Journal of Histochemistry and Cytochemistry, Vol. 46, 335-344, Copyright © 1998, The Histochemical Society, Inc.


ARTICLE

Asymmetrical Localization of mRNAs in Enterocytes of Human Jejunum

Jay A. Bartha,b,c, Wei Lia,b,c, Stephen D. Krasinskia,b,c, Robert K. Montgomerya,b,c, Menno Verhavea,b,c, and Richard J. Granda,b,c
a Division of Pediatric Gastroenterology and Nutrition, Department of Pediatrics, The Floating Hospital for Children, New England Medical Center Hospitals, Tufts University School of Medicine, Boston, Massachusetts
b School of Nutrition Science and Policy, Medford, Massachusetts
c Center for Gastroenterology Research on Absorptive and Secretory Processes, Boston, Massachusetts

Correspondence to: Richard J. Grand, Pediatric Gastroenterology and Nutrition, The Floating Hospital for Children, New England Medical Center Hospitals, 750 Washington St., Box 213, Boston, MA 02111-1533.


  Summary
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Intracellular localization of specific mRNAs is known to be a mechanism for targeting proteins to specific sites within the cell. Previous studies from this laboratory have demonstrated co-localization of mRNAs and proteins for a number of genes in absorptive enterocytes of fetal rat intestine. The present study was undertaken to examine in human enterocytes the intracellular localization patterns of mRNAs for the microvillous membrane proteins lactase–phlorizin hydrolase (LPH), sucrase–isomaltase (SI), and intestinal alkaline phosphatase (IAP), and the cytoskeletal protein ß-actin. In sections of human jejunum, mRNAs were localized by in situ hybridization using digoxigenin-labeled anti-sense RNA probes. Both LPH and SI mRNAs were localized to the apical region of villous enterocytes, whereas IAP and ß-actin mRNAs were detected both apically and basally relative to the nucleus. Therefore, in contrast to LPH, SI, and ß-actin mRNAs, which co-localize with their encoded proteins, that of IAP is present in the basal region of the cell where IAP protein has not directly been demonstrated to be present. Absorptive enterocytes from humans possess the mechanisms for intracellular mRNA localization, but not all mRNAs co-localize with their encoded proteins. (J Histochem Cytochem 46:335–343, 1998)

Key Words: mRNA localization, enterocytes, lactase–phlorizin hydrolase, sucrase–isomaltase, intestinal alkaline phosphatase, ß-actin


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The localization of certain mRNAs to specific regions in the cytoplasm has been demonstrated in a variety of cell types (Wilhelm and Vale 1993 ; St. Johnston 1995 ; Singer 1996 ). Cytoplasmic mRNA sorting was first shown in ascidian embryos, in which actin mRNA was found to be unevenly distributed (Jeffrey et al. 1983 ). Subsequent work revealed localized mRNAs in both Drosophila embryos (Frigerio et al. 1986 ; Dreiver and Nusslein-Volhard 1988 ) and Xenopus oocytes (Rebagliati et al. 1985 ; Weeks and Melton 1987 ), which generate localized protein concentrations critical for embryonic development. The localization of specific mRNAs in somatic cells was first described for cytoskeletal proteins in chicken embryonic myoblasts, where actin, vimentin, and tubulin mRNAs were each found to be compartmentalized in different regions of the cell (Lawrence and Singer 1986 ). Localized mRNA transcripts have now been found in a number of somatic cell types, including fibroblasts, neurons, and epithelial cells. The sorting of specific mRNAs to distinct intracellular regions is believed to be a mechanism of protein localization.

The 3'-untranslated region of all sorted mRNAs studied thus far contain cis-acting sequences that are responsible for localization (Wilhelm and Vale 1993 ; St. Johnston 1995 ; Singer 1996 ). For example, ß-actin mRNA localizes to the leading lamellae of chicken embryonic myoblasts (Lawrence and Singer 1986 ). The sequences responsible for this sorting were localized to a 54- and a 43-nucleotide sequence in the 3'-untranslated region (Kislauskis et al. 1983 ). Each of these regions contained both of the motifs GGACT and AATGC, all of which were required for full localization activity. When anti-sense oligonucleotides directed against these localization signals were added, ß-actin mRNA sorting was disrupted and ß-actin protein was no longer concentrated at the leading edge. This resulted in altered cellular morphology, demonstrating the functional importance of mRNA localization in somatic cells.

We have previously demonstrated that mRNAs encoding proteins with enzymatic function are localized to specific regions in the cytoplasm of absorptive enterocytes in fetal rat intestine (Rings et al. 1992a ) . In these cells, the mRNA for lactase–phlorizin hydrolase (LPH), a microvillous membrane disaccharidase, is targeted to the apical region, whereas the mRNA for carbamoyl-phosphate synthetase (CPS), a urea cycle enzyme, is sorted to the mitochondrial compartments in supra- and subnuclear regions of the cell. In contrast to the cytoplasmic partitioning of these mRNAs, the mRNA for the Krebs cycle enzyme phosphoenolpyruvate carboxykinase (PEPCK) is distributed evenly throughout the cytoplasm. Therefore, although not all mRNAs displayed an asymmetric distribution, all co-localized with their encoded proteins.

