Browsing by Subject "RNA, Messenger"
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Item Assessment of Gamma/Delta T Cell Functionality Following Pathogenic HIV/SIV and Non-Pathogenic SIV Infections(2007-05-22) Kosub, David Alan; Sodora, Donald L.Pathogenic HIV/SIV infection induces high viral loads, aberrant immune activation, and dysfunction in numerous immunologic cells (including gamma/delta (gamma delta ) T cells) leading to opportunistic infections. gamma delta T cells bridge the innate and adaptive immune responses primarily via cytokines produced in response to microbial phosphoantigens. gamma delta T cells have also been implicated in the control of an SIV challenge infection as evidenced by increased numbers and beta -chemokine expression at mucosal sites in vaccinated macaques. The goal of Aim 1 of this thesis was to assess the impact of an acute SIV infection on the levels of gamma delta T cells at mucosal and lymphoid sites in macaques utilizing quantitative PCR. At two days post-infection, a decrease in gamma delta T cell levels was observed at mucosa sites whereas increased levels were present at regional lymph nodes. Also, an increase in lymphoid homing molecules was observed at these lymph nodes, indicating a mechanism whereby gamma delta T cells migrate away from mucosal sites towards secondary lymphoid tissues following an acute SIV infection. The redistribution of gamma delta T cells may be important for the initiation of an anti-viral immune response and control of rapid viral spread. The goal of Aims 2 and 3 was to assess the ability of gamma delta T cells in HIV-infected patients to express cytokines and compare these results to analysis of the non-pathogenic SIV infection of sooty mangabeys. Following stimulation with the non-specific activators PMA/Ionomycin or the gamma delta TCR specific ligand isopentenyl pyrophosphate, a decrease in the percentages of gamma delta T cells expressing Th1 pro-inflammatory cytokines including TNF-alpha and IFN-gamma was observed in the HIV+ patients (regardless of CD4+ T cell levels). Highly active anti-retroviral therapy (HAART) partially restored the ability of gamma delta T cells from HIV+ patients to express Th1 cytokines. SIV infection of mangabeys results in high viral replication, low levels of immune activation, and generally no signs of progression to AIDS. Evidence for preserved or increased functionality of gamma delta T cells from SIV+ mangabeys (regardless of CD4+ T cell levels) was demonstrated by maintained percentages of gamma delta T cells that expressed Th1 cytokines following ex vivo stimulation. These data suggest that in the absence of aberrant immune activation, controlled Th1 responses by gamma delta T cells from mangabeys may assist in suppressing damage due to the SIV infection as well as inhibiting the onset of opportunistic infections. These data provide rationale for therapies aimed at increasing gamma delta T cell functionality in humans, particularly with regard to Th1 cytokine responses to augment protection against opportunistic infections and HIV disease progression.Item Chemical Intervention of Influenza Virus mRNA Nuclear Export(2020-07-15) Esparza, Matthew Aaron; Shay, Jerry W.; Fontoura, Beatriz; Grinnell, Frederick; Minna, John D.Influenza A viruses are human pathogens with limited therapeutic options, making it crucial to devise strategies for the identification of new classes of antiviral medications. The influenza A virus genome is constituted of 8 RNA segments. Two of these viral RNAs are transcribed into mRNAs that are alternatively spliced. The M1 mRNA encodes the M1 protein but is also alternatively spliced to yield the M2 mRNA during infection. M1 to M2 mRNA splicing occurs at nuclear speckles, and M1 and M2 mRNAs are exported to the cytoplasm for translation. M1 and M2 proteins are critical for viral trafficking, assembly, and budding. We show that influenza virus utilizes nuclear speckles to promote post-transcriptional splicing of its M1 mRNA. We assign previously unknown roles for the viral NS1 protein and cellular factors to an intranuclear trafficking pathway that targets the viral M1 mRNA to nuclear speckles, mediates splicing at these nuclear bodies, and exports the spliced M2 mRNA from the nucleus. In addition, gene knockout of