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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 5 1928-1936
Copyright © 2000 by The Endocrine Society

Original Studies

Spatio-Temporal Expression of the Trans-Acting Splicing Factors SF2/ASF and Heterogeneous Ribonuclear Proteins A1/A1B in the Myometrium of the Pregnant Human Uterus: A Molecular Mechanism for Regulating Regional Protein Isoform Expression in Vivo1

Alison J. Pollard, Colette Sparey, Stephen C. Robson, Adrian R. Krainer and G. Nicholas Europe-Finner

Department of Obstetrics and Gynecology, University of Newcastle upon Tyne, Royal Victoria Infirmary (A.J.P., C.S., S.C.R., G.N.E.-F.), Newcastle upon Tyne NE1 4LP, United Kingdom; and Cold Spring Harbor Laboratory (A.R.K.), Cold Spring Harbor, New York 11724

Address all correspondence and requests for reprints to: Drs. Alison J. Pollard and G. Nicholas Europe-Finner, Department of Obstetrics and Gynecology, University of Newcastle upon Tyne, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP, United Kingdom. E-mail: a.j.pollard{at}ncl.ac.uk and g.n.europe-finner@ncl.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Many of the human myometrial proteins associated with uterine quiescence and the switch to coordinated contractions at the onset of labor exist as alternatively spliced isoforms. There is now extensive evidence to indicate that the nuclear concentrations of the trans-acting splicing regulators SF2/ASF and hnRNP A1/A1B are fundamental in regulating the expression of specific protein isoforms derived from alternative splicing of single precursor messenger ribonucleic acid transcripts. The question thus arose as to whether these factors were also involved in regulating the expression of specific myometrial protein species within different uterine regions during human gestation and parturition. SF2/ASF and hnRNP A1/A1B expression was therefore determined in paired upper (corpus) and lower segment myometrial samples taken from individual women at term/during spontaneous labor and compared with nonpregnant control samples using specific monoclonal antibodies. We report that SF2/ASF levels were substantially increased in the lower uterine region, and this was associated with a parallel decrease in levels of hnRNP A1/A1B during gestation. Conversely, the opposite pattern was observed within the upper uterine region during pregnancy, where hnRNP A1/A1B was significantly up-regulated and SF2/ASF levels were much less than those found in the lower uterine segment. The differential expression of hnRNP A1/A1B and SF2/ASF in the upper and lower uterine segments may have a primary role in defining the formation of specific myometrial protein species associated with the known contractile and relaxatory properties of these regions before and during parturition.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MOLECULAR mechanisms controlling uterine quiescence during maturation of the fetus and the switch to powerful coordinated contractions at the onset of labor still remain to be fully characterized. However, it is now becoming increasingly evident that the myometrial processes regulating the activity of the uterus during gestation and parturition involve the differential expression of several specific genes. These regulatory gene products include oxytocin receptors (OTRs) (1, 2, 3), the gap junction connexin-43 protein (4, 5, 6), cyclooxygenase-1 and -2 (COX-1 and -2) (7, 8), G proteins (Gs{alpha}) (9), PGE2 receptors (EP1 and EP3), PGF2{alpha} receptors, thromboxane receptors (10, 11, 12, 13), progesterone receptors (14), estrogen receptors (15, 16), caldesmon (17), and CRH receptors (18, 19). Most of these proteins exist as alternatively spliced isoforms whose tissue-specific expression may be regulated by posttranscriptional premessenger ribonucleic acid (mRNA) processing. The adenylyl stimulatory G protein Gs{alpha} provides one such example, where two specific isoforms, Gs{alpha}-Large (with an additional serine after amino acid 86) and Gs{alpha}-Small (with an additional serine after amino acid 71) are up-regulated during pregnancy and subsequently down-regulated at the onset of labor (20).

Many of the proteins associated with smooth muscle myogenesis and contractility are also regulated by alternative splicing (reviewed in Ref. 21). These include ß-tropomyosin, {alpha}-tropomyosin, and the troponin genes (21, 22). The coding capacity of these genes is increased manyfold as a result of alternate pre-mRNA processing. Both ß-tropomyosin and troponin have been used as model proteins to study alternative splicing mechanisms and their regulation in different tissues (23, 24, 25, 26). In this respect there have also been comparable studies on {alpha}-tropomyosin (21).

