Single-Molecule Quantification of Translation-Dependent Association of mRNAs with the Endoplasmic Reticulum.

It is well established that mRNAs encoding secretory or membrane-bound proteins are translated on the surface of the endoplasmic reticulum (ER). The extent to which mRNAs that encode cytosolic proteins associate with the ER, however, remains controversial. To address this question, we quantified the number of cytosolic protein-encoding mRNAs that co-localize with the ER using single-molecule RNA imaging in fixed and living cells. We found that a small but significant number of mRNAs that encode cytosolic proteins associate with the ER and show that this interaction is translation dependent. Furthermore, we demonstrate that cytosolic protein-encoding transcripts can remain on the ER with dwell times consistent with multiple rounds of translation and have higher ribosome occupancies than transcripts translated in the cytosol. These results advance our understanding of the diversity and dynamics of localized translation on the ER.

mRNA encoding Sec61ß, a tail-anchored protein, is localized on the endoplasmic reticulum.

Although one pathway for the post-translational targeting of tail-anchored proteins to the endoplasmic reticulum (ER) has been well defined, it is unclear whether additional pathways exist. Here, we provide evidence that a subset of mRNAs encoding tail-anchored proteins, including Sec61ß and nesprin-2, is partially localized to the surface of the ER in mammalian cells. In particular, Sec61b mRNA can be targeted to, and later maintained on, the ER using both translation-dependent and -independent mechanisms. Our data suggests that this process is independent of p180 (also known as RRBP1), a known mRNA receptor on the ER, and the transmembrane domain recognition complex (TRC) pathway components, TRC40 (also known as ASNA1) and BAT3 (also known as BAG6). In addition, our data indicates that Sec61b mRNA might access translocon-bound ribosomes. Our results show that certain tail-anchored proteins are likely to be synthesized directly on the ER, and this facilitates their membrane insertion. Thus, it is clear that mammalian cells utilize multiple mechanisms to ensure efficient targeting of tail-anchored proteins to the surface of the ER.

Localization of mRNAs to the endoplasmic reticulum.

Almost all cells use mRNA localization to establish spatial control of protein synthesis. One of the best-studied examples is the targeting and anchoring of mRNAs encoding secreted, organellar, and membrane-bound proteins to the surface of the endoplasmic reticulum (ER). In this review, we provide a historical perspective on the research that elucidated the canonical protein-mediated targeting of nascent-chain ribosome mRNA complexes to the surface of the ER. We then discuss subsequent studies which provided concrete evidence that a subpopulation of mRNAs utilize a translation-independent mechanism to localize to the surface of this organelle. This alternative mechanism operates alongside the signal recognition particle (SRP) mediated co-translational targeting pathway to promote proper mRNA localization to the ER. Recent work has uncovered trans-acting factors, such as the mRNA receptor p180, and cis-acting elements, such as transmembrane domain coding regions, that are responsible for this alternative mRNA localization process. Furthermore, some unanticipated observations have raised the possibility that this alternative pathway may be conserved from bacteria to mammalian cells.

Identification of a region within the placental alkaline phosphatase mRNA that mediates p180-dependent targeting to the endoplasmic reticulum.

In both eukaryotic and prokaryotic cells, it has been recently established that mRNAs encoding secreted and membrane proteins can be localized to the surface of membranes via both translation-dependent and RNA element-mediated mechanisms. Previously, we showed that the placental alkaline phosphatase (ALPP) mRNA can be localized to the ER membrane independently of translation, and this localization is mediated by p180, an mRNA receptor present in the ER. In this article, we aimed to identify the cis-acting RNA element in ALPP. Using chimera constructs containing fragments of the ALPP mRNA, we demonstrate that the ER-localizing RNA element is present within the 3' end of the open reading frame and codes for a transmembrane domain. In addition, we show that this region requires p180 for efficient ER anchoring. Taken together, we provide the first insight into the nature of cis-acting ER-localizing RNA elements responsible for localizing mRNAs on the ER in mammalian cells.

RanBP2/Nup358 potentiates the translation of a subset of mRNAs encoding secretory proteins.