the cellular protein NS1-BP, a constituent of the M mRNA speckle-export pathway, inhibits M mRNA nuclear export without significantly altering bulk cellular mRNA export, providing an avenue to preferentially target influenza virus. We performed a high-content, image-based chemical screen using single-molecule RNA-FISH to label viral M mRNAs followed by multistep quantitative approaches to assess cellular mRNA and cell toxicity. We identified inhibitors of viral mRNA biogenesis and nuclear export that exhibited no significant activity towards bulk cellular mRNA at non-cytotoxic concentrations. Among the hits is a small molecule that inhibits nuclear export of a subset of viral and cellular mRNAs via the mRNA export factor UAP56 without altering bulk cellular mRNA nuclear export. These findings underscore specific nuclear export requirements for viral mRNA nuclear export. This RNA export inhibitor also impaired replication of diverse influenza virus strains at non-toxic concentrations. Thus, this screening strategy yielded compounds that alone or in combination may serve as leads to new ways of treating influenza virus infection and are novel tools for studying viral RNA trafficking in the nucleus.Item The Design, Synthesis, and Evaluation of Zwitterionic and Cationic Lipids for In Vivo RNA Delivery and Non-Viral CRISPR/Cas Gene Editing(2018-03-05) Miller, Jason Brian; Ready, Joseph M.; Gao, Jinming; Tambar, Uttam; Siegwart, Daniel J.The delivery of nucleic acids is an emerging therapeutic modality in clinical development for the treatment of many genetic diseases. The use of RNA interference (RNAi) as a therapeutic is an exciting and rapidly developing field that offers a promising alternative to small molecule drugs for the treatment of dysregulatory diseases, including cancer. Small interfering RNA (siRNA) can be designed against any mRNA target, and upon loading into the RNA-induced silencing complex (RISC) can enable sequence-specific target recognition and degradation. Meanwhile, messenger RNA is currently being utilized for protein replacement therapy and for the development of vaccines by expressing viral antigens on dendritic cells. However, because RNA molecules are unable to passively diffuse across plasma membranes due to a high molecular weight (~13 kDa for siRNA, >300 kDa for mRNA), hydrophilicity and strong anionic charge, while also being unstable and highly immunogenic when injected systemically, nucleic acid therapeutics require carriers for effective delivery. To date, many successful carriers have been designed using amphiphilic lipid-like compounds containing amine-rich cores, but the challenges of efficient endosomal release and delivery to organs outside of the liver remain major hurdles in the field of RNA therapeutics. This dissertation reports the design, synthesis and characterization of two new classes of lipids with unique chemical structures and in vivo RNA delivery capabilities to the lung: zwitterionic amino lipids (ZALs) and cationic sulfonamide amino lipids (CSALs). ZALs contain an amine rich core, hydrophobic tails introduced via conjugate addition or epoxide opening, and a zwitterionic sulfobetaine head group. ZALs were designed with a combination of cationic and zwitterionic lipid properties, to help stabilize and effectively deliver long RNA molecules. A lead compound, ZA3-Ep10, was effective for in vivo messenger RNA delivery and the first reported demonstration of in vivo non-viral gene editing by delivering mRNA components encoding the CRISPR/Cas gene editing platform. CSALs contain a unique chemical scaffold containing an internal quaternary ammonium group and a sulfonamide linker. A rational investigation of structure-activity relationships revealed that CSALs containing an acetate sidearm, a dimethyl amino head group and higher hydrophobic content were effective in delivery siRNA to human cancer cells in vitro. CSALs also demonstrated lung localization upon systemic delivery in vivo while also demonstrating the ability to redirect liver targeting ionizable lipid nanoparticles to the lung. These new classes of materials demonstrate the importance of structural consideration in material design for the development of nucleic acid therapeutics, while also providing structural templates for developing carriers