There is now extensive evidence indicating that alternative precursor mRNA splicing is regulated by trans-acting factors, which include small nuclear ribonuclear proteins (snRNPs) that are ubiquitously expressed, the serine-arginine protein family, and heterogeneous ribonuclear proteins (hnRNPs) (27, 28, 29, 30). Two of the major trans-acting factors that govern splice site selection, exon inclusion, and skipping within pre-mRNA transcripts have been shown to be the protein SF2/ASF, a member of the serine-arginine protein family, and the alternatively spliced hnRNP proteins hnRNP A1/A1B (31, 32, 33). Several studies have indicated that altering the concentrations of SF2/ASF and hnRNP A1/A1B results in the formation of different spliced isoforms of a number of precursor mRNAs (34, 35, 36, 37). The concentration ratios of hnRNP A1/A1B to SF2/ASF in vivo may therefore be critical in defining the expression of specific spliced protein isoforms in different tissues (38, 39).

As tissue-specific regulation of alternative splicing can be attributed to ratios of hnRNP A1/A1B to SF2/ASF, the question arises as to whether these trans-acting splicing factors are also involved in modulating the expression of specific isoforms of myometrial proteins involved in regulating uterine activity during pregnancy and labor. Consequently, the purpose of this present study was to address this question by investigating the expression of SF2/ASF, hnRNP A1/A1B, and several snRNP proteins (as controls) within the human myometrium during gestation and parturition using Western immunodetection/immunohistochemical staining with monoclonal antibodies of defined specificity. There is also increasing evidence to indicate that proteins involved in controlling uterine activity are not only temporally, but also spatially, regulated within different regions of the uterus (6, 18, 40). Fuchs et al. (40) have observed a fundus to cervix gradient of myometrial OTRs at term in the uterus. Similarly, Sparey et al. (6) have shown that levels of connexin-43 are more abundant in the upper region of the uterus than in the lower uterine segment, whereas the converse was observed for the expression of COX-1 and -2. A further aim of the study was to determine whether SF2/ASF and hnRNP A1/A1B were also spatially expressed within different regions of the uterus during gestation and parturition. To accomplish this, paired myometrial tissue samples from both the lower and upper (corpus) regions of the uterus from individual patients in and not in spontaneous labor at term were analyzed for the expression of both of these trans-acting splicing components in conjunction with the control snRNP proteins U1–70K, U1-A, and U2-B'', and a comparison was made with nonpregnant control tissue samples.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Monoclonal antibodies

The monoclonal antibodies used in this study were anti-SF2/ASF (mAb96) and anti-hnRNP A1/A1B (4B10). The specificity of these antibodies has been previously reported (39, 41). Expression of three snRNP proteins, U1–70K, U1-A, and U2-B'', were also included as positive controls, because they have been reported to have ubiquitous roles in both constitutive and alternative pre-mRNA splicing (29). Anti-U1–70K (H111), anti-U1-A (H304), and anti-U2-B'' (4G3) were all obtained from Euro-Diagnostica (Arnhem, The Netherlands). Mouse IgG1 (MOPC-21) was obtained from Sigma (Poole, UK) to use as a negative control monoclonal antibody.

Myometrial samples

Myometrium was obtained from nonpregnant, pregnant nonlaboring, and spontaneous laboring women undergoing surgery. Written consent was obtained from all women, and ethical approval was granted by the Newcastle and North Tyneside Health Authority ethics committee. Samples of myometrium from nonpregnant premenopausal women (age range, 32–46 yr) were obtained from hysterectomies performed for benign gynecological disorders. The uteri were excised longitudinally, and samples of myometrium were taken from the middle of the corpus (termed upper segment) and from close to the cervix (termed lower segment).

Myometrium was also obtained from 12 pregnant nonlaboring women undergoing elective lower segment cesarean section at term and from 12 women in spontaneous labor at term who required emergency cesarean section for fetal distress or failure to progress. Spontaneous labor was defined as regular uterine contractions and cervical dilatation of more than 3 cm on admission. Augmentation of labor with oxytocin was not a reason for exclusion, providing the onset of labor had been spontaneous. Cesarean sections were carried out under subarachnoid block or general anaesthetic. After delivery of the infant the placental site was confirmed manually, and the placenta was delivered.