In higher eukaryotes, most mRNAs that encode secreted or membrane-bound proteins contain elements that promote an alternative mRNA nuclear export (ALREX) pathway. Here we report that ALREX-promoting elements also potentiate translation in the presence of upstream nuclear factors. These RNA elements interact directly with, and likely co-evolved with, the zinc finger repeats of RanBP2/Nup358, which is present on the cytoplasmic face of the nuclear pore. Finally we show that RanBP2/Nup358 is not only required for the stimulation of translation by ALREX-promoting elements, but is also required for the efficient global synthesis of proteins targeted to the endoplasmic reticulum (ER) and likely the mitochondria. Thus upon the completion of export, mRNAs containing ALREX-elements likely interact with RanBP2/Nup358, and this step is required for the efficient translation of these mRNAs in the cytoplasm. ALREX-elements thus act as nucleotide platforms to coordinate various steps of post-transcriptional regulation for the majority of mRNAs that encode secreted proteins.

Visualization of endoplasmic reticulum localized mRNAs in mammalian cells.

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

p180 promotes the ribosome-independent localization of a subset of mRNA to the endoplasmic reticulum.

In metazoans, the majority of mRNAs coding for secreted and membrane-bound proteins are translated on the surface of the endoplasmic reticulum (ER). Although the targeting of these transcripts to the surface of the ER can be mediated by the translation of a signal sequence and their maintenance is mediated by interactions between the ribosome and the translocon, it is becoming increasingly clear that additional ER-localization pathways exist. Here we demonstrate that many of these mRNAs can be targeted to, and remain associated with, the ER independently of ribosomes and translation. Using a mass spectrometry analysis of proteins that associate with ER-bound polysomes, we identified putative mRNA receptors that may mediate this alternative mechanism, including p180, an abundant, positively charged membrane-bound protein. We demonstrate that p180 over-expression can enhance the association of generic mRNAs with the ER. We then show that p180 contains a lysine-rich region that can directly interact with RNA in vitro. Finally, we demonstrate that p180 is required for the efficient ER-anchoring of bulk poly(A) and of certain transcripts, such as placental alkaline phosphatase and calreticulin, to the ER. In summary, we provide, to our knowledge, the first mechanistic details for an alternative pathway to target and maintain mRNA at the ER. It is likely that this alternative pathway not only enhances the fidelity of protein sorting, but also localizes mRNAs to various subdomains of the ER and thus contributes to cellular organization.

Molecular characterization of Chinese hamster cells mutants affected in adenosine kinase and showing novel genetic and biochemical characteristics.

BACKGROUND: Two isoforms of the enzyme adenosine kinase (AdK), which differ at their N-terminal ends, are found in mammalian cells. However, there is no information available regarding the unique functional aspects or regulation of these isoforms. RESULTS: We show that the two AdK isoforms differ only in their first exons and the promoter regions; hence they arise via differential splicing of their first exons with the other exons common to both isoforms. The expression of these isoforms also varied greatly in different rat tissues and cell lines with some tissues expressing both isoforms and others expressing only one of the isoforms. To gain insights into cellular functions of these isoforms, mutants resistant to toxic adenosine analogs formycin A and tubercidin were selected from Chinese hamster (CH) cell lines expressing either one or both isoforms. The AdK activity in most of these mutants was reduced to <5% of wild-type cells and they also showed large differences in the expression of the two isoforms. Thus, the genetic alterations in these mutants likely affected both regulatory and structural regions of AdK. We have characterized the molecular alterations in a number of these mutants. One of these mutants lacking AdK activity was affected in the conserved NxxE motif thereby providing evidence that this motif involved in the binding of Mg2+ and phosphate ions is essential for AdK function. Another mutant, FomR-4, exhibiting increased resistance to only C-adenosine analogs and whose resistance was expressed dominantly in cell-hybrids contained a single mutation leading to Ser191Phe alteration in AdK. We demonstrate that this mutation in AdK is sufficient to confer the novel genetic and biochemical characteristics of this mutant. The unusual genetic and biochemical characteristics of the FomR-4 mutant suggest that AdK in this mutant might be complexed with the enzyme AMP-kinase. Several other AdK mutants were altered in surface residues that likely affect its binding to the adenosine analogs and its interaction with other cellular proteins. CONCLUSIONS: These AdK mutants provide important insights as well as novel tools for understanding the cellular functions of the two isoforms and their regulation in mammalian cells.

Analysis of mRNA nuclear export kinetics in mammalian cells by microinjection.

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes and genetically tractable organisms such as yeast and the Drosophila derived S2 cell line, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR). In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.