for effective delivery to tissues outside of the liver.Item Identifying Novel Regulators of LIN28B Through a Genome-Wide CRISPR/Cas9 Screen(2017-01-17) Budhipramono, Albert; Hao, Zhu; Nguyen, LiemLIN28 is a family of RNA-binding proteins that are well-conserved across species. It is well-known to regulate developmental timing by inhibiting the biogenesis of the let-7 microRNAs. Numerous studies have shown that LIN28 is dysregulated in a wide spectrum of cancer types, especially pediatric cancers such as hepatoblastoma and Wilms' tumor. Our laboratory previously showed that reactivation of LIN28B, one of the two LIN28 homologs, is sufficient to drive liver cancer, and that LIN28B deletion is detrimental to tumor development. LIN28B exerts its oncogenic function by inhibiting the maturation of let-7 precursors, as well as directly binding to and enhancing translation of growth-promoting mRNA targets, such as members of the IGF2BP family. While our work and others established that LIN28B functions as an oncogene, the identity of factors that regulate LIN28B expression during normal development and cancer remains elusive. As LIN28B is a driver of oncogenesis in various cancers, understanding its regulation in the process of oncogenesis will help uncover novel therapeutic targets. Here, we show an original approach for identifying regulators of the human LIN28B gene utilizing the CRISPR/Cas9 genome engineering system. Traditional transgenic approaches to study gene function often fail to capture transcriptional regulation at distal promoter and enhancer sequences. Using the CRISPR/Cas9 system, we knocked a GFP reporter sequence into the endogenous locus of LIN28B in human cancer cell lines, engineering a fusion LIN28B-GFP protein. This approach is unique in that GFP expression will be altered not only by changes in regulation at the coding sequence, mRNA and protein levels, but also changes at distal regulatory sequences. To identify unknown regulators of LIN28B, we will perform a genome-wide CRISPR/Cas9-mediated knockout screen in human cells expressing the fusion LIN28B-GFP protein. Using a genome-scale library with 76,441 sgRNAs, we will knock out 19,114 genes individually and assess their effects on LIN28B levels by measuring GFP expression. sgRNAs that are enriched in the high GFP-expressing population suggest genes that normally function as inhibitors of LIN28B. On the other hand, sgRNAs that are depleted suggest activators of LIN28B. Through this screen, we hope to gain further insight into how LIN28B is regulated in normal development and cancer. Furthermore, identifying regulators of LIN28B can provide novel avenues for developing cancer therapeutics.Item A Multi-Organ Role for Nocturnin in Post-Transcriptional Regulation of RNA(2018-04-16) Onder, Yasemin; Huber, Kimberly M.; Green, Carla B.; Takahashi, Joseph; Yu, Gang; Mishra, PrashantNocturnin is an RNA-specific nuclease, a circadian deadenylase first discovered in the retina of Xenopus laevis, and is conserved among eukaryotes. Nocturnin is widely expressed in the brain and in the periphery and is also an immediate early gene that is acutely induced in response to various stimuli. This study investigates Nocturnin's potential role in post-transcriptional regulation of mRNAs in the mitochondria and in two different tissues: brain and brown adipose tissue (BAT). Nocturnin has a predicted mitochondrial-localization signal (MLS) surrounded by two potential translation initiation sites (AUG codons) raising the possibility of dual translation initiation sites. Here we demonstrate that Nocturnin is present in the mitochondria and that it exhibits mitochondrial or cytoplasmic localization via the use of alternative translation initiation sites. I also show that Nocturnin is acutely induced in response to cold and mitochondrial-encoded mRNAs in Noc-/- BAT exhibit impaired stability upon cold exposure. Global analysis of cold-induced changes in the transcriptome of Noc-/- and wild type (WT) mice reveal down-regulation of glycan biosynthesis genes in the Noc-/- BAT. Strikingly, metabolomics analysis demonstrates robust alterations in key tricarboxylic acid metabolites like pyruvate and succinate in the Noc-/- mice BAT in response to a prolonged cold exposure. In summary, we propose a model that Nocturnin