ARTICLE |
Correspondence to: Richard J. Grand, Dept. of Pediatrics, Box 213, New England Medical Center, 750 Washington Street, Boston, MA 02111.
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Summary |
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To extend our recent observation that villin mRNA, encoding an apical microvillous protein, is dichotomously localized in the basal region of human enterocytes, we examined the localization of mRNAs for brush border myosin I (BBMI) and intestinal fimbrin (I-fim). In situ hybridization indicated that BBMI mRNA localized to the basal region of human enterocytes, whereas the mRNA for I-fim distributed diffusely. To facilitate study of potential mechanisms of mRNA targeting, we cloned a full-length cDNA for BBMI including its 5'- and 3'-untranslated regions (UTRs). This cDNA shares 86% sequence identity with bovine BBMI and 85% with rat BBMI. Sequence analysis revealed no obvious similarity between the 3'-UTRs of BBMI and villin. This study provides evidence of novel sorting pathways for intestinal microvillous cytoskeletal proteins. (J Histochem Cytochem 48:8994, 2000)
Key Words: brush border myosin I, BBMI, intestinal fimbrin, I-fimbrin (I-plastin), mRNA sorting, enterocyte
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Introduction |
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mRNA SORTING plays an important role in the establishment of protein gradients or cell polarity in Drosophila embryos and Xenopus oocytes (
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Materials and Methods |
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Materials
Digoxigenin-11-UTP (DIG-11-UTP) was purchased from Boehringer Mannheim (Indianapolis, IN). [32P]--UTP and [32P]-
-dCTP were from DuPontNew England Nuclear (Boston, MA). All restriction enzymes and DNA-dependent RNA polymerase were from Gibco BRL (Grand Island, NY). All other reagents were from Sigma (St Louis, MO). Human jejunal tissue was obtained from adults undergoing elective gastric bypass surgery with a protocol approved by the Human Investigation Review Committee of the New England Medical Center Hospitals.
In Situ Hybridization
A human BBMI (hBBMI) cDNA fragment (6031057 bp) was subcloned into BlueScript II KS (+) vector at Xho I/Hind III sites. A 510-bp fragment of human I-fim (7451254 bp) was subcloned into BlueScript II KS (+) vector at BamH I/Cla I sites. DIG-labeled RNA probes were prepared using DIG-11-UTP according to the manufacturer's instructions. Quantification and dot-blot assay of the RNA probes were performed as previously described (
Cloning of hBBMI
Human jejunal total RNA was isolated by homogenization of mucosal scrapings in guanidine isothiocyanate, purified through cesium chloride centrifugation as previously described (
The 5'-end of the mRNA was cloned by the 5'-Rapid Amplification of cDNA Ends Strategy (5' RACE) (
The 3'-end of the mRNA was cloned by 3' RACE. Briefly, PCR amplification was accomplished using an anchored primer (5'-TTCTAGAATTCAGCGGCCGCTTT-3'), and a gene-specific primer (GSP2: 5'-GCTCATTGAGCATAATCAGCGAGG-3'), which is located at 1529 bp of cDNA (position according to AF105424). The approximately 2.0-bp 3' RACE product was cloned into the pGEM-4 T Easy vector and sequenced. DNA sequencing was performed by the Protein Core Facility, Department of Physiology, Tufts University, Boston, MA. Sequence was determined on both strands and analyzed using Genetics Computer Group's computer software.
Northern Blotting
Northern blotting was performed as described previously (
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Results |
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mRNA Localization
All jejunal tissue samples were examined and demonstrated normal morphology and cellular architecture as shown previously (
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In control samples, human SI mRNA localized only in the apical domain of the enterocytes, with no mRNA detected in any other region of the cells, similar to our previously reported results (
In all of our in situ hybridization studies, we failed to see any signal if the sample was pretreated with ribonuclease A, indicating that the signals observed were due to hybridization to RNA targets (not shown). In addition, the specificity of the mRNA signals was also suggested by the absence of reaction product in the lamina propria.