We sought to extend these findings by studying the localization of specific mRNAs in absorptive enterocytes of adult humans. The intracellular distribution of mRNAs for the microvillous membrane proteins LPH and sucrase–isomaltase (SI), disaccharidases that are anchored to the cell membrane by short polypeptide sequences, and intestinal alkaline phosphatase (IAP), which hydrolyzes monophosphate esters and is anchored to the membrane by a phosphatidylinositol–glycan linkage (Domar et al. 1992 ), were compared to that of the cytoskeletal protein ß-actin. We found that LPH and SI mRNAs localize to the apical region of absorptive enterocytes, whereas IAP and ß-actin mRNAs are detected apically as well as in subnuclear regions of the cell.


  Materials and Methods
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Reagents
All restriction enzymes and DNA-dependent RNA polymerases were purchased from Gibco-Bethesda Research Laboratories (BRL), (Grand Island, NY), Promega-Biotec (Madison, WI), or Pharmacia-Biotec (Piscataway, NJ). RNase A and T1 were purchased from Sigma Chemical Company (St Louis, MO). Radioactive isotope was purchased from DuPont–New England Nuclear (Boston, MA). All other chemicals and reagents were purchased from Sigma, Gibco-BRL, or Fischer Scientific (Fair Lawn, NJ).

Tissue Preparation
Human jejunal tissue was obtained from adults undergoing elective gastric bypass surgery. Protocols were approved by the Human Investigation Review Committee of the New England Medical Center Hospitals. Specimens were obtained 20–120 cm distal to the ligament of Treitz and immediately placed in normal saline. The mucosa was dissected, mounted on filter paper, and fixed in 10% buffered formalin for 72 hr. After embedding in paraffin, the tissue was sectioned at 5 µm for histology and in situ hybridization. Tissue sections stained with hematoxylin and eosin were reviewed for orientation and integrity by standard light microscopy before in situ hybridization.

Preparation of Probe Templates
Templates for anti-sense and sense RNA probes were constructed for LPH, SI, IAP, and ß-actin using fragments of human cDNAs. The size of these fragments and the location of these sequences within the full-length cDNAs are indicated in Table 1. A 336-BP SphI/ClaI fragment of human LPH sequence, derived from a plasmid containing 2.4 KB of cDNA (gift from H. Naim; Naim et al. 1991 ) was cloned into the vector pGEM7Zf (Promega-Biotec). A 420-BP SalI/EcoRI fragment of human SI sequence (gift from P. Traber; Traber 1990 ), a 228-BP PstI/HindIII fragment of human IAP sequence [gift from P. Henthorn (Henthorn et al. 1987 ) and D. Alpers], and a 404-BP HinfI/Fnu4HI fragment of human ß-actin (gift from I. Herman) were all subcloned into pBluescript KS (Stratagene; La Jolla, CA) vectors. All constructs were confirmed by sequencing.


 
View this table:
[in this window]
[in a new window]
 
Table 1. Characteristics of RNA probe templates

Northern Blotting and RNase Protection Assays
To verify the specificity of the probes, Northern blots and RNase protection assays were carried out. Human jejunal RNA was isolated by homogenization of mucosal scrapings in guanidine isothiocyanate, purified through cesium chloride, and quantified by optical density at A260 by the method of Chirgwin et al. 1979 as described (Buller et al. 1990 ). After electrophoresis in a 1% denaturing agarose gel, the intestinal RNA was transferred to Genescreen (DuPont–New England Nuclear) membranes. Sharp ribosomal RNA bands in the agarose gel demonstrated that the RNA was not degraded. The blot was probed with labeled DNA fragments corresponding to the cDNA sequence used for in situ hybridization. The probes were random-labeled using [32P]-dCTP (3000 Ci/mmol) and an oligonucleotide labeling kit (Pharmacia-Biotec; Piscataway, NJ). The probes were hybridized to the membranes under high-stringency conditions [50% formamide, 42C, 5 x SSC (1 x SSC = 150 mM NaCl, 15 mM sodium citrate)] overnight. The membranes were washed and exposed to Kodak XAR film (Rochester, NY).

RNase protection assays were carried out as described (Krasinski et al. 1994 ) using both antisense and sense probes. RNA probes, labeled in the presence of [32P]-UTP (800 Ci/mmol), were gel-purified and hybridized to human intestinal RNA (5 µg) at 65C overnight. After hybridization and RNase A and T1 treatment, the protected probe fragments were separated in a 6% denaturing polyacrylamide gel. Undigested probes were loaded in adjacent lanes for size comparison. The gels were dried and exposed to film.

