ARTICLE |
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.
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Summary |
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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 lactasephlorizin hydrolase (LPH), sucraseisomaltase (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 46:335343, 1998)
Key Words: mRNA localization, enterocytes, lactasephlorizin hydrolase, sucraseisomaltase, intestinal alkaline phosphatase, ß-actin
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Introduction |
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The localization of certain mRNAs to specific regions in the cytoplasm has been demonstrated in a variety of cell types (
The 3'-untranslated region of all sorted mRNAs studied thus far contain cis-acting sequences that are responsible for localization (
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 (
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 sucraseisomaltase (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 phosphatidylinositolglycan linkage (
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Materials and Methods |
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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 DuPontNew 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 20120 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;
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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
RNase protection assays were carried out as described (
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
In Situ Hybridization
In situ hybridization was carried out as described by
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 (
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Results |
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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 (
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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).
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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 2CF). 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.
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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 (
Differential Expression of LPH and SI mRNA Along the CryptVillous Axis
The localization along the cryptvillous 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 cryptvillous 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.
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Discussion |
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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 (
The sorting of specific mRNAs to distinct cytoplasmic regions has been suggested as a mechanism of protein localization (
ß-Actin, a component of the cytoskeleton in nonmuscle cells (
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 (
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 (
In a previous study of human jejunum using a digoxigenin-labeled RNA probe,
Human LPH mRNA was expressed evenly along the length of the villus, a pattern similar to that found in fetal rats (
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 (
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 (
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Acknowledgments |
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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 ShephardBerry, Department of Pathology.
Received for publication July 17, 1997; accepted September 30, 1997.
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