Translation- and SRP-independent mRNA targeting to the endoplasmic reticulum in the yeast Saccharomyces cerevisiae

Abstract

mRNAs encoding secreted/membrane proteins (mSMPs) are believed to reach the endoplasmic reticulum (ER) in a translation-dependent manner to confer protein translocation. Evidence exists, however, for translation-and signal recognition particle (SRP)-independent mRNA localization to the ER, suggesting that there are alternate paths for RNA delivery. We localized endogenously expressed mSMPs in yeast using an aptamer-based RNA-tagging procedure and fluorescence microscopy. Unlike mRNAs encoding polarity and secretion factors that colocalize with cortical ER at the bud tip, mSMPs and mRNAs encoding soluble, nonsecreted, nonpolarized proteins localized mainly to ER peripheral to the nucleus (nER). Synthetic nontranslatable uracil-rich mRNAs were also demonstrated to colocalize with nER in yeast. This mRNA-ER association was verified by subcellular fractionation and reverse transcription-PCR, single-molecule fluorescence in situ hybridization, and was not inhibited upon SRP inactivation. To better understand mSMP targeting, we examined aptamer-tagged USE1, which encodes a tail-anchored membrane protein, and SUC2, which encodes a soluble secreted enzyme. USE1 and SUC2 mRNA targeting was not abolished by the inhibition of translation or removal of elements involved in translational control. Overall we show that mSMP targeting to the ER is both translation-and SRP-independent, and regulated by cis elements contained within the message and trans-acting RNA-binding proteins (e.g., She2, Puf2).

Diverse and pervasive subcellular distributions for both coding and long noncoding RNAs

“…Third, when examined in other highly polarized cell types, such as neurons, other studies have also shown extremely high rates of RNA subcellular localization (Bruckenstein et al 1990;Olink-Coux and Hollenbeck 1996;Mercer et al 2008). Finally, recent studies have shown that mRNAs encoding common organelle-specific gene products, such as those that function in the ER, mitochondria, and P bodies, can also be copurified bound to these organelles (Decker and Parker 2012;Reid and Nicchitta 2012;Kraut-Cohen et al 2013;Lesnik et al 2015). Indeed, these biochemical approaches have suggested that as much as 50% of cytosolic protein-encoding transcripts are translated on ER (Reid and Nicchitta 2012;Jagannathan et al 2014).…”

Use of the MS2 aptamer and coat protein for RNA localization in yeast: A response to “MS2 coat proteins bound to yeast mRNAs block 5′ to 3′ degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system”

“…In addition, it should be combined with quantitative analyses of mRNA granule size and/or colocalization with P-bodies to exclude suspicious subpopulations of granules from contributing to the interpretation of mRNA imaging results. Moreover, the use of colocalization procedures (e.g., co-FISH, as suggested by Garcia and Parker [2016]) or FISH-MS2 labeling (Kraut-Cohen et al 2013) would go far to rule out the introduction of RNA localization artifacts.…”

Multifunctional Roles for the Protein Translocation Machinery in RNA Anchoring to the Endoplasmic Reticulum

“…Identification of RNA-binding ER Membrane Proteins-Past and recent studies have provided evidence for the direct (ribosome-independent) association of endomembrane mRNAs with the ER (10,(12)(13)(14)(15)(16). Given the detergent solubilization properties of the endomembrane mRNAs reported above, we considered that this mRNA cohort might directly bind to resident integral membrane RBPs, although it is equally plausible that mRNA anchoring could occur via binding interactions between soluble RBPs and cognate ER RBP binding proteins.…”