Browsing by Author "Nicchitta, Christopher V"
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Item Open Access Complementary Roles of GADD34- and CReP-Containing Eukaryotic Initiation Factor 2α Phosphatases during the Unfolded Protein Response.(Molecular and cellular biology, 2016-07) Reid, David W; Tay, Angeline SL; Sundaram, Jeyapriya R; Lee, Irene CJ; Chen, Qiang; George, Simi E; Nicchitta, Christopher V; Shenolikar, ShirishPhosphorylation of eukaryotic initiation factor 2α (eIF2α) controls transcriptome-wide changes in mRNA translation in stressed cells. While phosphorylated eIF2α (P-eIF2α) attenuates global protein synthesis, mRNAs encoding stress proteins are more efficiently translated. Two eIF2α phosphatases, containing GADD34 and CReP, catalyze P-eIF2α dephosphorylation. The current view of GADD34, whose transcription is stress induced, is that it functions in a feedback loop to resolve cell stress. In contrast, CReP, which is constitutively expressed, controls basal P-eIF2α levels in unstressed cells. Our studies show that GADD34 drives substantial changes in mRNA translation in unstressed cells, particularly targeting the secretome. Following activation of the unfolded protein response (UPR), rapid translation of GADD34 mRNA occurs and GADD34 is essential for UPR progression. In the absence of GADD34, eIF2α phosphorylation is persistently enhanced and the UPR translational program is significantly attenuated. This "stalled" UPR is relieved by the subsequent activation of compensatory mechanisms that include AKT-mediated suppression of PKR-like kinase (PERK) and increased expression of CReP mRNA, partially restoring protein synthesis. Our studies highlight the coordinate regulation of UPR by the GADD34- and CReP-containing eIF2α phosphatases to control cell viability.Item Open Access Extracellular Hsp90 is Actively Trafficked and Internalized in Breast Cancer Cells(2016) Crowe, Lauren BurianekDespite its ubiquitous abundance, Hsp90 inhibitors have shown promise in anti-cancer clinical trials, suggesting that Hsp90 inhibitors selectively target tumor cells while exhibiting minimal effects in normal cells. Extracellular expression of heat shock protein 90 (eHsp90) by tumor cells is strongly correlated with malignancy. Development of small molecule probes that can specifically detect eHsp90 in vivo may therefore have utility in the early detection of malignancy. We synthesized a fluorescent cell impermeable Hsp90 inhibitor, HS-131, to target eHsp90 in vivo. HS-131 was characterized biochemically to ensure specificity for eHsp90, and an inactive analog was also synthesized to be used as an in vivo control.
Through confocal microscopy, eHsp90 can be visualized with cell impermeable, fluorophore-tagged Hsp90 inhibitors. High resolution confocal and real time lattice light sheet microscopy showed that probe-bound eHsp90 accumulates in punctate structures on the plasma membrane of breast tumor cells and is subsequently actively internalized. This internalization occurs in the presence and absence of inhibitors. The extent of internalization correlates with tumor cell aggressiveness, and this process can be induced in benign cells by over-expressing p110HER2, leading to malignant transformation of these cells. Internalization of eHsp90 is also increased after inhibition of Hsp70, suggesting that overcompensation of the heat shock response can also upregulate the eHsp90 trafficking mechanism. Whole body 3D cryo fluorescence imaging and histology of flank and spontaneous tumor-bearing mice strongly suggests that eHsp90 expression is a unique phenomenon in vivo.
Taken together, these results suggest that active and differential internalization of eHsp90 in aggressive cancer cells contributes to the selectivity observed upon Hsp90 inhibitor treatment and may provide a novel metastatic biomarker for solid tumors and may lead to the development of a tumor-specific drug delivery system.
Item Open Access Genetic and Functional Dissection of Age-Related Macular Degeneration(2016) Ahern, Perciliz Lumaban TanAge-related macular degeneration is one of the leading causes of vision loss in the world. While identification of various environmental risk factors including but not limited to smoking, ethnicity, and diet have been reported to contribute to the complex etiology of AMD, age and genetics remain the largest susceptibility factors in its pathogenesis. Initially, with the identification of the common Y402H variant in CFH, approximately 35% of the genetic determinants of AMD had been identified with the majority remaining unknown. Therefore, we set forth to A) identify additional AMD susceptibility genes that contribute to AMD through the use to next generation sequencing technologies and B) to assess associated alleles for pathogenicity in the attempt to interpret their functional contributions to AMD outcome as observed via patient serum and zebrafish analysis. In doing such, we have identified both common and rare variants that contribute to the heritability of AMD. Additionally, we report one of the first instances of a rare variant significantly increasing disease onset and a gene with increased rare mutational burden in AMD patients. All together adding to our understanding of the genetics of AMD and potentially leading to putative therapeutic targets.