Five nonfundal, upper segment (corpus) biopsies were taken from the side opposite the placental bed using laparoscopic biopsy forceps (Wolf) introduced through the lower segment incision. In addition, a 1- to 2-cm3 sample of myometrium was taken from the upper margin of the lower uterine segment using tissue forceps and scissors. Paired upper and lower segment samples were obtained from all women in each group. Myometrial samples were snap-frozen in liquid nitrogen and stored at -70 C until required. Blocks of tissue (0.5–1.0 cm3) to be used for immunohistochemical analysis were placed in Cryo-M.Bed compound (Brights Instrument Co., Huntington, UK), frozen in liquid nitrogen with cooled iso-pentane (BDH, Poole, UK), and stored at -70 C.

Preparation of myometrial homogenates

Samples were homogenized at a ratio of 1:10 in 25 mmol/L Tris buffer (pH 7.6) containing 0.25 mol/L sucrose and 1 mmol/L ethylenediamine tetraacetate in the presence of pepstatin, leupeptin, aprotinin, and phenylmethylsulfonylfluoride (Sigma). Homogenates were subsequently centrifuged at 1000 x g to remove tissue debris, and the resultant supernatants were stored at -70 C. The protein concentration was assayed using the DC protein assay kit (Bio-Rad Laboratories, Inc., Richmond, CA) with BSA as a protein standard.

Western blot immunodetection

SDS-PAGE was performed using 500 µg total protein from each myometrial homogenate solubilized in sample loading buffer (0.5 mol/L Tris, 5 mol/L urea, 2.5% SDS, and 3.5% ß-mercaptoethanol) and resolved on 12.5% or 7.5% polyacrylamide gels. Transfer of proteins was performed using a Bio-Rad Laboratories, Inc. Trans-Blot electrophoretic cell system. Samples were transferred onto either polyvinylidene difluoride (Hybond-P, Amersham Pharmacia Biotech, Aylesbury, UK) or nitrocellulose (Schleicher & Schuell, Inc., Keene, NH) at 90 V for 2.5 h. To check that similar levels of smooth muscle protein were loaded in each well, all nitrocellulose membranes were stained with Ponceau-S solution (Sigma) and densitometrically scanned before immunodetection. Membranes were preblocked with 10% Marvel milk powder/phosphate-buffered protein (PBS) for 1 h and then probed with primary mAbs (diluted 1:10–100 in PBS containing 1% Marvel) for 1–2 h at room temperature (mAb96, 4G3, H304) or overnight at 4 C (4B10, H111). After three 10-min washes, membranes were incubated for 1 h with horseradish peroxidase-conjugated antimouse IgG (DAKO Corp., High Wycombe, UK), diluted 1:2000, and detection was carried out using an enhanced chemiluminescence assay system (ECL, Amersham Pharmacia Biotech). In control experiments all antibodies were substituted with nonimmune mouse serum (data not shown). In all cases, immunodetected bands were specific. Data were obtained under conditions where a linear relationship existed between the amount of protein loaded and the intensity of the ECL signal from the immunoblots.

Quantification

ECL signals were quantified by scanning densitometry using a UMAX PS 2400 scanner at 700 dpi coupled to the Intelligent Quantifier software package from BioImage (Ann Arbor, MI). The data presented are the mean ± SEM (n = 12 for all nonpregnant, pregnant, and laboring samples). Quantification was performed using Prism 2.01 software (GraphPad Software, Inc., San Diego, CA), and data were subsequently analyzed using a one-way ANOVA with Bonferroni’s multiple comparison test.

