Browsing by Subject "Unfolded protein response"
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Item Open Access Macromolecular Organization of the Rough Endoplasmic Reticulum at Homeostasis and Stress(2019) Hoffman, Alyson MarieThe endoplasmic reticulum (ER) is an organelle that exists as a patchwork of
functional membrane domains with unique protein components. The rough ER is one
such domain characterized by associated ribosomal particles and is known as the site of
translation for secretory and membrane proteins (SMPs). This has since been extended
to include the translation of the entire transcriptome and other diverse functions
including autophagosome assembly and miRNA silencing, however, little is known
about the macromolecular organization of these complex biochemical processes. Using
a proximity labeling technique, BioID, four ribosome associated membrane proteins
(RAMPs) were selected to create a rudimentary map of translation on the rough ER by
combining the BioID nonspecific biotin‐labeling with streptavidin pulldowns and mass
spectrometry. This revealed distinct environments surrounding each RAMP in addition
to overlap that established spatial organization of these domains with each other. Of the
four selected, only two, LRRC59 and Sec61β of the Sec61 translocon, labeled ribosomes
in vivo. Identification of the mRNAs associated with these labeled ribosomes revealed
that translation was spatially distinct between the two sites. This supports the existence
of complex sorting mechanisms within this domain that extend to sites of optimized
protein synthesis for sub‐groups of mRNAs.
Since organisms exist within changing environments and stressors, the next step
was to determine if macromolecules associated with the ER during homeostasis change
their localization during stress, namely the unfolded protein response (UPR).
Combining DTT treatment for UPR stimulation with smFISH for ER chaperones
transcriptionally upregulated during this stress, it was determined that only specific
transcripts, e.g. GRP94, are localized to cytoplasmic granules during stress while others,
e.g. BiP, are not. Combining these observations with the existing literature, the working
hypothesis is that transcripts, such as BiP, able to recruit ribosomes during times of
inefficient cap‐dependent translation, can escape stress granules while the nascent
transcript RBP environment, such as that on GRP94, is responsible for its recruitment to
these granules. Whether this observation is generalizable to all transcripts being made
upon UPR stimulation is the subject of further study.
Item Open Access mRNA Partitioning to the Endoplasmic Reticulum(2021) Child, Jessica RaeThe signal recognition particle (SRP) pathway has long been regarded as the primary mechanism of transcriptome and translatome partitioning to the endoplasmic reticulum (ER). This co-translational targeting mechanism is conserved in all living organisms studied to date. By the SRP pathway, ribosomes translating secretory and membrane protein-encoding mRNAs in the cytosol are selected for recruitment to the ER following presentation of a peptide signal sequence early in translation. SRP recognizes and binds the peptide signal and targets the mRNA-ribosome-nascent chain complex to the ER membrane via interaction with the ER-resident SRP receptor (SR). The membrane-targeted signal peptide is then passed to the translocon and the secretory/membrane protein is co-translationally translocated into the ER lumen or inserted into the ER membrane. Via this positive selection strategy, mRNAs encoding secretory and membrane proteins are translated by ER-associated ribosomes, while non-signal encoding mRNAs are translated by free ribosomes in the cytosol. Yeast and bacterial species possess post-translational protein translocation mechanisms which allow SRP-independent translocation. Mammalian cells, by contrast, rely on the SRP pathway for protein targeting and translocation. The precise role of the SRP pathway in subcellular mRNA localization, however, remains elusive. In fact, studies which investigate RNA localization to the ER suggest many diverse strategies could anchor a broad representation of all cellular mRNAs to the ER membrane and regulate their translation. Here we evaluate the extent to which the SRP pathway contributes to transcriptome partitioning in mammalian cells via CRISPR/Cas9- and RNAi-mediated depletion of SR, followed by sequential detergent fractionation into cytosol and ER subcellular fractions and deep sequencing of the compartmentalized mRNAs. We found that disruption of the SRP pathway does not impact steady-state mRNA localization to the ER, though minor defects in protein expression were observed in a quantitative proteomic study, thereby decoupling SRP pathway function in protein biogenesis from general mRNA partitioning. Assessment of de novo subcellular localization patterns of newly synthesized mRNAs, via deep sequencing of 4-thiouridine metabolically labeled mRNAs in the cytosol and ER fractions of parental and SR-deficient cells, revealed that mRNAs are predominately localized to the ER membrane upon nuclear export, independently of a functional SRP pathway or encoded signal sequence. We further found that translation inhibition, through physiological stress or chemical inhibitors, enhanced the ER localization of mRNAs, especially non-signal encoding mRNAs. This suggests that translation-coupled events release this mRNA cohort into the cytosol to establish steady-state subcellular distributions. Additional investigation into RNA localization patterns by single molecule RNA imaging under conditions of stress-induced translation inhibition, which promotes the formation of ribonucleoprotein stress granules (SG), revealed that newly synthesized mRNAs serve as primary substrates for SG biogenesis, as transcription inhibition prevented mRNA recruitment into SGs. Furthermore, SG formation was found to occur in association with the ER membrane for both signal- and non-signal-encoding mRNAs. Collectively these data support a novel mRNA trafficking model by which newly synthesized mRNAs are exported from the nucleus and localized directly to the ER membrane independently of the SRP pathway, likely via interaction with ER-resident ribosome and/or RNA binding proteins, implicating the ER as a regulatory center for initiation of subcellar transcriptome partitioning.