Preparation of Digoxigenin-labeled RNA Probes
Digoxigenin-labeled RNA probes were prepared using the DIG RNA Labeling Mix (Boehringer Mannheim; Indianapolis, IN) according to the manufacturer's instructions with modifications described by Murray and DeSouza 1995 . Linearized templates were incubated with the buffered labeling mix, RNA polymerase, and RNase inhibitor for 2 hr at 37C. The transcription reactions were terminated by addition of EDTA to a final concentration of 0.2 M, and the digoxygenin-labeled RNA probes were precipitated in ethanol, dried, resuspended in diethyl pyrocarbonate-treated water, and purified through Select G-25 spin columns (5 Prime-3 Prime; Boulder, CO). The RNA was quantified spectrophotometrically at A260, and the size and the relative purity of the probes were verified by gel electrophoresis. A dot-blot assay was used to estimate the digoxigenin incorporation into the probe (Murray and DeSouza 1995 ). Aliquots in concentrations of 1 µg/ml were frozen at -20C until use.

In Situ Hybridization
In situ hybridization was carried out as described by Murray and DeSouza 1995 . All steps were performed at room temperature unless indicated otherwise. Sections were deparaffinized in xylene (three times for 20 min) followed by graded treatments in ethanol (100%, 95%, 70%; twice for 2 min each). After incubations in 0.2 N HCl in 1 x PBS (twice for 5 min), slides were treated with proteinase K (30 µg/ml) for 10 min at 37C, postfixed in 4% paraformaldehyde for 10 min, and washed in PBS. Sections were then washed in 0.1 M triethanolamine, acetylated with 0.25% acetic anhydride, washed in 2 x SSC, and dehydrated in graded treatments of ethanol. The sections were hybridized overnight at 50C in a solution containing 50% formamide and 1 ng/ml of digoxigenin-labeled RNA probe. Sections were then washed in 4 x SSC, incubated in RNase A, followed by sequential washings of increasing stringencies: 2 x SSC, 15 min; 1 x SSC, 15 min; 0.5 x SSC, 15 min; 0.1 x SSC, 30 min, 55C. Sections were then treated with a blocking buffer containing 0.05% Triton X-100 and 2% normal sheep serum. Alkaline phosphatase-conjugated anti-DIG antibody (Boehringer Mann-heim) was placed on the slides and incubated overnight in a humidified chamber at 4C. After washing, the sections were treated with 1 mg/ml levamisole to block labeling of any residual endogenous alkaline phosphatase. Slides were incubated in chromogen solution containing levamisole, 4-nitroblue tetrazolium chloride, and 5-bromo-4-chloro-3-indolyl-phosphate for 2–4 hr. The reactions were stopped by incubating the slides in a Tris–EDTA solution for 15 min. Sections were then lightly counterstained with methyl green. In situ hybridization was performed with each probe on tissue sections obtained from at least five different individuals. In addition to hybridization of sense probes to tissue sections, controls also consisted of treatment of sections with RNase before hybridization to anti-sense probes.

Sequence Analysis
Sequence analysis of the 3'-untranslated regions of all four cDNAs was carried out using the Genetics Computer Group's Sequence Analysis Software Package (Devereux et al. 1984 ). Sequence analysis included homology comparisons and identification of internal repeats as well as a search for the core motifs, GGACT and AATGC, which play a role in ß-actin mRNA sorting in chicken fibroblasts (Kislauskis et al. 1983 ).


  Results
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Specificity of Probes
The specificity of the probes used for the in situ hybridization assays was confirmed by Northern blotting and RNase protection assays using human jejunal RNA. On Northern blots (Figure 1), labeled LPH and SI probes hybridized to mRNAs that were approximately 6.5 KB in length, whereas the IAP and ß-actin probes hybridized to mRNAs that were 2.7 and 1.8 KB, respectively. The sizes of the mRNAs determined in the present study correspond to those previously determined by others (Ponte et al. 1983 ; Henthorn et al. 1988 ; Chantret et al. 1992 ; Escher et al. 1992 ), demonstrating that the probes used in the present study hybridize to their homologous authentic mRNAs, as anticipated. In RNase protection assays, all anti-sense probes protected a major fragment of predicted size, whereas no signal was detected when assays were performed using sense probes (not shown). These data demonstrate that the anti-sense RNA probes are specific for their corresponding mRNAs and that the sense probes can be used as negative controls.



View larger version (43K):
[in this window]
[in a new window]
 
Figure 1. Northern blot analysis demonstrating the specificity of the probes. The probes (indicated across the top of the gels) were constructed and labeled as described in Materials and Methods. Ten or 25 µg of human jejunal RNA was loaded in lanes as indicated. The mobility of the 28S and 18S ribosomal RNA bands is shown at left. The membranes were exposed to film for 6–40 hr.

