Late Endosomes Act as mRNA Translation Platforms and Sustain Mitochondria in Axons.

Local translation regulates the axonal proteome, playing an important role in neuronal wiring and axon maintenance. How axonal mRNAs are localized to specific subcellular sites for translation, however, is not understood. Here we report that RNA granules associate with endosomes along the axons of retinal ganglion cells. RNA-bearing Rab7a late endosomes also associate with ribosomes, and real-time translation imaging reveals that they are sites of local protein synthesis. We show that RNA-bearing late endosomes often pause on mitochondria and that mRNAs encoding proteins for mitochondrial function are translated on Rab7a endosomes. Disruption of Rab7a function with Rab7a mutants, including those associated with Charcot-Marie-Tooth type 2B neuropathy, markedly decreases axonal protein synthesis, impairs mitochondrial function, and compromises axonal viability. Our findings thus reveal that late endosomes interact with RNA granules, translation machinery, and mitochondria and suggest that they serve as sites for regulating the supply of nascent pro-survival proteins in axons.

Beyond the Triplet Code: Context Cues Transform Translation.

The elucidation of the genetic code remains among the most influential discoveries in biology. While innumerable studies have validated the general universality of the code and its value in predicting and analyzing protein coding sequences, established and emerging work has also suggested that full genome decryption may benefit from a greater consideration of a codon's neighborhood within an mRNA than has been broadly applied. This Review examines the evidence for context cues in translation, with a focus on several recent studies that reveal broad roles for mRNA context in programming translation start sites, the rate of translation elongation, and stop codon identity.

The m6A epitranscriptome: transcriptome plasticity in brain development and function.

The field of epitranscriptomics examines the recently deciphered form of gene expression regulation that is mediated by type- and site-specific RNA modifications. Similarly to the role played by epigenetic mechanisms - which operate via DNA and histone modifications - epitranscriptomic modifications are involved in the control of the delicate gene expression patterns that are needed for the development and activity of the nervous system and are essential for basic and higher brain functions. Here we describe the mechanisms that are involved in the writing, erasing and reading of N6-methyladenosine, the most prevalent internal mRNA modification, and the emerging roles played by N6-methyladenosine in the nervous system.

Thermal adaptation of mRNA secondary structure: stability versus lability.

Macromolecular function commonly involves rapidly reversible alterations in three-dimensional structure (conformation). To allow these essential conformational changes, macromolecules must possess higher order structures that are appropriately balanced between rigidity and flexibility. Because of the low stabilization free energies (marginal stabilities) of macromolecule conformations, temperature changes have strong effects on conformation and, thereby, on function. As is well known for proteins, during evolution, temperature-adaptive changes in sequence foster retention of optimal marginal stability at a species' normal physiological temperatures. Here, we extend this type of analysis to messenger RNAs (mRNAs), a class of macromolecules for which the stability-lability balance has not been elucidated. We employ in silico methods to determine secondary structures and estimate changes in free energy of folding (ΔGfold) for 25 orthologous mRNAs that encode the enzyme cytosolic malate dehydrogenase in marine mollusks with adaptation temperatures spanning an almost 60 °C range. The change in free energy that occurs during formation of the ensemble of mRNA secondary structures is significantly correlated with adaptation temperature: ΔGfold values are all negative and their absolute values increase with adaptation temperature. A principal mechanism underlying these adaptations is a significant increase in synonymous guanine + cytosine substitutions with increasing temperature. These findings open up an avenue of exploration in molecular evolution and raise interesting questions about the interaction between temperature-adaptive changes in mRNA sequence and in the proteins they encode.

Differential fates of introns in gene expression due to global alternative splicing.

The discovery of introns over four decades ago revealed a new vision of genes and their interrupted arrangement. Throughout the years, it has appeared that introns play essential roles in the regulation of gene expression. Unique processing of excised introns through the formation of lariats suggests a widespread role for these molecules in the structure and function of cells. In addition to rapid destruction, these lariats may linger on in the nucleus or may even be exported to the cytoplasm, where they remain stable circular RNAs (circRNAs). Alternative splicing (AS) is a source of diversity in mature transcripts harboring retained introns (RI-mRNAs). Such RNAs may contain one or more entire retained intron(s) (RIs), but they may also have intron fragments resulting from sequential excision of smaller subfragments via recursive splicing (RS), which is characteristic of long introns. There are many potential fates of RI-mRNAs, including their downregulation via nuclear and cytoplasmic surveillance systems and the generation of new protein isoforms with potentially different functions. Various reports have linked the presence of such unprocessed transcripts in mammals to important roles in normal development and in disease-related conditions. In certain human neurological-neuromuscular disorders, including myotonic dystrophy type 2 (DM2), frontotemporal dementia/amyotrophic lateral sclerosis (FTD/ALS) and Duchenne muscular dystrophy (DMD), peculiar processing of long introns has been identified and is associated with their pathogenic effects. In this review, we discuss different mechanisms involved in the processing of introns during AS and the functions of these large sections of the genome in our biology.

