particular cell type (Caceres et al., 1994) and influence the selection of alternative splice sites in a concentration-dependent manner (Fu et al., 1992; Caceres et al., 1994).
The concept that correct expression of splicing regulatory factors is crucial for proper cell function and development has been illustrated in a number of physiological and pathological conditions including blastocyst formation in the mouse (Jumaa et al., 1999) and control of the proliferative lifespan and senescence of cells (Ito et al., 1998). Furthermore, irregular expression of splicing regulatory factors might be one of the causes for aberrant expression of alternatively spliced mRNAs in tumour cells (Maeda et al., 1999). Changes in the expression of the SR family members has been demonstrated during mammary tumorigenesis, and this leads to alterations in the normal splicing of a number of pre-mRNAs (Stickeler et al., 1999). The expression of SC35 splicing factor is also altered after human immunodeficiency virus (HIV) infection (Maldarelli et al., 1998), indicating that HIV modifies the expression of genes in a normal cell by affecting the splicing machinery. Thus, it is clear that splicing of pre-mRNA can be an important controlling point in the regulation of cell function and pathogenesis.
The high expression of SC35 splicing factor mRNA at implantation sites during early pregnancy correlates well with the expression of many implantation-related genes (Huet-Hudson et al., 1990; Stewart et al., 1992; Bigsby and Li, 1994; Kraus et al., 1994; Everett et al., 1997; Das et al., 1997, 1999; Robb et al., 1998; Zhu et al., 1998). As many of these genes contain introns, correct processing of their pre-mRNAs by splicing mechanisms is clearly important for the accurate expression of vital proteins during early pregnancy. Thus, it can be predicted that correct expression of SC35 splicing factor in the uterus at implantation sites is pivotal, controlling the correct expression of other proteins important for implantation. In addition, due to the concentration-dependent action of SC35 splicing factor in the splicing event (Fu et al., 1992), higher expression of this splicing factor at implantation sites may also influence the pattern of the alternative splicing of other genes.
This study also attempted to determine the full cDNA sequence of mouse uterine SC35 splicing factor by screening a cDNA library using the DDPCR derived short cDNA as a probe. Four longer positive clones were fully sequenced and analysed. Strikingly, although all of these had common 3' ends, they were not identical. When these four clones were compared with the newly available sequence of mouse SC35 splicing factor, MS-SC35 (Yang et al., 1998), a complex but interesting alignment map was obtained. This map strongly suggested that these four library-derived clones represented alternatively spliced forms of SC35 splicing factor pre-mRNA. Based on the sequence analyses of all clones related to mouse SC35 splicing factor, we proposed a splicing structure for the pre-mRNA of this protein which was then successfully validated by Northern blotting with region specific probes. The multiple bands seen on the Northern blots were confirmed to be alternatively spliced forms of the same pre-mRNA. To the best of our knowledge, no previous publication has addressed the phenomenon of alternative splicing for this protein in the mouse. Interestingly, this resembles very closely the situation in the human. Several bands were similarly detected on Northern blots for human SC35 splicing factor and were established to result from alternative splicing of the pre-mRNA (Sureau and Perbal, 1994). These alternatively spliced mRNAs, also with various lengths of the 3' UTR of SC35 splicing factor, were further demonstrated to exhibit significantly different half-lives (Sureau and Perbal, 1994). This feature is likely to also hold in the mouse. Expression of multiple mRNAs of this splicing factor with different stability may be one of the mechanisms by which cells modulate the amount of SC35 splicing factor and, thereby, allow specific concentration-dependent splicing reactions. Our preliminary data (not shown) also suggests the same characteristic of splicing events for SC35 splicing factor in non-human primates. Therefore, it seems that alternative splicing of its pre-mRNA is a common feature for this splicing factor across species.
This is the first report demonstrating alterations in expression of a factor critical for pre-mRNA splicing at the time of embryo implantation. It also provides the first evidence that splicing factor SC35 mRNA is regulated both by blastocyst signals and ovarian hormones. How steroid hormones might regulate the expression of splicing factor SC35 is not clear, as no perfect hormone response elements for oestrogen or progesterone can be found on the limited short promoter sequence available for this factor. Such hormonal regulation can be both cell-type and/or species specific. Several alternatively spliced variants of SC35 mRNA are present in the uterus and it is apparent that not all of these are modulated in the same manner between implantation and inter-implantation sites during early pregnancy. The dominant band at 2.0 kb appears to be subject to the greatest regulation, both at implantation sites and also by ovarian steroids. The exact mechanism and significance of the regulation of transcription including alternative splicing of this splicing factor in establishment of pregnancy remains to be determined.
Acknowledgments
We are grateful to Jin Zhang and Anne Hampton for their technical assistance and Sue Panckridge for assistance with the illustrations. This work was funded by the Rockefeller Foundation Contraceptive 21 Initiative Project and the Wellcome Trust (grant 52666), the Rockefeller/World Health Organization (WHO) Initiative on Implantation. LAS and JKF were supported by the NH&MRC of Australia (grant 971292 and 983212 respectively). JW was a visiting research officer from SIPPR, supported by a Twinning Programme of the WHO Special Programme for Research Development and Research Training, Geneva, Switzerland. BG was an Honours student supported by PHIMR and Monash University. The nucleotide sequences reported in this paper have been submitted to the GenBank database with accession numbers AF250132–AF250135.
Notes
2 Current address: Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China 
3 To whom correspondence should be addressed at: Prince Henry's Institute of Medical Research P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: guiying.nie{at}med.monash.edu.au
References
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Submitted on April 25, 2000; accepted on September 4, 2000.
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