Histology
All jejunal tissue samples were examined and demonstrated normal morphology and cellular architecture, as shown in Figure 2A. There was no increase in inflammatory cells, crypt lengthening, or villous shortening in any of the tissue samples studied. Therefore, the integrity of the tissue section was not affected by the length of time the tissue remained in normal saline after surgery (average 30 min), or by the fixation, embedding, and sectioning procedure. Higher magnification confirmed the character of the enterocytes (Figure 2B).



View larger version (113K):
[in this window]
[in a new window]
 
Figure 2. Intracellular localization of specific mRNAs in absorptive enterocytes. (A,B) H&E staining of sections demonstrating the integrity of the crypt–villus structure (A) and character of the absorptive enterocytes (B). (C–N) In situ hybridization assays of proximal jejunum using digoxigenin-labeled probes. Anti-sense probes for LPH (C,D) and SI (E,F) mRNAs revealed that these mRNAs are localized to the apical region of the cell. No signal was detected when sense probes for LPH (G) and SI (H) were used. An anti-sense probe for IAP (I) revealed staining in both apical and basal regions of the cell (arrows). (J) Section adjacent to that shown in I hybridized to the sense IAP probe, revealing nonspecific background staining in the lamina propria. (K) High magnification of cells demonstrating that staining for IAP mRNA is more intense in apical regions (arrows). An anti-sense probe for human ß-actin demonstrated apical and basal localization (arrows) in absorptive enterocytes on villi (L) as well as in cells in crypts (M). (N) Section adjacent to that shown in M hybridized to a sense ß-actin probe, showing absence of background staining. Bars: A = 100 µm; C,E I,J,M,N = 200 µm; B,D,FH,K,L = 400 µm.

Intracellular Localization of Specific mRNAs
The intracellular localization of specific mRNAs was studied in absorptive enterocytes of human jejunum using digoxigenin-labeled RNA probes. LPH and SI mRNAs both localized to the apical region of the enterocytes with little or no detectable signal in any other region of the cells (Figure 2C–F). No significant reaction product was found in the tissue sections exposed to LPH or SI sense probes (Figure 2G and Figure 2H, respectively). In contrast, IAP mRNA was detected both apically and basally relative to the nucleus (Figure 2I). The anti-sense probe used for the identification of IAP mRNA yielded signal in nonepithelial cells of the lamina propria. However, a similar signal was also noted when the IAP sense probe was used (Figure 2J), suggesting that the staining in the lamina propria was nonspecific background. The apical staining appeared to be more intense than that of the basal, suggesting that the IAP mRNA is distributed asymmetrically (Figure 2K). IAP mRNA was not present in crypt cells (not shown). ß-Actin mRNA, like that of IAP, was detected in positions apical and basal to the nucleus (Figure 2L). However, the basal staining for ß-actin mRNA was concentrated at the basal membrane (Figure 2L), whereas that of IAP appeared more diffuse (Figure 2K). Further, the basal staining for ß-actin mRNA was more intense than its apical staining (Figure 2L), suggesting an asymmetrical mRNA localization that differs from that of LPH, SI, and IAP. ß-actin mRNA was also detected in crypt cells where an apical and basal localization similar to that observed in villous enterocytes was found (Figure 2M). The intensity of staining of ß-actin and IAP mRNAs was not uniform from cell to cell. No significant reaction product was detected in the tissue sections hybridized to ß-actin sense probes (Figure 2N). Moreover, no staining was observed with any of the anti-sense probes when the slides were pretreated with RNase (not shown), demonstrating that the signals obtained were due to hybridization to RNA.

Sequence Analysis of the 3'-untranslated Regions
Computer-assisted sequence analysis of the 3'-untranslated regions of all four mRNAs was carried out (Figure 3A). Sequence analysis of the human LPH mRNA revealed a 10-base motif (Figure 3B) that repeated itself identically in adjacent sequence. Within this 10-base motif, a 5-base sequence, TTAAG, is present in three copies in both the human LPH and the SI 3'-untranslated region, but it is not present in that of either IAP or ß-actin. A sequence similar to the 10-base human LPH motif (80% identical) is also present in the SI mRNA 3'-untranslated region (Figure 3B). The 10-base SI homologue contains a 5-base sequence, TCAAT, which is repeated four other times in the SI 3'-untranslated region (Figure 3A); this sequence was also present in a single copy in the IAP 3'-untranslated region.