acts as a metabolic switch in response to cold by diverting glucose and free fatty acids (FFA) to the mitochondria. In this study, Nocturnin's potential role in post-transcriptional regulation of synaptic plasticity is also investigated. Activity-dependent local protein translation in the dendrites is thought to be an important component of synaptic plasticity. The mechanism of how translation of different mRNAs is differentially regulated in the dendrites is yet not clear. Here I demonstrate that Nocturnin is present in the dendrites as well as in the post-synaptic density as observed in cultured neurons and brain slices from cortex and hippocampus. I investigated mGluR-LTD in hippocampal slices from Noc-/- and WT littermates and observed an attenuation of mGluR-LTD in Noc-/- mice in the first cohort, but this result was not replicated in a second cohort. Further studies are needed to elucidate Nocturnin's role in the synapse.Item The Regulation of hTERT by Alternative Splicing(2017-07-27) Yuan, Laura Yu; Yu, Hongtao; Shay, Jerry W.; Wright, Woodring E.; Fontoura, Beatriz; Cobb, Melanie H.Telomeres are non-coding DNA hexameric repeats (TTAGGG in mammals) located at the ends of linear chromosomes that, along with their associated proteins, protect against the loss of genomic material during cell division and prevent the recognition of chromosome ends as double-strand breaks. Human telomeres shorten with continued cell proliferation but are maintained by human telomerase reverse transcriptase (hTERT), an enzyme that synthesizes telomeric repeats using an RNA template. The regulation of telomerase has been studied at many levels--from epigenetic and transcriptional regulation to the alternative splicing of hTERT pre-mRNA into catalytically inactive splice variants. Our hypothesis is that if the regulation of telomerase reverse transcriptase splicing is necessary for telomere length homeostasis, altering telomerase splicing to decrease the production of full-length hTERT and will result in decreased telomerase activity and subsequently telomere shortening. We focused our efforts on identifying splicing factors are involved in hTERT splicing and characterized the role of two splicing factors, NOVA1 and PTBP1, in regulation of hTERT splicing in non-small cell lung cancer cells. We show that these splicing factors are important for full-length hTERT, telomerase activity and telomere length maintenance in vitro. Xenograft studies suggest that NOVA1 is also important for tumor growth in vivo. We found that these splicing factors are able to directly interact with hTERT in a region our group previously identified to be important for hTERT splicing. Altogether, our work suggests that splicing factors are important for hTERT regulation and telomerase activity in cancer. Since telomerase activity is undetectable in most somatic tissues but is increased in the vast majority of human cancers, dependence on telomerase represents a key vulnerability in cancer tissues which could be therapeutically targetable.Item Regulatory RNAs at the Heart of Sugar Metabolism: New Mechanisms and Novel Discoveries(2011-02-01) Irnov; Winkler, Wade C.Bacteria are adept at using a variety of posttranscriptional strategies to regulate gene expression. Specifically, various RNA-mediated genetic control elements have been discovered in the past decade through a combination of genetics, bioinformatics, and transcriptomic approaches. Together, these RNA elements control the expression of many genes involved in diverse cellular processes such as energy metabolism, stress response, biofilm formation, and pathogenesis. In the Gram-positive bacterium Bacillus subtilis, several RNA elements have been shown to be required for the precise coordination of genes involved in various sugar utilization pathways. These genetic switches typically regulate gene expression by modulating the formation of a transcription termination element in a ligand-dependent manner. Interestingly, two unique elements, the glmS ribozyme and the eps-associated RNA (EAR), are missing the signature elements required for control of transcription termination or translation initiation. The latter mechanism is more commonly found in Gram-negative bacteria. Our objective is to study the mechanisms by which these two RNAs