Inverted translational control of eukaryotic gene expression by ribosome collisions.

The canonical model of eukaryotic translation posits that efficient translation initiation increases protein expression and mRNA stability. Contrary to this model, we find that increasing initiation rate can decrease both protein expression and stability of certain mRNAs in the budding yeast Saccharomyces cerevisiae. These mRNAs encode a stretch of polybasic residues that cause ribosome stalling. Our computational modeling predicts that the observed decrease in gene expression at high initiation rates occurs when ribosome collisions at stalls stimulate abortive termination of the leading ribosome or cause endonucleolytic mRNA cleavage. Consistent with this prediction, the collision-associated quality-control factors Asc1 and Hel2 (orthologs of human RACK1 and ZNF598, respectively) decrease gene expression from stall-containing mRNAs only at high initiation rates. Remarkably, hundreds of S. cerevisiae mRNAs that contain ribosome stall sequences also exhibit lower translation efficiency. We propose that inefficient translation initiation allows these stall-containing endogenous mRNAs to escape collision-stimulated reduction in gene expression.

Nanoparticle-based local translation reveals mRNA as a translation-coupled scaffold with anchoring function.

The spatial regulation of messenger RNA (mRNA) translation is central to cellular functions and relies on numerous complex processes. Biomimetic approaches could bypass these endogenous complex processes, improve our comprehension of the regulation, and allow for controlling local translation regulations and functions. However, the causality between local translation and nascent protein function remains elusive. Here, we developed a nanoparticle (NP)-based strategy to magnetically control mRNA spatial patterns in mammalian cell extracts and investigate how local translation impacts nascent protein localization and function. By monitoring the translation of the magnetically localized mRNAs, we show that mRNA-NP complexes operate as a source for the continuous production of proteins from defined positions. By applying this approach to actin-binding proteins, we triggered the local formation of actin cytoskeletons and identified the minimal requirements for spatial control of the actin filament network. In addition, our bottom-up approach identified a role for mRNA as a translation-coupled scaffold for the function of nascent N-terminal protein domains. Our approach will serve as a platform for regulating mRNA localization and investigating the function of nascent protein domains during translation.

Reduction of mRNA export unmasks different tissue sensitivities to low mRNA levels during Caenorhabditis elegans development.

Animal development requires the execution of specific transcriptional programs in different sets of cells to build tissues and functional organs. Transcripts are exported from the nucleus to the cytoplasm where they are translated into proteins that, ultimately, carry out the cellular functions. Here we show that in Caenorhabditis elegans, reduction of mRNA export strongly affects epithelial morphogenesis and germline proliferation while other tissues remain relatively unaffected. Epithelialization and gamete formation demand a large number of transcripts in the cytoplasm for the duration of these processes. In addition, our findings highlight the existence of a regulatory feedback mechanism that activates gene expression in response to low levels of cytoplasmic mRNA. We expand the genetic characterization of nuclear export factor NXF-1 to other members of the mRNA export pathway to model mRNA export and recycling of NXF-1 back to the nucleus. Our model explains how mutations in genes involved in general processes, such as mRNA export, may result in tissue-specific developmental phenotypes.

Integrated microRNA and transcriptome profiling reveal key miRNA-mRNA interaction pairs associated with seed development in Tartary buckwheat (Fagopyrum tataricum).