Item Open Access Identification of Essential Functions of GRP94 in Metazoan Growth Control and Epithelial Homeostasis(2009) Maynard, Jason ChristopherGRP94, the endoplasmic reticulum Hsp90, is a metazoan-restricted chaperone essential for early development in mammals, yet dispensable for mammalian cell viability. These data suggest that GRP94 is required for important developmental processes relying on cell-cell communication and cell-cell interaction. Consistent with this hypothesis, loss of GRP94 expression in mouse is embryonic lethal yet tissue culture cells expressing no GRP94 are viable. To date, functional studies of GRP94 have relied on cell-autonomous model systems, the use of which has lead to discoveries of proteins that GRP94 chaperones also called client proteins. These systems give limited insight into the essential role(s) played by GRP94 in metazoan biology. The dichotomy that GRP94 is necessary for metazoan life, but dispensable for cellular viability suggests that the chaperone is required for the functional expression of secretory and/or membrane proteins that enable cells to function in the context of tissues.
To explore this hypothesis, the Drosophila ortholog of GRP94, Gp93, was identified and Gp93 mutants were created using imprecise P-element excision. Gp93 was found to be an essential gene in Drosophila. Loss of zygotic Gp93 expression is late larval lethal and causes prominent defects in the larval midgut, the sole endoderm-derived larval tissue. Gp93 mutant larvae display pronounced defects in the midgut epithelium, with aberrant copper cell structure, markedly reduced gut acidification, atypical septate junction structure, depressed gut motility, and deficits in intestinal nutrient uptake. The metabolic consequences of the loss of Gp93-expression are profound; Gp93 mutant larvae exhibit a starvation-like metabolic phenotype, including suppression of insulin signaling and extensive mobilization of amino acids and triglycerides. The defects in copper cell structure/function accompanying loss of Gp93 expression resemble those reported for mutations in labial, an endodermal homeotic gene required for copper cell specification, and α-spectrin, thus suggesting an essential role for Gp93 in the functional expression of secretory/integral membrane protein-encoding labial protein target genes and/or integral membrane protein(s) that interact with the spectrin cytoskeleton.
The creation of Gp93 mutant Drosophila has allowed for the study of GRP94 function in vivo and will be of upmost importance to future studies examining the function of this chaperone in all aspects of metazoan biology. This dissertation focuses on the morphological and physiological defects that accompany loss of Gp93 expression in Drosophila larvae. It will also outline future studies utilizing this model.
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 Mechanisms of Messenger RNA Localization to the Endoplasmic Reticulum: Purification of Native Polyribosomes for Proteomic Analysis of RNA-Binding Protein Composition(2010) Cooper, MelissaThe accepted mechanism for mRNA partitioning to the endoplasmic reticulum (ER) is the signal recognition particle (SRP) pathway. Although this model is well established, substantial experimental evidence exists to suggest additional mechanisms of localizing mRNAs to the ER. For example, evidence for ribosome independent mRNA association with the ER, as well as signal sequence and translation independent mRNA localization to the ER, has been reported. It has been hypothesized that there exist mRNA-binding proteins that, through interactions with mRNA-intrinsic localization elements, serve essential roles in the partitioning of mRNAs between cytosolic and ERbound ribosomes.
A method was devised for comparing the RNA-binding proteins associated withentire populations of cytosolic and ER-bound polysomes. In this method, cytosolic and ER-bound polysomes are isolated from tissue culture cells via a sequential detergent extraction method: digitonin permeabilization of the plasma membrane to release cytosolic polysomes followed by lysis of the ER membrane to obtain ER-bound polysomes. Purified polysomes are then subjected to mild digestion with micrococcal nuclease, the ribosomes pelleted via ultra-centrifugation, and the supernatants containing RNA-binding proteins submitted for proteomic analysis via mass spectrometry. Candidate proteins for a role in mRNA localization to the ER will be those enriched in ER-bound polysomes. However, while characterizing this method for generating RNA-binding protein enriched and ribosomal protein depleted samples for proteomic analysis, many concerns have been identified that need further optimization and careful consideration prior to investing the time and resources into a mass spectrometry-based analysis of such samples.
An immunoaffinity isolation approach was taken to interrogate mRNA-specific RNA-binding protein composition. In this approach, an anti-lambda light chain antibody was used for the isolation of lambda light chain polysomes from J558 plasmacytoma cells via immunoprecipitation of the nascent polypeptide chains.
