Small Upstream Open Reading Frame Research Articles

Translational regulation enhances distinction of cell types in the nervous system.

Multicellular organisms are composed of specialized cell types with distinct proteomes. While recent advances in single-cell transcriptome analyses have revealed differential expression of mRNAs, cellular diversity in translational profiles remains underinvestigated. By performing RNA-seq and Ribo-seq in genetically defined cells in the Drosophila brain, we here revealed substantial post-transcriptional regulations that augment the cell-type distinctions at the level of protein expression. Specifically, we found that translational efficiency of proteins fundamental to neuronal functions, such as ion channels and neurotransmitter receptors, was maintained low in glia, leading to their preferential translation in neurons. Notably, distribution of ribosome footprints on these mRNAs exhibited a remarkable bias toward the 5' leaders in glia. Using transgenic reporter strains, we provide evidence that the small upstream open-reading frames in the 5' leader confer selective translational suppression in glia. Overall, these findings underscore the profound impact of translational regulation in shaping the proteomics for cell-type distinction and provide new insights into the molecular mechanisms driving cell-type diversity.

Translational regulation enhances distinction of cell types in the nervous system

Multicellular organisms are composed of specialized cell types with distinct proteomes. While recent advances in single-cell transcriptome analyses have revealed differential expression of mRNAs, cellular diversity in translational profiles remains underinvestigated. By performing RNA-seq and Ribo-seq in genetically defined cells in the Drosophila brain, we here revealed substantial post-transcriptional regulations that augment the cell-type distinctions at the level of protein expression. Specifically, we found that translational efficiency of proteins fundamental to neuronal functions, such as ion channels and neurotransmitter receptors, was maintained low in glia, leading to their preferential translation in neurons. Notably, distribution of ribosome footprints on these mRNAs exhibited a remarkable bias toward the 5′ leaders in glia. Using transgenic reporter strains, we provide evidence that the small upstream open-reading frames in the 5’ leader confer selective translational suppression in glia. Overall, these findings underscore the profound impact of translational regulation in shaping the proteomics for cell-type distinction and provide new insights into the molecular mechanisms driving cell-type diversity.

Refinement of Leishmania donovani Genome Annotations in the Light of Ribosome-Protected mRNAs Fragments (Ribo-Seq Data).

Advances in next-generation sequencing methodologies have facilitated the assembly of an ever-increasing number of genomes. Gene annotations are typically conducted via specialized software, but the most accurate results require additional manual curation that incorporates insights derived from functional and bioinformatic analyses (e.g., transcriptomics, proteomics, and phylogenetics). In this study, we improved the annotation of the Leishmania donovani (strain HU3) genome using publicly available data from the deep sequencing of ribosome-protected mRNA fragments (Ribo-Seq). As a result of this analysis, we uncovered 70 previously non-annotated protein-coding genes and improved the annotation of around 600 genes. Additionally, we present evidence for small upstream open reading frames (uORFs) in a significant number of transcripts, indicating their potential role in the translational regulation of gene expression. The bioinformatics pipelines developed for these analyses can be used to improve the genome annotations of other organisms for which Ribo-Seq data are available. The improvements provided by these studies will bring us closer to the ultimate goal of a complete and accurately annotated L. donovani genome and will enhance future transcriptomics, proteomics, and genetics studies.

Translational control of E2f1 regulates the Drosophila cell cycle

E2F transcription factors are master regulators of the eukaryotic cell cycle. In Drosophila, the sole activating E2F, E2F1, is both required for and sufficient to promote G1→S progression. E2F1 activity is regulated both by binding to RB Family repressors and by posttranscriptional control of E2F1 protein levels by the EGFR and TOR signaling pathways. Here, we investigate cis-regulatory elements in the E2f1 messenger RNA (mRNA) that enable E2f1 translation to respond to these signals and promote mitotic proliferation of wing imaginal disc and intestinal stem cells. We show that small upstream open reading frames (uORFs) in the 5' untranslated region (UTR) of the E2f1 mRNA limit its translation, impacting rates of cell proliferation. E2f1 transgenes lacking these 5'UTR uORFs caused TOR-independent expression and excess cell proliferation, suggesting that TOR activity can bypass uORF-mediated translational repression. EGFR signaling also enhanced translation but through a mechanism less dependent on 5'UTR uORFs. Further, we mapped a region in the E2f1 mRNA that contains a translational enhancer, which may also be targeted by TOR signaling. This study reveals translational control mechanisms through which growth signaling regulates cell cycle progression.