BACKGROUND: Tartary buckwheat seed development is an extremely complex process involving many gene regulatory pathways. MicroRNAs (miRNAs) have been identified as the important negative regulators of gene expression and performed crucial regulatory roles in various plant biological processes. However, whether miRNAs participate in Tartary buckwheat seed development remains unexplored. RESULTS: In this study, we first identified 26 miRNA biosynthesis genes in the Tartary buckwheat genome and described their phylogeny and expression profiling. Then we performed small RNA (sRNA) sequencing for Tartary buckwheat seeds at three developmental stages to identify the miRNAs associated with seed development. In total, 230 miRNAs, including 101 conserved and 129 novel miRNAs, were first identified in Tartary buckwheat, and 3268 target genes were successfully predicted. Among these miRNAs, 76 exhibited differential expression during seed development, and 1534 target genes which correspond to 74 differentially expressed miRNAs (DEMs) were identified. Based on integrated analysis of DEMs and their targets expression, 65 miRNA-mRNA interaction pairs (25 DEMs corresponding to 65 target genes) were identified that exhibited significantly opposite expression during Tartary buckwheat seed development, and 6 of the miRNA-mRNA pairs were further verified by quantitative real-time polymerase chain reaction (qRT-PCR) and ligase-mediated rapid amplification of 5' cDNA ends (5'-RLM-RACE). Functional annotation of the 65 target mRNAs showed that 56 miRNA-mRNA interaction pairs major involved in cell differentiation and proliferation, cell elongation, hormones response, organogenesis, embryo and endosperm development, seed size, mineral elements transport, and flavonoid biosynthesis, which indicated that they are the key miRNA-mRNA pairs for Tartary buckwheat seed development. CONCLUSIONS: Our findings provided insights for the first time into miRNA-mediated regulatory pathways in Tartary buckwheat seed development and suggested that miRNAs play important role in Tartary buckwheat seed development. These findings will be help to study the roles and regulatory mechanism of miRNAs in Tartary buckwheat seed development.

Comprehensive analysis of ceRNA networks reveals prognostic lncRNAs related to immune infiltration in colorectal cancer.

BACKGROUND: Competing endogenous RNA (ceRNA) represents a class of RNAs (e.g., long noncoding RNAs [lncRNAs]) with microRNA (miRNA) binding sites, which can competitively bind miRNA and inhibit its regulation of target genes. Increasing evidence has underscored the involvement of dysregulated ceRNA networks in the occurrence and progression of colorectal cancer (CRC). The purpose of this study was to construct a ceRNA network related to the prognosis of CRC and further explore the potential mechanisms that affect this prognosis. METHODS: RNA-Seq and miRNA-Seq data from The Cancer Genome Atlas (TCGA) were used to identify differentially expressed lncRNAs (DElncRNAs), microRNAs (DEmiRNAs), and mRNAs (DEmRNAs), and a prognosis-related ceRNA network was constructed based on DElncRNA survival analysis. Subsequently, pathway enrichment, Pearson correlation, and Gene Set Enrichment Analysis (GSEA) were performed to determine the function of the genes in the ceRNA network. Gene Expression Profiling Interactive Analysis (GEPIA) and immunohistochemistry (IHC) were also used to validate differential gene expression. Finally, the correlation between lncRNA and immune cell infiltration in the tumor microenvironment was evaluated based on the CIBERSORT algorithm. RESULTS: A prognostic ceRNA network was constructed with eleven key survival-related DElncRNAs (MIR4435-2HG, NKILA, AFAP1-AS1, ELFN1-AS1, AC005520.2, AC245884.8, AL354836.1, AL355987.4, AL591845.1, LINC02038, and AC104823.1), 54 DEmiRNAs, and 308 DEmRNAs. The MIR4435-2HG- and ELFN1-AS1-associated ceRNA subnetworks affected and regulated the expression of the COL5A2, LOX, OSBPL3, PLAU, VCAN, SRM, and E2F1 target genes and were found to be related to prognosis and tumor-infiltrating immune cell types. CONCLUSIONS: MIR4435-2HG and ELFN1-AS1 are associated with prognosis and tumor-infiltrating immune cell types and could represent potential prognostic biomarkers or therapeutic targets in colorectal carcinoma.

Identification of circRNA-miRNA-mRNA Regulatory Network in Bladder Cancer by Integrated Analysis.