Item Open Access Mechanisms of Molecular Chaperone Surface Binding and Endocytosis: Insights into the Molecular Basis for GRP94 Immune Function(2010) Jockheck-Clark, Angela RobertaExtracellular GRP94 can elicit both innate and adaptive immune responses by interacting with endocytic and signaling receptors on professional antigen presenting cells (pAPCs). CD91 was the first receptor proposed to facilitate GRP94-mediated immune responses. Using a GRP94 affinity matrix, a CD91 fragment was isolated from the detergent-solubilized membranes of a pAPC cell line. It was then demonstrated that CD91 ligands could inhibit GRP94-mediated peptide cross-presentation, suggesting that CD91 played a critical role in this process. While these studies implied that CD91 could function as a GRP94 endocytic receptor, later works suggested that CD91 may not recognize GRP94 at the cell surface. These opposing observations have lead to a significant controversy surrounding the identity of CD91 as an endocytic receptor for GRP94. Because the ability of CD91 to directly mediate GRP94 surface binding and uptake has not been established, the studies included in this dissertation have focused on evaluating the ability of CD91 to facilitate three processes that are necessary for GRP94-mediated peptide cross-presentation: surface binding, internalization, and processing.
These studies utilized a recombinantly-expressed N-terminal domain of GRP94 (GRP94.NTD), which was previously shown to have nearly identical biological activity to full length GRP94. The ability of CD91 to directly bind and internalize GRP94.NTD was examined using murine embryonic fibroblast (MEF) cell lines whose expression of CD91 was either reduced via siRNA, or eliminated by genetic disruption of the CD91 locus. Binding competition experiments were also conducted. Together, these studies reveal that CD91 does not directly interact with GRP94 at the cell surface. The ability of CD91 to directly facilitate GRP94 internalization was examined using various internalization and internalization competition assays. These studies demonstrated that GRP94.NTD and the CD91 ligand RAP were internalized through spatially and kinetically distinct pathways, that CD91 was not necessary for GRP94.NTD internalization, and that RAP did not inhibit GRP94 endocytosis. Together, these studies strongly suggest that CD91 does not directly facilitate GRP94 internalization. When these studies were extended to DC2.4 mouse dendritic cells, the CD91 ligand RAP reduced GRP94.NTD internalization/process by ~15%. This suggests that CD91 may indirectly facilitate GRP94 internalization in pAPC cell lines. Lastly, cross-presentation studies were utilized to examine the ability of various CD91 ligands to influence GRP94.NTD-mediated peptide cross-presentation through a post-uptake mechanism using the DC2.4/OT-1 system. Although it was discovered that DC2.4 cells can internalize and process GRP94.NTD/peptide complexes through fluid-phase endocytosis, CD91 ligands did not significantly inhibit GRP94-mediated peptide cross-presentation by DC2.4 cells. These studies demonstrate that CD91 does not play a primary role in GRP94-mediated peptide cross-presentation.
In the course of these studies, cell surface heparan sulfate proteoglycans (HSPGs) were identified as novel cell surface binding sites for GRP94.NTD on MEF cells. This conclusion was established using three distinct experimental approaches. GRP94.NTD surface binding was significantly decreased following heparin pre-treatment, following incubation with the sulfation inhibitor sodium chlorate, and following digestion with extracellular heparinase II. Conversely, these treatments did not significantly influence GRP94.NTD binding to RAW264.7 mouse macrophage-like cells. This suggested that GRP94.NTD-HSPG cell surface interactions may require the expression of a specific type of cell surface HSPG that is not expressed by RAW264.7 cells. However, additional studies strongly suggested that GRP94.NTD-HSPG cell surface interactions were mediated by the heparan sulfate-containing side chains rather than the presence of a specific cell surface HSPG core protein.
This dissertation focuses on the critical re-examination of CD91 functions in GRP94 surface binding, uptake, and cross-presentation. Together, these results clarify conflicting data on CD91 function in GRP94 surface binding and endocytosis. This dissertation also describes the identification of cell surface HSPGs as GRP94 binding sites on MEF cells. These studies extend the diversity of surface receptors that recognize of GRP94, and suggest that cell surface HSPG-dependent interactions may contribute to the biology of GRP94-elicited immune responses.
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.