Immunohistochemical analysis

Six-micron serial sections were cut from the frozen blocks in an Anglia Scientific Cryotome-620 cryostat at -20 C, mounted on 3-amino-propytriethoxy-silane (Sigma)-pretreated slides, and set aside to dry at room temperature for over 1 h. Tissue sections were fixed in cold acetone (BDH) for 10 min and either stained immediately or stored at -20 C. Primary antibodies diluted in PBS (1:100) were applied to each tissue section and incubated at room temperature for 1–2 h. To quench any endogenous peroxidase activity, tissue sections were incubated with 0.6% hydrogen peroxide in methanol for 3 min and then rinsed three times in PBS. Immunodetection was performed with a Histostain-Plus antimouse SP (peroxidase) staining kit (Zymed Laboratories, Inc., San Francisco, CA) using the recommended labeled biotinylated streptavidin (LAB-SA) method with a horseradish peroxidase-aminoethyl-carbazole substrate/chromogen kit.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunoblotting with the specific anti-SF2/ASF mAb96 monoclonal antibody detected a 35-kDa protein band in all tissue samples (see Fig. 1AGo). Quantification by densitometric analysis indicated that levels of SF2/ASF were similar in upper and lower uterine myometrium in nonpregnant women. However, during pregnancy myometrial SF2/ASF expression increased significantly in the lower uterine segment (P < 0.001), whereas there was a small, although not significant, increase in the upper uterine region compared with similar nonpregnant tissues (see Fig. 1BGo). During parturition, levels of SF2/ASF in the lower uterine segment decreased approximately 2-fold (P < 0.05); the data also suggest that there was a small concurrent reduction in levels of SF2/ASF in the upper uterine segment (see Fig. 1BGo).



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  		Figure 1. SF2/ASF protein expression within the human myometrium. A, Detection of SF2/ASF by Western blot analysis. Myometrial homogenates (500 µg), using paired samples from the lower uterine (LS) and upper (US) segments of the uterus, were separated by SDS-PAGE under reducing conditions. SF2/ASF protein was detected using mAb96. B, Densitometric analysis of SF2/ASF protein expression. SF2/ASF is up-regulated in the lower uterine segment during pregnancy compared with the upper uterine segment and decreases significantly in this region during labor. Data are shown as the mean ± SEM [n = 12 for all nonpregnant (NP), pregnant (P), and laboring (L) samples].*, NP/LS vs. P/LS, P < 0.001; NP/LS vs. L/LS, P < 0.05; NP/US vs. L/US, P < 0.05; P/LS vs. L/US, P < 0.01 (by Bonferroni’s multiple comparison test).

 
Western blotting with the 4B10 anti-hnRNP A1/A1B monoclonal antibody detected two protein bands of 34 and 38 kDa, representing the two hnRNP A1 isoforms, hnRNP A1 and A1B, respectively (Fig. 2AGo). Quantification demonstrated that levels of hnRNP A1/A1B were similar in both lower and upper nonpregnant myometrium. However, hnRNP A1/A1B expression increased during gestation in the upper uterine region compared with that in nonpregnant tissues (P < 0.01). Levels of expression of hnRNP A1/A1B proteins appeared to increase further within the upper uterine region when comparing laboring with pregnant tissues (see Fig. 2BGo), although the difference was not significant (P > 0.05). A substantial decrease in the levels of expression of hnRNP A1/A1B also occurred in lower segment myometrium during both pregnancy (P < 0.001) and spontaneous labor (P < 0.001) compared to levels in nonpregnant myometrium.



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  		Figure 2. hnRNP A1/A1B protein expression within the human myometrium. A, Detection of hnRNP A1/A1B by Western blot analysis. Detection of hnRNP A1 and A1B proteins (34 and 38 kDa) was undertaken using 4B10 mAb. B, Densitometric analysis of hnRNP A1/A1B protein expression. hnRNP A1/A1B is most abundant in the upper uterine segment, with only negligible levels detected in the lower segments in both pregnancy and labor. hnRNP A1 does not significantly change during labor. Data are shown as the mean ± SEM [n = 12 for all nonpregnant (NP), pregnant (P), and laboring (L) samples]. *, NP/US vs. P/US, P < 0.05; NP/US vs. L/US, P < 0.001; P/LS vs. P/US, P < 0.001; L/LS vs. L/US, P < 0.001.