INTRODUCTION: Bladder cancer (BC) is a common malignant tumor in the urinary system with high mortality and recurrence rates. This study sought to identify crucial circular RNAs (circRNAs) associated with BC. METHODS: The mRNA, miRNA, and circRNA expression profiles of BC were downloaded from GEO database. The differentially expressed mRNAs (DEmRNAs), miRNAs (DEmiRNAs), and circRNAs (DEcircRNAs) were identified using bioinformatics method. Combining circRNA-miRNA pairs with miRNA-mRNA pairs, the competing endogenous RNA (ceRNA; DEcircRNA-DEmi-RNA-DEmRNA) regulatory network was constructed. Functional annotation of host gene of DEcircRNAs and DEmRNAs in ceRNA regulatory network were performed. qRT-PCR validation was performed. RESULTS: A total of 4,003 DEmRNAs, 25 DEmiRNAs, and 119 DEcircRNAs were obtained. The ceRNA network contained 18 circRNA-miRNA pairs and 699 mi-RNA-mRNA pairs, including 17 circRNAs, 4 miRNAs, and 624 mRNAs. Functional annotation of DEmRNAs in ceRNA regulatory network revealed that these DEmRNAs were significantly enriched in glycerolipid metabolism, p53 signaling pathway, and oocyte meiosis. Except for hsa_circ_0028173, expression of the others in the qRT-PCR results was consistent with that in our integrated analysis, generally. CONCLUSION: We speculate that hsa_circ_0008035/hsa-miR-107/MSRB3 and hsa_circ_0028173/hsa-miR-338-3p/TPX2/GATA3 interaction pairs may play a vital role in BC.

Dissecting MicroRNA-mRNA Regulatory Networks Underlying Sulfur Assimilation and Cadmium Accumulation in Poplar Leaves.

The process of cadmium (Cd) accumulation and detoxification under different sulfur levels remains largely unknown in woody plants. To investigate the physiological and transcriptomic regulation mechanisms of poplars in response to different sulfate (S) supply levels and Cd exposure, we exposed Populus deltoides saplings to one of the low, moderate and high S levels together with either 0 or 50 µM Cd. Cd accumulation was decreased in low S-treated poplar leaves, and it tended to be increased in high S-supplied leaves under the Cd exposure condition. Sulfur nutrition was deficient in low S-supplied poplars, and it was improved in high S-treated leaves. Cd exposure resulted in lower sulfur level in the leaves supplied with moderate S, it exacerbated a Cd-induced sulfur decrease in low S-treated leaves and it caused a higher sulfur concentration in high S-supplied leaves. In line with the physiological changes, a number of mRNAs and microRNAs (miRNAs) involved in Cd accumulation and sulfur assimilation were identified and the miRNA-mRNA networks were dissected. In the networks, miR395 and miR399 members were identified as hub miRNAs and their targets were ATP sulfurylase 3 (ATPS3) and phosphate 2 (PHO2), respectively. These results suggest that Cd accumulation and sulfur assimilation are constrained by low and enhanced by high S supply, and Cd toxicity is aggravated by low and relieved by high S in poplar leaves, and that miRNA-mRNA regulatory networks play pivotal roles in sulfur-mediated Cd accumulation and detoxification in Cd-exposed poplars.

Post-transcriptional control of cytokine production.

The cytokine-encoding messenger RNA (mRNA) molecules transcribed in the nucleus acquire a protein coat that facilitates nuclear export, influences cytoplasmic localization, and determines stability and translational competence. The composition of this coat is determined by sequence elements that recruit proteins that influence the rate of translation and/or mRNA decay. Some of these regulatory proteins direct their associated mRNA molecules to discrete cytoplasmic foci (stress granules and processing bodies) that are essential in 'programming' mRNA 'metabolism'. Studies have begun to identify how these various mechanisms are integrated and regulated to determine the amount of cytokine production in cells involved in immune responses. Understanding of these mechanisms has identified targets for the development of new classes of immunomodulatory drugs.

Intragenic enhancers act as alternative promoters.

A substantial amount of organismal complexity is thought to be encoded by enhancers which specify the location, timing, and levels of gene expression. In mammals there are more enhancers than promoters which are distributed both between and within genes. Here we show that activated, intragenic enhancers frequently act as alternative tissue-specific promoters producing a class of abundant, spliced, multiexonic poly(A)(+) RNAs (meRNAs) which reflect the host gene's structure. meRNAs make a substantial and unanticipated contribution to the complexity of the transcriptome, appearing as alternative isoforms of the host gene. The low protein-coding potential of meRNAs suggests that many meRNAs may be byproducts of enhancer activation or underlie as-yet-unidentified RNA-encoded functions. Distinguishing between meRNAs and mRNAs will transform our interpretation of dynamic changes in transcription both at the level of individual genes and of the genome as a whole.

Intercellular mRNA trafficking via membrane nanotube-like extensions in mammalian cells.