control gene expression. Additionally, we would like to identify other regulatory RNAs that are important for sugar metabolism in Bacillus subtilis. Both the glmS ribozyme and EAR are positioned at the center of the sugar metabolism pathways in B. subtilis. The glmS RNA is a glucosamine-6-phosphate responsive element that regulates the expression of the GlmS enzyme, which directs sugar precursors from glycolysis into the cell wall biosynthesis pathway. The EAR element resides within the 16-kb eps operon that is required for biofilm exopolysaccharide production. Our data demonstrates that both RNAs employ novel mechanisms: the glmS ribozyme utilizes a ligand-specific RNase-mediated degradation event, while EAR uses a processive antitermination mechanism for complete synthesis of the long operon. Furthermore, by using high-throughput sequencing approach we have successfully identified many new regulatory RNA candidates, including various long 5`-UTR, toxin-antitoxin systems, prophage-encoded RNAs, and several developmentally regulated small RNAs. Their functions are still under investigation. Collectively, our studies provide important insights into the different aspects of bacterial physiology, including RNA decay pathways, transcription of long operons and cellular differentiation. We argue that posttranscriptional regulation is of greater importance to Bacillus subtilis (and probably all bacteria) than previously realized.Item The Role of NS1-BP in Influenza Virus Replication(2012-08-13) Tsai, Pei-Ling; Fontoura, BeatrizInfluenza A viruses are negative-sense, segmented RNA viruses which cause about 500,000 deaths worldwide per year. Genomic studies have shown that the non-structural protein (NS1) of influenza A virus is a major virulence factor that is essential for pathogenesis. NS1 is a multifunctional protein localized in the nucleus and in the cytoplasm. In the cytoplasm, NS1 inhibits host signaling pathways that result in down-regulation of interferon expression and innate immune response. In the nucleus, NS1 represses host gene expression. I have shown that NS1 binds an mRNA complex containing NXF1/TAP, NXT/p15, Rae1, and E1B-AP5, which are key components of the mRNA export machinery. By targeting this complex, NS1 blocks host mRNA export, and cells become highly permissive to viral replication. Another intranuclear pool of NS1 was found to interact with a host protein termed NS1-BP, which has been suggested to play a role in pre-mRNA splicing. However, the functions and mechanisms of NS1-BP involved in influenza life cycle remain to be elucidated. To investigate the function of NS1-BP, I first identified its binding partners by immunoprecipitation followed by mass spectrometry. I found interactions of NS1-BP with viral polymerase complex and host RNA polymerase II indicating that NS1-BP has a role in regulating viral RNA transcription and replication. I further showed that low levels of NS1-BP led to a decrease in viral polymerase activity resulting in inhibition of virus replication. Thus, I identified NS1-BP as a novel pro-viral factor required for proper replication of influenza virus. Since NS1 is a key contributor to the virulence of influenza viruses, discovering the function of NS1 interacting partners has major implications for antiviral therapy.Item The Roles of Codon Usage in Translational and Transcriptional Regulation on Gene Expression(2020-11-12) Yang, Qian; Tu, Benjamin; Liu, Yi; Cobb, Melanie H.; Thomas, Philip J.; Buszczak, MichaelCodon usage bias refers to the universal feature of genome that synonymous codons are used at different frequency. Codon usage plays critical roles in determining gene expression levels through impacting multiple fundamental cellular processes. Although the correlation between codon usage and gene expression level has long been observed, the underlying mechanisms are still largely unclear. In the first part, I demonstrate the mechanism of codon usage determining translation efficiency. The slow decoding rate of nonoptimal codon causes ribosome stalling on mRNA, which leads to premature termination of translation and reduced protein production. This process is conserved from Neurospora to Drosophila. In addition, I demonstrate that the premature termination of translation is mediated by the canonical