 
Expression of three snRNP proteins was also evaluated by immunoblotting, in the paired myometrial tissue samples described above. The monoclonal antibody to snRNP U2-B'' (4G3) detected a protein band of 31 kDa in all tissues (see Fig. 3AGo). Similar levels of expression were observed in lower and upper uterine samples from nonpregnant, pregnant, and laboring samples (Fig. 3BGo). Immunodetection using the H111 monoclonal antibody to a core U1 snRNP protein, U1–70K, detected bands of 70 kDa in all tissues (Fig. 4AGo). Quantification of U1–70K confirmed that similar levels of U1–70K were expressed in both upper and lower samples of myometrial tissues from all three patient groups (Fig. 4BGo).



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  		Figure 3. U2-B'' snRNP protein expression within the human myometrium. A, Detection of U2-B'' by Western blot analysis. Detection of U2-B'' was undertaken using 4G3 mAb. B, Densitometric analysis of U2-B'' protein expression. There were no significant differences in the levels of U2-B'' between the upper and lower uterine segments in all three patient groups. Data are shown as the mean ± SEM [n = 12 for all nonpregnant (NP), pregnant (P), and laboring (L) samples].

 


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  		Figure 4. U1–70K snRNP protein expression in the human myometrium. A, Detection of U1–70K snRNP protein by Western blotting. UI-70K protein was detected using H111 mAb. B, Densitometric analysis of U1–70K protein expression. There is no significant difference in the levels of U1–70K in the upper and lower uterine regions in nonpregnant, pregnant nonlaboring, and spontaneous laboring states. Data are shown as the mean ± SEM [n = 12 for all nonpregnant (NP), pregnant (P), and laboring (L) samples].

 
Immunodetection using the monoclonal antibody specific to U1-A (H304), a U1 snRNP-specific protein, recognized protein bands of 34 kDa (see Fig. 5AGo). Quantification of U1-A protein levels indicated that U1-A expression decreased significantly during pregnancy and parturition in both the lower (P < 0.01) and upper (P < 0.01) segments of the myometrium (Fig. 5BGo) compared to that in nonpregnant samples.



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  		Figure 5. U1-A snRNP protein expression within the human myometrium. A, Detection of U1-A by Western blot analysis. U1-A protein was detected using H304 mAb. B, Densitometric analysis of U1-A protein expression. A significant decrease in the level of U1-A was observed in both the upper and lower uterine regions in all pregnant and laboring samples (B, lanes 5–12). Data are shown as the mean ± SEM [n = 12 for all nonpregnant (NP), pregnant (P), and laboring (L) samples]. *, NP/LS vs. P/LS, P < 0.01; NP/US vs. P/US, P < 0.05; NP/US vs. L/US, P < 0.05.

 
As fluctuations in the concentration ratio of hnRNP A1/A1B to SF2/ASF are an important factor in splice site selection, and thus protein isoform expression, the relative ratios of hnRNP A1/A1B to SF2/ASF were determined and are shown in Fig. 6Go. The data clearly indicate that a switch in the ratios of hnRNP A1/A1B to SF2/ASF occurs in both lower and upper uterine segments during gestation and parturition. The increase in the ratio of hnRNP A1/A1B to SF2/ASF during labor in the upper region reflects the further increase in hnRNP A1/A1B during labor and the corresponding decrease in SF2/ASF.



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  		Figure 6. Ratio of hnRNP A1/A1B to SF2/ASF, showing the switch in the ratios of these regulators in the upper and lower regions of the uterus during pregnancy. The further increase in the ratio of hnRNP A1 to SF2/ASF during labor relates to the corresponding decrease in SF2/ASF.