RNAs have been shown to undergo transfer between mammalian cells, although the mechanism behind this phenomenon and its overall importance to cell physiology is not well understood. Numerous publications have suggested that RNAs (microRNAs and incomplete mRNAs) undergo transfer via extracellular vesicles (e.g., exosomes). However, in contrast to a diffusion-based transfer mechanism, we find that full-length mRNAs undergo direct cell-cell transfer via cytoplasmic extensions characteristic of membrane nanotubes (mNTs), which connect donor and acceptor cells. By employing a simple coculture experimental model and using single-molecule imaging, we provide quantitative data showing that mRNAs are transferred between cells in contact. Examples of mRNAs that undergo transfer include those encoding GFP, mouse ß-actin, and human Cyclin D1, BRCA1, MT2A, and HER2. We show that intercellular mRNA transfer occurs in all coculture models tested (e.g., between primary cells, immortalized cells, and in cocultures of immortalized human and murine cells). Rapid mRNA transfer is dependent upon actin but is independent of de novo protein synthesis and is modulated by stress conditions and gene-expression levels. Hence, this work supports the hypothesis that full-length mRNAs undergo transfer between cells through a refined structural connection. Importantly, unlike the transfer of miRNA or RNA fragments, this process of communication transfers genetic information that could potentially alter the acceptor cell proteome. This phenomenon may prove important for the proper development and functioning of tissues as well as for host-parasite or symbiotic interactions.

Emerging Role for Linear and Circular Spermine Oxidase RNAs in Skeletal Muscle Physiopathology.

Skeletal muscle atrophy is a pathological condition so far without effective treatment and poorly understood at a molecular level. Emerging evidence suggest a key role for circular RNAs (circRNA) during myogenesis and their deregulation has been reported to be associated with muscle diseases. Spermine oxidase (SMOX), a polyamine catabolic enzyme plays a critical role in muscle differentiation and the existence of a circRNA arising from SMOX gene has been recently identified. In this study, we evaluated the expression profile of circular and linear SMOX in both C2C12 differentiation and dexamethasone-induced myotubes atrophy. To validate our findings in vivo their expression levels were also tested in two murine models of amyotrophic lateral sclerosis: SOD1G93A and hFUS+/+, characterized by progressive muscle atrophy. During C2C12 differentiation, linear and circular SMOX show the same trend of expression. Interestingly, in atrophy circSMOX levels significantly increased compared to the physiological state, in both in vitro and in vivo models. Our study demonstrates that SMOX represents a new player in muscle physiopathology and provides a scientific basis for further investigation on circSMOX RNA as a possible new therapeutic target for the treatment of muscle atrophy.

Assembly of Proteins by Free RNA during the Early Phase of Proteostasis Stress.

At any stage of their lifecycle, mRNAs are coated by specialized proteins. One of few circumstances when free mRNA appears in the cytosol is the disassembly of polysomes during the stress-induced shutdown of protein synthesis. Using quantitative mass spectrometry, we sought to identify the free RNA-interacting cellular machinery in heat-shocked mammalian cells. Free RNA-associated proteins displayed higher disorder and larger size, which supports the role of multivalent interactions during the initial phase of the association with RNAs during stress. Structural features of the free RNA interactors defined them as a subset of RNA-binding proteins. The interaction between these assembled proteins in vivo required RNA. Reconstitution of the association process in vitro indicated a multimolecular basis for increased binding to RNA upon heat shock in the cytosol. Our study represents a step toward understanding how free RNA is processed in the cytosol during proteostasis stress.

Synchronous profiling and analysis of mRNAs and ncRNAs in the dermal papilla cells from cashmere goats.

BACKGROUND: Dermal papilla cells (DPCs), the "signaling center" of hair follicle (HF), delicately master continual growth of hair in mammals including cashmere, the fine fiber annually produced by secondary HF embedded in cashmere goat skins. Such unparalleled capacity bases on their exquisite character in instructing the cellular activity of hair-forming keratinocytes via secreting numerous molecular signals. Past studies suggested microRNA (miRNAs) and long non-coding RNAs (lncRNAs) play essential roles in a wide variety of biological process, including HF cycling. However, their roles and related molecular mechanisms in modulating DPCs secretory activities are still poorly understood. RESULTS: Here, we separately cultivated DPCs and their functionally and morphologically distinct dermal fibroblasts (DFs) from cashmere goat skins at anagen. With the advantage of high throughput RNA-seq, we synchronously identified 2540 lncRNAs and 536 miRNAs from two types of cellular samples at 4th passages. Compared with DFs, 1286 mRNAs, 18 lncRNAs, and 42 miRNAs were upregulated, while 1254 mRNAs, 53 lncRNAs and 44 miRNAs were downregulated in DPCs. Through overlapping with mice data, we ultimately defined 25 core signatures of DPCs, including HOXC8 and RSPO1, two crucial activators for hair follicle stem cells (HFSCs). Subsequently, we emphatically investigated the impacts of miRNAs and lncRNAs (cis- and trans- acting) on the genes, indicating that ncRNAs extensively exert negative and positive effects on their expressions. Furthermore, we screened lncRNAs acting as competing endogenous RNAs (ceRNAs) to sponge miRNAs and relief their repressive effects on targeted genes, and constructed related lncRNAs-miRNAs-HOXC8/RSPO1 interactive lines using bioinformatic tools. As a result, XR_310320.3-chi-miR-144-5p-HOXC8, XR_311077.2-novel_624-RSPO1 and others lines appeared, displaying that lncRNAs might serve as ceRNAs to indirectly adjust HFSCs status in hair growth. CONCLUSION: The present study provides an unprecedented inventory of lncRNAs, miRNAs and mRNAs in goat DPCs and DFs. We also exhibit some miRNAs and lncRNAs potentially participate in the modulation of HFSCs activation via delicately adjusting core signatures of DPCs. Our report shines new light on the latent roles and underlying molecular mechanisms of ncRNAs on hair growth.