View larger version (20K):
[in this window]
[in a new window]
 
Figure 3. Computer-assisted sequence analysis of the 3'-untranslated regions of human LPH, SI, IAP, and ß-actin mRNAs. The numbers coincide with the numbers of the sequence in the corresponding cDNAs. (A) Identification of repeated sequence or sequence known to play a role in mRNA sorting. Sequences a and b were repeated motifs found in LPH and/or SI, and sequences c and d are elements known to play a role in the localization of ß-actin mRNA in chicken fibroblasts (Kislauskis et al. 1983 ); (B) Identification of a 10-base direct repeat in the 3'-untranslated region of the human LPH cDNA. A homologous sequence (80% identity, boxes) was also found in the 3'-untranslated region of the human SI cDNA as indicated. Brackets above the sequence indicate the 10-base repeat and TTAAG core motifs.

The two 5-base core elements in the 3'-untranslated region of the chicken ß-actin mRNA, GGACT, and AATGC, have been shown to play a role in its localization in chicken myoblasts (Kislauskis et al. 1983 ). Of the four genes under study, only the ß-actin 3'-untranslated region contains both motifs (Figure 3A); IAP has two copies of only the GGACT sequence and SI has a single copy of the AATGC element.

Differential Expression of LPH and SI mRNA Along the Crypt–Villous Axis
The localization along the crypt–villous axis differed for LPH and SI mRNAs. LPH mRNA was present uniformly throughout the villus (Figure 4A), whereas SI mRNA was present primarily in the lower half of the villus, with little detectable signal in the upper half (Figure 4B). Neither mRNA was detected in crypt cells. This crypt–villous distribution of LPH and SI mRNA was consistent for all individuals studied. Furthermore, there was no evidence of a patchy or mosiac pattern of expression of either LPH or SI mRNA.



View larger version (106K):
[in this window]
[in a new window]
 
Figure 4. Crypt/villous distribution of LPH and SI mRNAs. LPH mRNA (A) is present along the entire length of the villus, whereas SI mRNA (B) is localized to the lower half of the villus. The crypts are free of reaction product. Bars = 100 µm.


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The present study reveals different intracellular localization patterns for specific mRNAs in absorptive enterocytes of human jejunum. The mRNAs for the microvillous membrane proteins LPH and SI localize to the apical region, whereas the mRNAs for IAP, also a microvillous membrane protein, and the cytoskeletal protein ß-actin are sorted both apically and basally to the nucleus. Previously, we showed that mRNAs encoding proteins with enzymatic function (LPH, CPS, and PEPCK) are localized to specific regions in the cytoplasm of absorptive enterocytes in fetal rat intestine (Rings et al. 1992a ), demonstrating that intestinal cells are capable of intracellular mRNA sorting. Apical sorting of LPH mRNA has also been shown in suckling and adult rat intestine by others (Duluc et al. 1993 ; Freund et al. 1995 ). Here we show for the first time that absorptive enterocytes from humans possess the mechanisms for intracellular mRNA localization. The presence of mRNA sorting systems across species suggests that this process may be an important intra-cellular mechanism in intestinal cells.

The sorting of specific mRNAs to distinct cytoplasmic regions has been suggested as a mechanism of protein localization (Wilhelm and Vale 1993 ; Rings et al. 1994a ; St. Johnston 1995 ; Singer 1996 ). Both LPH and SI mRNAs localize to the apical region of human enterocytes, adjacent to the microvillous membrane in which the proteins encoded by these mRNAs are situated. However, in contrast, IAP mRNA is not localized to the apical region alone but instead appears diffuse in the cytoplasm of the human enterocyte, and is present in both apical and basal regions of the cell. In previous experiments using radiolabeled probes, IAP mRNA was found to be distributed diffusely in the cytoplasm of absorptive enterocytes of newborn rats (Estrada et al. 1996 ), a finding also recently reported by Xie et al. 1997 for adult rats, and similar to the distribution of human IAP mRNA shown in the present study. In rats, IAP protein is found both in the microvillous membrane and diffusely in the cytoplasm of absorptive enterocytes (Shields et al. 1982 ). In contrast, human IAP protein has been reported to be abundantly expressed in the microvillous membrane (Domar et al. 1992 ). IAP is involved in transepithelial lipid transport in rat and human enterocytes, indicating that some of the protein is directed to other enterocyte membranes (Xie et al. 1997 ). Thus, in rats, IAP mRNA localization parallels that of IAP protein, whereas in humans, IAP mRNA is found in a region of the enterocyte (basal to the nucleus) likely containing IAP protein but in which it has not yet been directly shown to be present. These data suggest that mRNA sorting may play a role in the intracellular localization of certain proteins but may not be a mechanism that is necessary for the targeting of other proteins.