 
Immunohistochemical analysis was undertaken to confirm that SF2/ASF and hnRNP A1/A1B expression was localized within myometrial cells. Immunostaining of laboring tissue sections was similar to that of pregnant nonlaboring sections for all antibodies used (data not shown). Localization of U2-B'' was also included in the analysis as a positive control. The staining patterns for SF2/ASF, hnRNP A1/A1B, and U2-B'' are consistent with and confirm the results obtained from the Western blot experiments. Briefly, staining for SF2/ASF using mAb96 was negligible in nonpregnant tissues (Fig. 7Go, A and B, ii) and weak in sections taken from the upper uterine regions during pregnancy (Fig. 7DGo). However, strong nuclear staining for SF2/ASF was observed in the lower uterine regions prepared from pregnant samples (Fig. 7CGo), providing further verification that SF2/ASF is spatially regulated within the myometrium during pregnancy (Fig. 7CGo, ii). hnRNP A1/A1B staining, using 4B10 mAb, was moderate in lower and upper nonpregnant samples (Fig. 7Go, A and B, i). Very strong nuclear staining for hnRNP A1/A1B was observed in upper uterine sections from pregnant samples (Fig. 7DGo), consistent with the Western blot data; negligible staining for hnRNP A1/A1B was observed in sections taken from the lower uterine region during pregnancy. Immunostaining for U2-B'', using 4G3 mAb, confirmed that U2-B'' protein levels were similar in upper and lower uterine regions in nonpregnant and pregnant-nonlaboring states and that U2-B'' expression was confined to the nucleus of myometrial cells. Control reactions for each tissue section using a negative control IgG1 mAb (MOPC 21) were all negative (Fig. 7Go, A–D, iv).



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  		Figure 7. Immunohistochemical staining of the myometrium for hnRNP A1/A1B, SF2/ASF, and U2-B''. A and C, Section of lower uterine segment stained for 1) hnRNP A1/A1B (using 4B10 mAb), 2) SF2/ASF (using mAb 96), 3) U2-B'' (using 4G3 mAb), and 4) IgG mAb negative control (MOPC 21). B and D, Section of the upper uterine segment stained for 1) hnRNP A1/A1B, 2) SF2/ASF, 3) U2-B'', and 4) IgG mAb negative control. A and B, Nonpregnant; C and D, pregnant nonlaboring. Results confirm the expression patterns described in Figs. 1–3GoGoGo, showing nuclear staining for all three trans-acting regulators within the myometrial cells; i.e. SF2/ASF staining was strong in lower uterine tissue sections taken from pregnant samples and was negligible in sections from both the upper segments and nonpregnant tissue. Conversely, hnRNP A1/A1B staining was abundant in the upper uterine tissue sections from pregnant samples, moderate in nonpregnant tissue sections, and negligible in sections taken from the lower uterine region during pregnancy. Immunohistostaining for U2-B'' was consistent with the data presented in Fig. 3Go, with moderate to strong staining in all upper and lower uterine tissue sections. Magnification, x400.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we provide the first report describing the expression of the trans-acting splicing regulators SF2/ASF and hnRNP A1/A1B in a developmentally regulated human organ. Collectively, the data demonstrate that SF2/ASF and hnRNP A1/A1B are both spatially and temporally regulated within the myometrium of the human uterus during pregnancy and parturition. This is evidenced by the substantial increase in SF2/ASF levels in the lower uterine segment, which is associated with a parallel decrease in levels of hnRNP A1/A1B during gestation. Conversely, the opposite pattern is found within the upper uterine region during pregnancy, where hnRNP A1/A1B is significantly up-regulated and SF2/ASF levels are much lower than those found in the lower uterine segment. It is also interesting that during labor there appears to be a further increase in the levels of hnRNP A1/A1B in the upper uterine region, whereas SF2/ASF levels significantly decrease in the lower uterine segment; both of these latter effects may be associated with the progression of labor and the subsequent delivery of the infant. This switch in the pattern of myometrial SF2/ASF and hnRNP A1/A1B expression within different regions of the uterus from the nonpregnant to the pregnant/spontaneous laboring state, which is represented graphically in Fig. 8Go, is further confirmed by the change in ratios of hnRNP A1/A1B to SF2/ASF.



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  		Figure 8. Spatio-temporal expression of trans-acting splicing factors in the human myometrium during gestation. The switch in the pattern of myometrial SF2/ASF and hnRNP A1/A1B expression within the upper (corpus) and lower uterine regions from the nonpregnant to the pregnant state is indicated. M, Myometrium; P, placenta. The diagram of the uterus is adapted from Ref. 47.