Item Open Access Post-transcriptional Regulation of Membrane-associated RNAs(2013) Jagannathan, SujathaRNA localization provides the blueprint for compartmentalized protein synthesis in eukaryotic cells. Current paradigms indicate that RNAs encoding secretory and membrane proteins are recruited to the endoplasmic reticulum (ER), via positive selection of a `signal peptide' tag encoded in the protein. Thus RNA sorting to the ER follows protein sorting and the RNA is considered a passive player. However, RNAs have been shown to access the ER independent of the signal peptide and display a wide range of affinities to the ER that does not correlate with signal peptide strength. How and why mRNAs localize to the ER to varying extents and whether such localization serves a purpose besides protein sorting is poorly understood. To establish the cause and consequence of RNA binding to the ER membrane, I pose three primary questions: 1. How are mRNAs targeted to the ER? 2. Once targeted, how are mRNAs anchored to the ER membrane? 3. Are ER localized mRNAs subject to transcript-specific regulation?
I address cytosolic mRNA targeting to the ER by comparing the partitioning profiles of cytosolic/nuclear protein-encoding mRNA population (mRNACyto) to that of mRNAs encoding a signal peptide (mRNAER). I show that, at a population level, mRNACyto display a mean ER enrichment that is proportional to the amount of ER-bound ribosomes. Thus, I propose that targeting of mRNACyto to the ER is stochastic and over time, the specific interactions engaged by an individual mRNACyto with the ER determines its steady state partitioning profile between the cytoplasm and the ER.
To address the modes of direct binding of mRNA to the ER, I examined the association of various RNA populations with the ER after disrupting membrane-bound ribosome's interaction with its ER receptor. mRNACyto and most of mRNAs encoding secretory proteins (mRNACargo) are released upon disruption of ribosome-receptor interactions, indicating no direct mRNA-ER interactions. However, the population of mRNAs that encode resident proteins of the endomembrane organelles such as the ER, lysosome, endosome and the Golgi apparatus (mRNARes) maintain their association with the ER despite the disruption of ribosome-receptor interactions. These results indicate direct binding of mRNARes to the ER, further suggesting that the function of the encoded proteins dictates the mode of association of corresponding mRNA with the ER.
To uncover the mode of mRNARes binding directly to ER, I performed differential proteomic analysis of cytosolic and membrane bound RNA-protein complexes, which revealed a network of RNA binding proteins that interact uniquely with the ER-anchored mRNAs. The anchoring of endomembrane resident protein-encoding RNAs to the ER through these RNA binding proteins may reflect an imprinting of the ER with the information necessary for the continued biogenesis of the endomembrane organelle system even in situations where translation-dependent ER targeting of an mRNA is compromised.
Finally, I address whether ER-bound mRNAs can be regulated differentially by comparing the fates of two signal peptide-encoding RNAs, B2M and GRP94, during the unfolded protein response (UPR). I show that in response to ER stress, GRP94 mRNA, but not B2M, relocates to stress-induced RNA granules, thus escaping an RNA decay program that operates at the ER membrane during the UPR. Hence, I propose that the mode of RNA association to the ER is subject to regulation and influences the fate of RNAs during cellular stress. Thus, by demonstrating diverse modes of mRNA localization to the ER and differential regulation of ER bound mRNAs during cellular stress, my work has helped establish an emerging role for the ER as a post-transcriptional gene regulatory platform.