Human BBMI cDNA
To date, most of the cis-acting elements responsible for mRNA sorting have been found in the 3'-UTR (
Sequencing of hBBMI revealed a single open reading frame of 3132 bp. The ATG start codon was found 210 bp downstream from the 5'-end of the cDNA. An in-frame termination codon was present 48 bp upstream of the translation start codon. The 3'-UTR was 237 bp before the poly(A) tail. A polyadenylation signal was also present in the 3'-UTR. A search of the GenBank revealed that our cDNA is highly homologous to another recently cloned hBBMI cDNA containing only the full-length coding region (C at 174 in AF009961, A1546
C1337, G1547
C1338, A1616
G1407, C2343
G2134, and G2726
A2517. The coding sequence showed 86%, 85%, 86%, and 65% homology with bovine (
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The deduced hBBMI amino acid sequence encodes a 1043-residue polypeptide with a deduced molecular weight of 118,387 D. It differs only in two amino acid residues from AF009961: Q446P446 and R712
G712. It shares 87% identity and 90% similarity with bovine BBMI, 88% identity and 91% similarity with rat BBMI, and 64% identity and 70% similarity with chicken BBMI. hBBMI has a putative ATP binding sequence at its N-terminus and an actin binding domain in the middle region (
Northern Blot Analysis
Using the full-length hBBMI cDNA as a probe in Northern blot analyses, we demonstrated that hBBMI was detected as a ~3.6-kb single band species in total RNA extracts from human jejunum and Caco-2 cells (Figure 4). This size is consistent with our cloned cDNA and also suggests that no alternative splicing occurs in the tissue and cells examined, as suggested for rat BBMI (
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Discussion |
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In the past decade, intensive investigation of protein transport has yielded important insights into the mechanisms involved in protein localization in epithelial cells (
The mRNA distribution of I-fim, another microvillous core cytoskeletal protein, was also examined by nonradioactive in situ hybridization. In contrast to the basal localization of villin and BBMI mRNAs, I-fim mRNA distributes diffusely throughout the cytoplasm in enterocytes. This distinction also indicates that the mRNAs for microvillous core cytoskeletal proteins distribute in enterocytes in different patterns. Because of the previous demonstration of apical localization of I-fim protein in the microvillous core region (
The mechanism of mRNA sorting remains largely unknown, although some studies have linked sequence repeats in the 3'-UTR to the anchoring site for mRNA binding to cytosolic proteins. For example,
Although sequence analysis of the hBBMI cDNA revealed several repeats in its 3'-UTR (ACCCTT, TCCTCCAA and CCCCTCTG) (Figure 3), the significance of these repeats for mRNA asymmetrical distribution is unclear. These repeats are not found in the 3'-UTR of ß-actin mRNA nor in the 3'-UTR of human villin mRNA although, as discussed above, different repeated sequences are found in the ß-actin 3'-UTR (
The cloning of the hBBMI cDNA revealed some unique features, as well as conserved sequences shared with bovine, rat, and chicken BBMIs. The hBBMI cDNA has the highest homology to bovine BBMI, followed by rat and then chicken BBMI; a similar pattern is found in analysis of the deduced amino acid sequence. Similar to bovine, rat, and chicken BBMI, hBBMI has a putative ATP binding domain at its N-terminus followed by an actin binding site and an IQ domain containing three putative calmodulin binding motifs. However, hBBMI possesses an additional putative ATP/GTP binding domain between the actin binding site and calmodulin binding sites. The functional significance of this site is unknown. It will be interesting to examine the functional difference of mutated hBBMI lacking the second ATP/GTP binding site.
The mechanisms controlling mRNA sorting in intestinal epithelial cells and their role in the establishment and maintenance of epithelial polarity remain to be elucidated. The distinct localizations of hBBMI and I-fim mRNAs, as well as the basal localization of villin mRNA in enterocytes and the cloning of the hBBMI cDNA, including its 3'-UTR, offer the opportunity to expand our understanding of the functional significance of mRNA sorting in enterocytes.
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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, and Pediatric Gastroenterology Research Training Grant T32-DK-07471.
We wish to thank Drs Robert Montgomery, Stephen Krasinski, Menno Verhave, and Diana Bianchi for scientific discussion, Dr Scott Shikora for human specimens, and Dr Anne Kane (Director of the Microbiology Core, GRASP Center) for plasmid preparations.
Received for publication May 11, 1999; accepted August 16, 1999.
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