release factor eRF1, which recognizes ribosomes stalled at nonoptimal codons. Together, I propose a model that explains the impact of codon usage on translation efficiency. In the second part, I investigate the role of promoter in codon- dependent gene expression at transcription level. I show that codon usage and sequence downstream of core promoter act in concert to determine the transcription level. Moreover, I identify the regulatory element in Hsp70 promoter that upregulates the transcription of genes containing nonoptimal codons. The differential transcriptional level is achieved through epigenetic regulations affecting nucleosome density and H3K27ac level and premature termination pathway mediated by Ars2, NEXT complex and nuclear exosome. Collectively, these results show that codon usage and promoter impact transcription through multilayer regulatory mechanisms.Item Roles of miR-1246, miR-146, and miR-155 and Their Targets in Human Trophoblast Differentiation(2016-01-19) Nambiar, Anjali; Guo, Wei; Mendelson, Carole R.BACKGROUND: Abnormal placental implantation has been implicated in preeclampsia (PE), a devastating hypertensive disorder of pregnancy that is a leading cause of maternal and neonatal morbidity and mortality. Placental development depends upon fetal cytotrophoblast (CytT) invasion into the maternal decidua and enlargement of the uterine arterioles with increased blood flow and O2 availability to chorionic villi. CytT fuse and differentiate into syncytiotrophoblast (SynT), which mediate gas and nutrient exchange between mother and fetus, and produce several key steroid and polypeptide hormones of pregnancy. Recent studies from our lab suggest that SynT differentiation is regulated by differentially expressed microRNAs (miRNA/miR). By miRNA microarray of RNA from mid-gestation human CyT before and after differentiation to SynT in culture, miR-1246, miR-146, and miR-155 were found to be highly and significantly upregulated. Interestingly, miR-1246 and miR-155 have been predicted and proven, respectively, to target Jarid2, which recruits the polycomb repressive complex (PRC) 2 to developmentally-regulated genes. PRC2 catalyzes methylation of histone H3 on lysine 27. This chromatin mark promotes recruitment of the PRC1 complex, resulting in repressed chromatin. Moreover, miR-1246, miR-155 and miR-146 all are predicted to modulate components of the Wnt signaling pathway, which plays an integral role in placental development. OBJECTIVE: To analyze expression of miR-155, miR-1246, miR-146 and their targets during differentiation of human trophoblasts in culture and effects of hypoxia. METHODS: CytT were isolated from mid-gestation human placenta and placed in monolayer culture for 24, 48 and 72 h, under normoxic (20 O2%) and hypoxic (2% O2) conditions. RNA was isolated and analyzed for expression of Jarid2, miR-1246, -146, and -155 using Taqman-based RT-qPCR and for markers of SynT differentiation using SYBR Green RT-qPCR. RESULTS: As observed in the microarray, miR-1246, -146, and -155 were dramatically upregulated during SynT differentiation. Conversely, Jarid2 decreased markedly with SynT differentiation. Hypoxia had no effect of expression of any of these miRNAs or on Jarid2 mRNA or protein. CONCLUSION: This research establishes the expression patterns of miR-1246, miR-155, miR-146, and Jarid2 during differentiation of human CytT to SynT in culture. Further studies will evaluate effects of overexpression and inhibition of these miRNAs on trophoblast differentiation and expression of targets, and in PE placentas, where differentiation is poorly regulated. Increased understanding of these miRNAs will provide insight into mechanisms that regulate trophoblast differentiation and the pathogenesis of PE.Item Switching the Fate of mRNAs for Mitochondrial Biogenesis(2017-03-02) Lee, Chien-Der; Liu, Yi; Tu, Benjamin; McKnight, Steven L.; Conrad, NicholasmRNAs encoding mitochondrial biogenesis proteins are co-regulated in a manner closely linked to metabolism. In yeast growing in glucose, mitochondrial biogenesis is repressed, but must be induced upon glucose depletion to enable energy production using alternative carbon sources such as ethanol or acetate through mitochondrial respiration. Yeast cells growing in glucose constitutively