ß-Actin, a component of the cytoskeleton in nonmuscle cells (Sakiyama et al. 1989 ) is distributed in the microvilli, terminal web, and near the basal and lateral membranes of rat absorptive enterocytes (Hagen and Trier 1988 ). The intracellular distribution of ß-actin in human enterocytes has not been studied extensively. We found ß-actin mRNA to be localized to the apical and basal poles of human intestinal cells within crypts and on villi, with a more intense staining in basal regions. In contrast, previous studies in mice using radiolabeled probes revealed an asymmetric distribution of ß-actin mRNA in villous enterocytes, with an accumulation of mRNA in the apical portion of the cell (Cheng and Bjerknes 1989 ). In mouse crypts, a uniform distribution of the mRNA was described, which also contrasts with the asymmetric distribution we found in human crypt cells. Although it is possible that the localization patterns of ß-actin mRNA are species-specific, it is likely that the differences in resolution between the use of radioactive vs digoxigenin-labeled probes could account for the differences in localization patterns observed in these studies.

Computer-assisted sequence analysis of the 3'-untranslated regions revealed similarities between LPH and SI. A TTAAG motif is present in three copies in both the LPH and SI 3'-untranslated regions but is not present in those of IAP and ß-actin. Although there is no evidence that this sequence plays a role in mRNA sorting, it is interesting that this motif is present in multiple copies only in the mRNAs that are targeted exclusively to the apical region. Five copies of a TCAAT motif were present in the SI 3'-untranslated region but were not widely distributed among the other mRNAs. Two previously characterized targeting signals in the chicken ß-actin mRNA, GGACT and AATGC, are required for full localization activity in chicken myoblasts (Kislauskis et al. 1983 ). Of the four mRNAs under study, only the human ß-actin 3'-untranslated region contains both motifs. However, it is not yet known if these motifs play a role in the localization of human ß-actin mRNA in absorptive enterocytes.

The mRNAs for microvillous membrane hydrolases were not detected in crypt cells, supporting previous data obtained by us and others for LPH and IAP mRNA in rat intestine (Rings et al. 1992a , Rings et al. 1992b ; Duluc et al. 1993 ; Rings et al. 1994a , Rings et al. 1994b ; Freund et al. 1995 ; Estrada et al. 1996 ; Xie et al. 1997 ) and for SI mRNA in rat and human intestine (Traber 1990 ; Traber et al. 1992 ). Previous reports of LPH and IAP mRNAs in crypts (Freeman 1995 ) cannot be reconciled with the present results.

In a previous study of human jejunum using a digoxigenin-labeled RNA probe, Maiuri et al. 1994 demonstrated a patchy pattern of expression of LPH mRNA in humans with adult-type hypolactasia. In the present study we found no evidence of a patchy pattern of expression, suggesting that the individuals in our study were lactase-sufficient. Although these investigators were not studying intracellular mRNA localization directly, their photomicrographs showed staining for LPH mRNA in both apical and basal regions of the cells as well as throughout the lamina propria. Staining in the lamina propria, where LPH is known not to be expressed, suggests that their probe or hybridization conditions allowed nonspecific background staining.

Human LPH mRNA was expressed evenly along the length of the villus, a pattern similar to that found in fetal rats (Rings et al. 1992a , Rings et al. 1992b ). After birth in rats, LPH mRNA becomes restricted to the lower half of the villus (Rings et al. 1992b ; Duluc et al. 1993 ; Freund et al. 1995 ), a pattern also seen in mature rabbits (Freeman 1995 ) but contrasting with that in adult humans observed in the present study. Our data suggest that expression of LPH mRNA along the crypt–villous axis is different in the human from that in the rat or rabbit.

In contrast to LPH, SI mRNA in human jejunum was expressed mainly in the lower half of the villus, a pattern previously reported in both rat and human duodenum (Traber 1990 ; Traber et al. 1992 ). The difference between this pattern and that observed for human LPH mRNA suggests that the regulation of gene expression along the villus differs for LPH and SI. Indeed, we have previously shown that LPH and SI are independently regulated in individual enterocytes along the villus during intestinal development (Rings et al. 1994b ). However, it is possible that the different patterns reflect differences in the limits of detection or relative abundance of these mRNAs in villous enterocytes.

The present study demonstrates that intracellular mRNA localization occurs in human enterocytes. The mRNAs of two microvillous membrane enzymes, LPH and SI, are localized to the apical region of the cells, suggesting that mRNA sorting may play a role in targeting these proteins to their site of function. In contrast, human IAP mRNA is present throughout the cell. Human IAP protein is predominantly localized to the microvillous membrane but is also targeted to other enterocyte membranes in its role in transepithelial lipid transport (Xie et al. 1997 ). These findings indicate that not all localized enterocyte proteins are subjected to control at the mRNA sorting level. The signal for mRNA targeting likely resides in the 3'-untranslated regions of the mRNAs. Although some homologies between human LPH and SI 3'-untranslated regions were identified, it remains to be determined whether these sequences play a role in the intracellular localization of these mRNAs.