Item Open Access Premature Translational Termination and the Rapidly Degraded Polypeptide Pathway(2012) Lacsina, Joshua ReneNearly thirty percent of all newly synthesized polypeptides are targeted for rapid proteasome-mediated degradation. These rapidly degraded polypeptides (RDPs) are the primary source of antigenic substrates for the major histocompatibility complex (MHC) class I presentation pathway, allowing for the immunosurveillance of newly synthesized proteins by cytotoxic T lymphocytes. Despite the recognized role of RDPs in MHC class I presentation, it remains unclear what molecular characteristics distinguish RDPs from their more stable counterparts. It has been proposed that premature translational termination products may constitute a form of RDP; indeed, in prokaryotes translational drop-off products are normal by-products of protein synthesis and are subsequently rapidly degraded.
To study the cellular fate of premature termination products, the antibiotic puromycin was used to modulate prematurely terminated polypeptide production in human cells. At low concentrations, puromycin doubled the fraction of rapidly degraded polypeptides, with enhanced degradation predominantly affecting small polypeptides, consistent with rapid degradation of truncated translation products. Immunoprecipitation experiments using anti-puromycin antisera demonstrated that the majority of peptidyl-puromycins are rapidly degraded in a proteasome-dependent manner. Low concentrations of puromycin increased the recovery of cell surface MHC class I-peptide complexes, indicating that prematurely terminated polypeptides can be processed for presentation via the MHC I pathway. In the continued presence of puromycin, MHC I export to the cell surface was inhibited, coincident with the accumulation of polyubiquitinated proteins. The time- and dose-dependent effects of puromycin suggest that the pool of peptidyl-puromycin adducts differ in their targeting to various proteolytic pathways which, in turn, differ in the efficiency with which they access the MHC class I presentation machinery. These studies highlight the diversity of cellular proteolytic pathways necessary for the metabolism and immunosurveillance of prematurely terminated polypeptides which are, by their nature, highly heterogeneous.
Item Embargo Redefining Criteria for RNA-Binding Activity Through Signal-to-Noise (S:N)-Based Analysis of RNA-Bound Proteomes(2023) Kristofich, JohnCarlo LouisRNA-binding proteins (RBPs) play an important role in post-transcriptional gene regulation by binding mRNAs to regulate splicing, stability, nuclear export, and target them for translation at specific subcellular compartments. While the role of RBPs in localization of mRNA to different subcellular compartments has been expanding in recent years, little has been done to understand their role in targeting mRNA for translation at the ER. The recent introduction of UV-based RNA-centric methods for proteome-wide identification of RNA-interactors has revealed numerous candidate RBPs on the ER. However, validation for most of these candidate integral ER RBPs is lacking, and the biological function of their RNA-binding activity is unknown. To address this, we introduce signal-to-noise (S:N)-based analytical approaches for direct validation of RNA-binding and reveal previously unrecognized sources of experimental noise inherit to UV-based RNA-centric methods which confound meaningful interpretation and introduce false positives. To overcomes these issues, we devise a novel biochemical method termed LEAP-RBP (Liquid-Emulsion-Assisted-Purification of RNA-Bound Protein) for the selective isolation of UV-crosslinked RNA-protein adducts and introduce new signal-to-noise (S:N)-based metrics for distinguishing relevant vs non-specific RNA-interactors. Using this approach, we identify integral ER RBPs with potential RNA-regulatory roles and provide a solid foundation for the proper identification and characterization of RNA-protein interactomes.
Item Open Access RNA Localization and Translational Regulation on the Endoplasmic Reticulum(2016) Hsu, Chun-ChiehmRNA localization is emerging as a critical cellular mechanism for the spatiotemporal regulation of protein expression and serves important roles in oogenesis, embryogenesis, cell fate specification, and synapse formation. Signal sequence-encoding mRNAs are localized to the endoplasmic reticulum (ER) membrane by either of two mechanisms, a canonical mechanism of translation on ER-bound ribosomes (signal recognition particle pathway), or a poorly understood direct ER anchoring mechanism. In this study, we identify that the ER integral membrane proteins function as RNA-binding proteins and play important roles in the direct mRNA anchoring to the ER. We report that one of the ER integral membrane RNA-binding protein, AEG-1 (astrocyte elevated gene-1), functions in the direct ER anchoring and translational regulation of mRNAs encoding endomembrane transmembrane proteins. HITS-CLIP and PAR-CLIP analyses of the AEG-1 mRNA interactome of human hepatocellular carcinoma cells revealed a high enrichment for mRNAs encoding endomembrane organelle proteins, most notably encoding transmembrane proteins. AEG-1 binding sites were highly enriched in the coding sequence and displayed a signature cluster enrichment downstream of encoded transmembrane domains. In overexpression and knockdown models, AEG-1 expression markedly regulates translational efficiency and protein functions of two of its bound transcripts, MDR1 and NPC1. This study reveals a molecular mechanism for the selective localization of mRNAs to the ER and identifies a novel post-transcriptional gene regulation function for AEG-1 in membrane protein expression.