transcribe nuclear-encoded mitochondrial ribosomal mRNAs at a basal level. However, instead of sharing a common upstream activating sequence for transcription, those mRNAs all harbor a common sequence motif within their 3'UTRs. Puf3p, an RNA-binding protein, can directly bind to this class of mRNA transcripts to promote degradation in glucose medium. However, the function of Puf3p upon glucose depletion is not clear. In the first part of this study, I show how Puf3p responds to glucose availability to switch the fate of its bound transcripts that encode proteins required for mitochondrial biogenesis. This regulation allows cell to quickly respond to glucose depletion by switching the degradation fate of those mRNAs to translation. Thus, yeast can activate pre-existing mRNA without relying on de novo transcription for mitochondrial biogenesis. I then show Puf3p is subjected to phosphorylation downstream of a glucose sensing pathway. Puf3p is hypophosphorylated in glucose medium; however, upon glucose depletion, Puf3p becomes heavily phosphorylated within its N-terminal region of low complexity, associates with polysomes, and promotes translation of its target mRNAs. In the second part of this study, I show that phosphorylation of Puf3p is required for translational activation of its bound mRNAs. Strikingly, a Puf3p mutant that prevents its phosphorylation no longer promotes mRNA translation but also becomes trapped in intracellular foci in an mRNA-dependent manner. These findings suggest how the inability to properly resolve Puf3p-containing RNA-protein granules via a phosphorylation-based mechanism might be toxic to a cell. The toxicity might be due to sequestration of translational factors in the Puf3p RNA protein granule in a manner reminiscent of neurodegenerative disease-related protein aggregation.Item Translational Control by the Ribosome-Associated Complex in the Unfolded Protein Response(2020-12-01T06:00:00.000Z) Wu, I-Hui; Shay, Jerry W.; Thomas, Philip J.; Mendell, Joshua T.; Tu, BenjaminRibosome-associated chaperones are ubiquitous and highly conserved. There are two classes of ribosome-associated chaperones in eukaryotes, the nascent polypeptide-associated complex (NAC) and the ribosome-associated complex (RAC). Mammalian RAC consists of Hsp70L1, an Hsp70 chaperone homologue, and Mpp11, a DnaJ cofactor. RAC interacts with the nascent chain near the polypeptide exit tunnel and the decoding center on the 60S and 40S ribosomal subunits, respectively. Its unique position on the ribosome implies the coordinating role of de novo protein folding with translation. Deletion of RAC causes growth defects and sensitizes to osmotic, cold, and aminoglycoside stresses in yeast. Furthermore, studies have shown that Mpp11 is over-expressed in head and neck squamous cell cancer and leukemia. However, the function of RAC in stress responses and its role in oncogenesis remain obscure. The current hypothesis predicts that RAC supports co-translational folding of nascent cytosolic polypeptides. To directly test this hypothesis, I altered levels of RAC components and monitored the cytosolic heat shock response (HSR) and the unfolded protein response (UPR) in the ER, two stress pathways known to be activated by accumulation of misfolded proteins. Contrary to its presumptive role in cytosolic protein folding, the reduction of RAC expression did not activate the cytosolic HSR. Unexpectedly, reduction of RAC sensitizes cells to ER stress by selectively attenuating activation of the IRE1 branch of UPR. When RAC is reduced, Xbp1 mRNA splicing is inhibited upon ER stress. Consistent with this activity, ER stress induces changes in the subcellular distribution of RAC, which coincides with the localization of Xbp1 mRNA. Mechanistically, reduction of RAC affects the pathway at a very early step, as IRE1 self-association is inhibited. Additionally, this study shows that the reduction of RAC enhances cellular mRNA translation, including Xbp1 mRNA translation. Interestingly, reduction of Pelo, a protein involved in recognizing stalled ribosomes, counters the inhibition of Xbp1 mRNA splicing, and IRE1 foci formation due to RAC knockdown. Collectively, these results suggest that RAC plays a central role in the IRE1 branch of the UPR tuning IRE1 clustering and mRNA translation.