  Acknowledgments

Supported by National Institutes of Health Research Grant R01-DK-32658, Digestive Disease Core Center Grant P30-DK-34928, Pediatric Gastroenterology Research Training Grant T32-DK-07471, Clinical Investigator Award K08-DK-02182 (MV), an American Gastroenterological Association Industry Scholar Award (MV), and by grants from the Charles H. Hood Foundation and March of Dimes Birth Defects Foundation (SDK).

We are grateful to Beverly Rubin, PhD, Mary Murray, PhD, and Yan Wei of the Center for Reproductive Biology (NIH P30 HD28897), Department of Anatomy and Cellular Biology, Tufts University School of Medicine, for assistance with the in situ hybridization protocols and the use of the Imaging Core. We are also indebted to the following colleagues for participation in various aspects of the study: Peter Benotti, MD, and Scott Shikora, MD, Department of Surgery; and Annette Shephard–Berry, Department of Pathology.

Received for publication July 17, 1997; accepted September 30, 1997.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Büller HA, Kothe MJC, Goldman DA, Grubman SA, Sasak WV, Matsudaira PT, Montgomery RK, Grand RJ (1990) Coordinate expression of lactase phlorizin hydrolase mRNA and enzyme levels in rat intestine during development. J Biol Chem 265:6978-6983[Abstract/Free Full Text]

Chantret I, Lacasa M, Chevalier G, Ruf J, Islam I, Mantei N, Edwards Y, Swallow D, Rousset M (1992) Sequence of the complete cDNA and the 5' structure of the human sucrase-isomaltase gene. Biochem J 285:915-923[Medline]

Cheng H, Bjerknes M (1989) Asymmetric distribution of actin mRNA and cytoskeletal pattern generation in polarized epithelial cells. J Mol Biol 210:541-549[Medline]

Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299[Medline]

Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387-395[Abstract]

Domar U, Nilsson B, Baranov V, Gerdes U, Stigbrand T (1992) Expression of intestinal alkaline phosphatase in human organs. Histochemistry 98:359-364[Medline]

Dreiver W, Nusslein-Volhard C (1988) The bicoid protein determines position in the Drosophila embryo in a concentration-dependent manner. Cell 54:95-104[Medline]

Duluc I, Jost B, Freund JN (1993) Multiple levels of control of the stage- and region-specific expression of rat intestinal lactase. J Cell Biol 123:1577-1586[Abstract]

Escher JC, de Koning ND, van Engen CGJ, Arora S, Büller HA, Montgomery RK, Grand RJ (1992) Molecular basis of lactase levels in adult humans. J Clin Invest 89:480-483[Medline]

Estrada G, Krasinski SD, Rings EHHM, Büller HA, Grand RJ, López–Tejero MD (1996) Prenatal ethanol exposure alters the expression of intestinal hydrolase mRNAs in newborn rats. Alcoholism: Clin Exp Res 20:1662-1668[Medline]

Freeman TC (1995) Parallel patterns of cell-specific gene expression during the enterocyte differentiation and maturation in the small intestine of the rabbit. Differentiation 59:179-192[Medline]

Freund JN, Jost B, Duluc I, Morel G (1995) Ultrastructural study of intestinal gene expression. Biol Cell 83:211-217[Medline]

Frigerio G, Burri M, Bopp S, Baumgartner S, Noll M (1986) Structure of the segmentation gene paired and the Drosophila PRD gene set as part of a gene network. Cell 47:735-746[Medline]

Hagen SJ, Trier JS (1988) Immunocytochemical localization of actin in epithelial cells of rat small intestine by light and electron microscopy. J Histochem Cytochem 36:717-727[Abstract]

Henthorn PS, Raducha M, Edwards YH, Weiss MJ, Slaughter C, Lafferty MA, Harris H (1987) Nucleotide and amino acid sequences of human intestinal alkaline phosphatase: close homology to placental alkaline phosphatase. Proc Natl Acad Sci USA 84:1234-1238[Abstract]

Henthorn PS, Raducha M, Kadesch T, Weiss MJ, Harris H (1988) Sequence and characterization of the human intestinal alkaline phosphatase gene. J Biol Chem 263:12011-12019[Abstract/Free Full Text]

Jeffrey WR, Tomlinson CR, Brodeur RD (1983) Localization of actin messenger RNA during early ascidian development. Dev Biol 99:408-417[Medline]

Kislauskis EH, Li Z, Singer RH, Taneja KL (1983) Isoform-specific 3'-untranslated sequences sort {alpha}-cardiac and ß-cytoplasmic actin messenger RNAs to different cytoplasmic compartments. J Cell Biol 123:165-172[Abstract]

Krasinski SD, Estrada G, Yeh K-Y, Yeh M, Traber PG, Rings EHHM, Büller HA, Verhave M, Montgomery RK, Grand RJ (1994) Transcriptional regulation of intestinal hydrolase biosynthesis during postnatal development in rats. Am J Physiol 267:G584-594[Abstract/Free Full Text]

