mRNA stability, mRNA translation and spatial localization of mRNA species within a cell can be governed by signals in the 3′-UTR (3′-untranslated region). Local translation of proteins is essential for the development of many eukaryotic cell types, such as the Drosophila embryo, where the spatial and temporal localization of bicoid and gurken mRNAs, among others, is required to establish morphogen gradients. More recent studies have suggested that mRNA localization also occurs with transcripts coding for membrane-based or secreted proteins, and that localization at organelles such as the endoplasmic reticulum directs translation more efficiently to specific subdomains, so as to aid correct protein localization. In human epithelial cells, the mRNA coding for SGLT1 (sodium–glucose co-transporter 1), an apical membrane protein, has been shown to be localized apically in polarized cells. However, the nature of the signals and RNA-binding proteins involved are unknown. Ongoing work is aimed at identifying the localization signals in the SGLT1 3′-UTR and the corresponding binding proteins. Using a protein extract from polarized Caco-2 cells, both EMSAs (electrophoretic mobility-shift assays) and UV cross-linking assays have shown that a specific protein complex is formed with the first 300 bases of the 3′-UTR sequence. MFold predictions suggest that this region folds into a complex structure and ongoing studies using a series of strategic deletions are being carried out to identify the precise nature of the motif involved, particularly the role of the sequence or RNA secondary structure, as well as to identify the main proteins present within the complex. Such information will provide details of the post-transcriptional events that lead to apical localization of the SGLT1 transcript and may reveal mechanisms of more fundamental importance in the apical localization of proteins in polarized epithelia.

Introduction

In eukaryotic cells, many mRNAs are subject to post-transcriptional regulation. Different regulatory processes can affect an mRNA's stability, govern the transport of a specific transcript within the cell or regulate the translation of a transcript. Each of these processes is controlled by specific trans-acting proteins that recognize and bind to signal motifs. Many of these motifs are present in the 3′-UTR (3′-untranslated region), which can contain multiple motifs. These processes can control when and where an mRNA is localized within the cell and, therefore, along with translational regulation, dictate where a protein is translated. The process of mRNA localization can have numerous functions in the cell [1], but essentially aids the targeting of specific proteins to their sites of function. This targeting has been shown to be vital in the successful maturation of developing cells, where an asymmetrical distribution of key proteins is required at specific time points.

Cytoplasmic mRNA localization

Until recently, most of the mRNA localization studies have been concerned with cytoplasmic mRNAs [2]. β-Actin mRNA has been shown to be co-localized with the protein at the leading edge of endothelial cells due to conserved sequence motifs in the 3′-UTR and, furthermore, the mutation of these motifs resulted in a distinct effect on cell morphology [3]. Much work has been done with the Drosophila embryo where the spatial and temporal localizations of bicoid and oskar mRNAs, among others, have been shown to be required for establishing morphogen gradients [4].

Over time, a large library of varied mRNA localization signals has been complemented with a growing number of functioning trans-acting RNA-binding proteins. Both signals and binding proteins highlight the variation seen between localized mRNAs [5]. The number of proteins involved in an RNA–protein localization complex can be quite large. For example, the Staufen protein in Drosophila is part of a complex shown to be required for the targeting of mRNAs coding for oskar and bicoid [6], with at least three more proteins found to be present in the localization complex [7] and more proteins being implicated as studies continue [8].

mRNA localization within the ER (endoplasmic reticulum)

Recent studies have indicated that mRNA localization can also occur with transcripts coding for membrane-associated or secreted proteins and that, in some instances, this appears to be within organelles such as the ER [9]. mRNA localization to distinct subdomains of the ER has been observed previously in developing rice seeds in which the prolamine storage protein mRNA is localized at the prolamine protein bodies ER and the glutelin RNA on the cisternal ER, both requiring signals in the 3′-UTR [10]. In Xenopus oocytes, Vg1 mRNA, which codes for a secreted protein, is localized, and Vera protein, which co-fractionates with the ER, is one of the proteins required for Vg1 mRNA localization [11]. Thus, again, an interaction of the mRNA with the ER is implicated in localization. Vg1 mRNA localization is directed by a signal in the 3′-UTR and Vera has been shown to bind to this region [11]. It is not yet established fully whether mRNA localization to the ER requires the signal sequence in the nascent polypeptide chain to be synthesized first or whether mRNA is somehow localized before this event. mRNA localization to a subcompartment of the ER could aid the localization of the encoded protein (Figure 1).