Development of insulin resistance in Nischarin mutant female mice.

BACKGROUND/OBJECTIVES: NISCH-STAB1 is a newly identified locus correlated to human waist-hip ratio (WHR), which is a risk indicator of developing obesity-associated diabetes. Our previous studies have shown that Nisch mutant male mice increased glucose tolerance in chow-fed conditions. Thus we hypothesized that Nisch mutant mice will have changes in insulin resistance, adipocytes, hepatic steatosis when mice are fed with high-fat diet (HFD). METHODS: Insulin resistance was assessed in Nisch mutant mice and WT mice fed with high-fat diet (60% by kCal) or chow diet. Whole-body energy metabolism was examined using an indirect calorimeter. Adipose depots including inguinal white adipose tissue (WAT), perigonadal WAT, retroperitoneal WAT, and mesenteric WAT were extracted. Area and eqdiameter of each adipocyte were determined, and insulin signaling was examined as well. Paired samples of subcutaneous and omental visceral adipose tissue were obtained from 400 individuals (267 women, 133 men), and examined the expression of Nischarin. RESULTS: We found that insulin signaling was impaired in major insulin-sensitive tissues of Nisch mutant female mice. When mice were fed with HFD for 15 weeks, the Nisch mutant female mice not only developed severe insulin resistance and decreased glucose tolerance compared with wild-type control mice, but also accumulated more white fat, had larger adipocytes and developed severe hepatic steatosis than wild-type control mice. To link our animal studies to human diseases, we further analyzed Nischarin expression in the paired human samples of visceral and subcutaneous adipose tissue from Caucasians. In humans, we found that Nischarin expression is attenuated in adipose tissue with obesity. More importantly, we found that Nischarin mRNA inversely correlated with parameters of obesity, fat distribution, lipid and glucose metabolism. CONCLUSIONS: Taken together, our data revealed sexual dimorphism of Nischarin in body fat distribution, insulin resistance, and glucose tolerance in mice.

Induction of an IFN-Mediated Antiviral Response by a Self-Amplifying RNA Vaccine: Implications for Vaccine Design.

RNA-based vaccines have recently emerged as a promising alternative to the use of DNA-based and viral vector vaccines, in part because of the potential to simplify how vaccines are made and facilitate a rapid response to newly emerging infections. SAM vaccines are based on engineered self-amplifying mRNA (SAM) replicons encoding an Ag, and formulated with a synthetic delivery system, and they induce broad-based immune responses in preclinical animal models. In our study, in vivo imaging shows that after the immunization, SAM Ag expression has an initial gradual increase. Gene expression profiling in injection-site tissues from mice immunized with SAM-based vaccine revealed an early and robust induction of type I IFN and IFN-stimulated responses at the site of injection, concurrent with the preliminary reduced SAM Ag expression. This SAM vaccine-induced type I IFN response has the potential to provide an adjuvant effect on vaccine potency, or, conversely, it might establish a temporary state that limits the initial SAM-encoded Ag expression. To determine the role of the early type I IFN response, SAM vaccines were evaluated in IFN receptor knockout mice. Our data indicate that minimizing the early type I IFN responses may be a useful strategy to increase primary SAM expression and the resulting vaccine potency. RNA sequence modification, delivery optimization, or concurrent use of appropriate compounds might be some of the strategies to finalize this aim.