 
Expression of the snRNP proteins, U1–70K, U1-A, and U2-B'', was also determined. U1–70K and U2-B'' protein levels were uniformly expressed within the lower and upper uterine regions from all three patient groups, which is consistent with the ubiquitous roles of snRNPs in constitutive and alternative pre-mRNA splicing (29). However in contrast, U1-A protein levels decreased significantly in both uterine regions during pregnancy and labor. This may indicate that U1-A is more involved in regulating the expression of specific myometrial proteins related to the nonpregnant condition, including the processes involved in menstruation. The decrease in U1-A protein may, in addition, be related to another step in RNA processing, i.e. mRNA 3'-end formation. Apart from being a member of the U1 snRNP complex, U1-A has also been implicated in the regulation of polyadenylation (42, 43).

There have been numerous reports demonstrating that altering the ratios of hnRNP A1/A1B to SF2/ASF in vitro promotes the formation of different spliced mRNA isoforms from the same pre-mRNA transcript (33, 34, 36). Subtle fluctuations in the concentrations or ratios of SF2/ASF and hnRNP A1/A1B can alter the splicing patterns; notably, these two regulators have an antagonistic relationship and can counteract each other in a concentration-dependent manner in selecting 5'- and 3'-splice sites (34, 36, 37, 44). These studies included the use of several reporter gene constructs and have demonstrated by overexpression of SF2/ASF and/or hnRNP A1/A1B that different alternatively spliced mRNA isoforms could be generated. Furthermore, SF2/ASF and hnRNP A1/A1B levels have been shown to fluctuate in different cell types (39, 45), and two studies have looked at the variability of hnRNP A1/A1B and SF2/ASF expression in different tissues in the mouse (38) and rat (39). However, no previous studies have as yet looked at SF2/ASF and hnRNP A1/A1B levels within different human organs and tissues.

The present study supports the previously published findings that fluctuations in the concentration and/or ratios of hnRNP A1/A1B to SF2/ASF govern splice site selection in vitro. The data presented in this study clearly complement results of the in vitro studies and also provide definitive evidence to demonstrate that this posttranscriptional regulatory process occurs in vivo. The polarization of SF2/ASF and hnRNP A1/A1B within the lower and upper segments of the myometrium, respectively, may be important in defining the functions of these different regions of the uterus at the onset of parturition. Different regions of the uterus have distinct functions; i.e. the upper region governs contractility, and the lower region governs dilatation. Sparey et al. (6) have recently shown that the myometrial gap junction protein connexin-43, which is associated with cell-cell communication, is up-regulated in the upper region at term, and in conjunction with the fundus to cervix gradient of OTRs (40) is probably involved in the propagation of contractions during parturition. Conversely, these investigators observed the increased expression of COX-1 and COX-2. COX-2 exists as two spliced isoforms in the lower uterine segment. The high levels of COX-1 and -2 in the lower uterine region may be responsible for the increased synthesis of prostacyclin/PGE2, resulting in cervical ripening and dilatation before and during parturition, respectively (46). Stevens et al. (18) also reported that CRH receptor R1 levels increased significantly in the lower region compared to the upper uterine region at preterm and term labor. Another study comparing the regional variations of PG receptors in the baboon myometrium (12) demonstrated that EP3 receptor mRNA was significantly decreased in the lower region compared to the upper uterine region, whereas EP2 receptor mRNA was more abundant in the lower region than the upper region during pregnancy. It is worthy of note that EP1, EP3, PGF2{alpha}, and thromboxane receptors can all exist as alternatively spliced isoforms (11). In this context it is also probable that SF2/ASF and hnRNP A1/A1B are involved in regulating the spatial expression of specific protein isoforms of many more genes that are involved in controlling uterine activity during gestation and labor. Future studies will be undertaken to further elucidate the involvement of SF2/ASF and hnRNP A1/A1B in regulating the expression of specific myometrial proteins within the uterus.


   Acknowledgments
 
Anti-hnRNP A1/A1B monoclonal antibody 4B10 was a kind gift from Dr. Gideon Dreyfuss (University of Pennsylvania, Philadelphia, PA).


   Footnotes
 
1 This work was supported by a grant from Action Research and in part by NCI Grant CA-13106 (to A.R.K.). Back

Received September 29, 1999.

Revised December 15, 1999.

Accepted December 28, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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