Schematic diagram describing possible ER mRNA apical localization mechanisms

Figure 1
Schematic diagram describing possible ER mRNA apical localization mechanisms

Subsequent to mRNA–protein binding, the complex is targeted to a specific subdomain of the ER for localized synthesis that will facilitate protein targeting. By this mechanism, the signal peptide would be inserted into the correct ER subdomain more efficiently, thus aiding apical protein localization.

Figure 1
Schematic diagram describing possible ER mRNA apical localization mechanisms

Subsequent to mRNA–protein binding, the complex is targeted to a specific subdomain of the ER for localized synthesis that will facilitate protein targeting. By this mechanism, the signal peptide would be inserted into the correct ER subdomain more efficiently, thus aiding apical protein localization.

Such a mechanism may apply to apical or basal proteins in polarized cells. Crb (Crumbs) is a transmembrane protein localized on the apical domain in Drosophila follicular cells and is a regulator of epithelial polarity. The crb 3′-UTR is required for apical mRNA localization of the transcript, a process shown to be vital for Crb to function effectively [12].

SGLT1 (sodium–glucose co-transporter 1) mRNA localization in polarized epithelial cells

In situ hybridization studies in enterocytes have shown that the mRNAs for the apical membrane proteins LPH (lactase–phlorizin hydrolase) and SI (sucrase–isomaltase) are co-localized apically [13], as is the mRNA for SGLT1 [14]. SGLT1 is a membrane protein found in the apical membrane of polarized epithelial cells of the small intestine and kidney. Its apical localization is vital in its function of transporting dietary glucose across the brush border membrane during nutrient absorption. Although this apical localization of the SGLT1 protein in polarized enterocytes is well established, much less is known about the nature and function of the mRNA localization. Localization signals and associated proteins are yet to be identified in the human SGLT1 mRNA sequence; however, studies have revealed a stability element at nucleotides 2596–2642 within the 3′-UTR of the pig SGLT1 sequence, bound by HuR protein as part of a cAMP-dependent stabilization of the mRNA [15].

Protein binding to 3′-UTR of human SGLT1 mRNA

We have investigated protein binding to the 3′-UTR sequence of the human SGLT1 mRNA transcript. The main approach was to investigate protein binding to regions of the human SGLT1 3′-UTR, to both identify regions of binding within the sequence and to identify and isolate the proteins involved.

Sequence alignment between a number of mammalian SGLT1 3′-UTR sequences has highlighted a region of high homology within the first ∼300 bases (nucleotides 2003–2312) of the 800 base 3′-UTR sequence. This region is upstream in the 3′-UTR compared with the stability element identified in the pig SGLT1 3′-UTR, so it may be that this unusually conserved region could provide us with additional motifs and binding sites. On this basis, MAXIscript 32P-radiolabelled RNA transcripts were made of the 2003–2312 region and combined with a protein extract derived from Caco-2 cells in EMSAs (electrophoretic mobility-shift assays). Preliminary data have shown complex formation and the addition of unlabelled RNA competitors in these assays indicated a specific RNA–protein binding complex with the 2003–2312 SGLT1 3′-UTR sequence. Use of additional unlabelled RNA competitors, with deletions in the 3′-UTR, has shown that only part of this region is required for binding. UV cross-linking assays suggest that three major proteins are involved. Work using biotinylated probe sequences to capture binding proteins is continuing in order to identify these proteins and to observe in more detail the binding signals involved. From these data, we aim to gain an increased understanding of the mechanisms involved in the apical localization of the SGLT1 mRNA in epithelial cells. Such mechanisms might be of fundamental importance in apical membrane protein targeting in polarized epithelia.

RNA UK 2008: Independent Meeting held at The Burnside Hotel, Bowness on Windermere, Cumbria, U.K., 18–20 January 2008. Organized and Edited by David Elliot (Newcastle, U.K.), Sarah Newbury (Sussex, U.K.) and Alison Tyson-Capper (Newcastle, U.K.).

Abbreviations

     
  • Crb

    Crumbs

  •  
  • ER

    endoplasmic reticulum

  •  
  • SGLT1

    sodium–glucose co-transporter 1

  •  
  • UTR

    untranslated region

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