Lawrence JB, Singer RH (1986) Intracellular localization for messenger RNAs for cytoskeletal proteins. Cell 445:407-415

Maiuri L, Rossi M, Raia V, Garipoli V, Hughes LA, Swallow D, Norén O, Sjöström H, Auricchio S (1994) Mosaic regulation of lactase in human adult-type hypolactasia. Gastroenterology 107:54-60[Medline]

Mantei N, Villa M, Enzler T, Wacker H, Boll W, James P, Hunziker W, Semenza G (1988) Complete primary structure of human and rabbit lactase-phlorizin hydrolase: implications for biosynthesis, membrane anchoring and evolution of the enzyme. EMBO J 7:2705-2713[Abstract]

Murray MK, DeSouza MM (1995) Messenger RNA encoding an estrogen-dependent oviduct secretory protein in the sheep is localized in the apical tips and basal compartments of fimbria and ampulla epithelial cells implying translation at unique cytoplasmic foci. Mol Reprod Dev 42:268-283[Medline]

Naim HY, Lacey SW, Sambrook JF, Gething MJH (1991) Expression of a full-length cDNA coding for human intestinal lactase-phlorizin hydrolase reveals an uncleaved, enzymatically active, and transport-competent protein. J Biol Chem 266:12313-12320[Abstract/Free Full Text]

Ponte P, Gunning P, Blau H, Kedes L (1983) Human actin genes are single copy for {alpha}-skeletal and {alpha}-cardiac actin but multicopy for ß- and {gamma}-cytoskeletal genes: 3' untranslated regions are isotype specific but are conserved in evolution. Mol Cell Biol 3:1783-1791[Medline]

Ponte P, Ng S-Y, Engel J, Gunning P, Kedes L (1984) Evolutionary conservation in the untranslated regions of actin mRNAs: DNA sequence of a human beta-actin cDNA. Nucleic Acids Res 12:1687-1696[Abstract]

Rebagliati MR, Weeks DL, Harvey RP, Melton DA (1985) Identification and cloning of localized maternal RNAs from Xenopus eggs. Cell 42:769-777[Medline]

Rings EHHM, Büller HA, de Boer PAJ, Grand RJ, Montgomery RK, Lamers WH, Charles R, Moorman AFM (1992a) Messenger RNA sorting in enterocytes: co-localization with encoded proteins. FEBS Lett 300:183-187[Medline]

Rings EHHM, Büller HA, Neele AM, Dekker J (1994a) Protein sorting versus messenger RNA sorting? Eur J Cell Biol 63:161-171[Medline]

Rings EHHM, de Boer PAJ, Moorman AFM, van Beers EH, Dekker J, Montgomery RK, Grand RJ, Büller HA (1992b) Lactase gene expression during early development of rat small intestine. Gastroenterology 103:1154-1161[Medline]

Rings EHHM, Krasinski SD, van Beers EH, Moorman AFM, Dekker J, Montgomery RK, Grand RJ, Büller HA (1994b) Restriction of lactase gene expression along the proximal to distal axis of rat small intestine occurs around weaning. Gastroenterology 106:1223-1232[Medline]

Sakiyama S, Nakamura Y, Tokunaga K, Takazawa H, Ohwaki Y, Nagano T (1989) Stage-specific localization of cytoskeletal actin mRNA in murine seminiferous tubules and intestinal epithelia as demonstrated by in-situ hybridization. Cell Tissue Res 258:225-231[Medline]

Shields HM, Bair FA, Bates ML, Yedlin ST, Alpers DH (1982) Localization of immunoreactive alkaline phosphatase in the rat small intestine at the light microscopic level by immunocytochemistry. Gastroenterology 82:39-45[Medline]

Singer RH (1996) RNA: traffic report. Trends Cell Biol 6:486-489

St. Johnston D (1995) The intracellular localization of messenger RNAs. Cell 81:161-171[Medline]

Traber PG (1990) Regulation of sucrase-isomaltase gene expression along the crypt-villus axis of rat small-intestine. Biochem Biophys Res Commun 173:765-773[Medline]

Traber PG, Yu L, Wu GD, Judge TA (1992) Sucrase-isomaltase gene expression along the crypt-villus axis of human small intestine is regulated at the level of mRNA abundance. Am J Physiol 262:G123-130[Abstract/Free Full Text]

Weeks DL, Melton DA (1987) A maternal mRNA localized to the vegetal hemisphere in Xenopus eggs codes for a growth factor related to TGF-ß. Cell 51:861-867[Medline]

Wilhelm JE, Vale RD (1993) RNA on the move: the mRNA localization pathway. J Cell Biol 123:269-274[Medline]

Xie Q-M, Zhang Y, Mahmood S, Alpers DH (1997) Rat intestinal alkaline phosphatase II messenger RNA is present in duodenal crypt and villus cells. Gastroenterology 112:376-386[Medline]