Item Open Access Segregation of Protein Synthesis Between the Cytoplasm and Endoplasmic Reticulum of Eukaryotic Cells(2014) Reid, David WilliamThe partitioning of translation to the outer membrane of the endoplasmic reticulum is a problem that has been the subject of inquiry since the discovery of the ribosome. The large degree to which ribosomes were found to be tethered to the membrane led to intense investigation of a series of related questions regarding the identity of those mRNAs that are translated on the endoplasmic reticulum, and the functions of that localization in cell stress. In this dissertation, I approach each of these questions in turn and work to reconcile my observations with those models that have been previously proposed. A theme of this work is the application of modern methods, particularly deep sequencing technology, to address problems that had largely been considered solved. The most prominently featured method is ribosome profiling, which is paired with classical biochemical and cell biological techniques. I arrive at several conclusions: 1) a significant fraction of all mRNAs is well represented on the endoplasmic reticulum membrane, 2) the properties of translation diverge substantially between membrane-associated and free ribosomes, and 3) the compartmentalization of translation can serve as an important variable in cell stress.
Item Open Access Simple and inexpensive ribosome profiling analysis of mRNA translation.(Methods (San Diego, Calif.), 2015-12) Reid, David W; Shenolikar, Shirish; Nicchitta, Christopher VThe development and application of ribosome profiling has markedly advanced our understanding of ribosomes and mRNA translation. The experimental approach, which relies on deep sequencing of ribosome-protected mRNA fragments generated by treatment of polyribosomes with exogenous nucleases, provides a transcriptome-wide assessment of translation. The broad application of ribosome profiling has been slowed by the complexity and expense of the protocol. Here, we provide a simplified ribosome profiling method that uses micrococcal nuclease to generate ribosome footprints in crude cellular extracts, which are then purified simply by size selection via polyacrylamide gel electrophoresis. This simplification removes the laborious or expensive purification of ribosomes that has typically been used. This direct extraction method generates gene-level ribosome profiling data that are similar to a method that includes ribosome purification. This protocol should significantly ease the barrier to entry for research groups interested in employing ribosome profiling.Item Open Access The Role of mRNA Translation in the Regulation of Ribosome Trafficking to the Endoplasmic Reticulum(2014) Ponn, Alison ElyseThe goal of this research is to identify the trafficking patterns that direct ribosomes to the endoplasmic reticulum (ER). It is widely believed that the SRP pathway is the only mechanism that cells use to localize mRNA and ribosomes to the ER, but this has been found not to be a sufficient explanation for the patterns of RNA localization in cells, namely that non-signal sequence-containing mRNA are translated on the ER and that ribosomes retain their membrane association after translation termination. First, a summary of the history of the field is presented to provide context for the key, unanswered questions in the field. Then, experiments employing [32Pi] pulse-chase labeling of HeLa cells over a time course to follow nascent ribosome trafficking are presented. The purpose of the cell labeling was to track rRNA processing and assembly into nascent ribosomes, followed by their export into the cytoplasm and recruitment into active polysomes. A detergent-based cell fractionation procedure was also utilized to separate the cytosol and ER compartments in order to observe ribosomes on their path as they exit the nucleus and either localize to the ER or cytosolic cellular compartment. Through this method, it was seen that ribosomes appear in both compartments at the same time, suggesting a mechanism may be occurring in addition to SRP-dependent ribosome trafficking. This research provides an understanding toward a mechanism that is not currently known, but will one day more fully explain the patterns of ribosomal localization.
Item Open Access The unfolded protein response triggers selective mRNA release from the endoplasmic reticulum.(Cell, 2014-09) Reid, David W; Chen, Qiang; Tay, Angeline S-L; Shenolikar, Shirish; Nicchitta, Christopher VThe unfolded protein response (UPR) is a stress response program that reprograms cellular translation and gene expression in response to proteotoxic stress in the endoplasmic reticulum (ER). One of the primary means by which the UPR alleviates this stress is by reducing protein flux into the ER via a general suppression of protein synthesis and ER-specific mRNA degradation. We report here an additional UPR-induced mechanism for the reduction of protein flux into the ER, where mRNAs that encode signal sequences are released from the ER to the cytosol. By removing mRNAs from the site of translocation, this mechanism may serve as a potent means to transiently reduce ER protein folding load and restore proteostasis. These findings identify the dynamic subcellular localization of mRNAs and translation as a selective and rapid regulatory feature of the cellular response to protein folding stress.