Probabilistic models of uORF-mediated ATF4 translation control

ATF4 is a key transcription factor that activates transcription of genes needed to respond to cellular stress. Although the mRNA encoding ATF4 is present at constant levels in the cell during the initial response, translation of ATF4 increases under conditions of cellular stress while the global translation rate decreases. We study two models for the control system that regulates the translation of ATF4, both based on the Vattem-Wek hypothesis. This hypothesis is based on a race to reload, following the translation of a small upstream open reading frame (uORF), the ternary complex that brings the initiator tRNA to the ribosome as the 40S subunit scans along the mRNA, encountering first a start codon for an inhibitory uORF whose reading frame overlaps the start of the ATF4 coding sequence. We develop a pair of simple, analytic, probabilistic models, one of which assumes all nucleotide triplets have identical kinetic properties, while the other recognizes the existence of triplets at which the ternary complex loads more efficiently. We also consider two different functions representing the dependence of the rate of initiation at uORF1 on the ternary complex concentration. In keeping with the theme of this Special Issue, we studied the properties of these models in a Maple document, which can easily be modified to consider different parameters, translation rate initiation functions, and so on.

Translation of GGC repeat expansions into a toxic polyglycine protein in NIID defines a novel class of human genetic disorders: The polyG diseases

SummaryNeuronal intranuclear inclusion disease (NIID) is a neurodegenerative disease characterized by the presence of intranuclear inclusions of unknown origin. NIID is caused by an expansion of GGC repeats in the 5′ UTR of the NOTCH2NLC (N2C) gene. We found that these repeats are embedded in a small upstream open reading frame (uORF) (uN2C), resulting in their translation into a polyglycine-containing protein, uN2CpolyG. This protein accumulates in intranuclear inclusions in cell and mouse models and in tissue samples of individuals with NIID. Furthermore, expression of uN2CpolyG in mice leads to locomotor alterations, neuronal cell loss, and premature death of the animals. These results suggest that translation of expanded GGC repeats into a novel and pathogenic polyglycine-containing protein underlies the presence of intranuclear inclusions and neurodegeneration in NIID.

Alternative transcription start site selection in Mr-OPY2 controls lifestyle transitions in the fungus Metarhizium robertsii

Metarhizium robertsii is a versatile fungus with saprophytic, plant symbiotic and insect pathogenic lifestyle options. Here we show that M. robertsii mediates the saprophyte-to-insect pathogen transition through modulation of the expression of a membrane protein, Mr-OPY2. Abundant Mr-OPY2 protein initiates appressorium formation, a prerequisite for infection, whereas reduced production of Mr-OPY2 elicits saprophytic growth and conidiation. The precise regulation of Mr-OPY2 protein production is achieved via alternative transcription start sites. During saprophytic growth, a single long transcript is produced with small upstream open reading frames in its 5′ untranslated region. Increased production of Mr-OPY2 protein on host cuticle is achieved by expression of a transcript variant lacking a small upstream open reading frame that would otherwise inhibit translation of Mr-OPY2. RNA-seq and qRT-PCR analyses show that Mr-OPY2 is a negative regulator of a transcription factor that we demonstrate is necessary for appressorial formation. These findings provide insights into the mechanisms regulating fungal lifestyle transitions.

Differential regulation of hepatitis B virus core protein expression and genome replication by a small upstream open reading frame and naturally occurring mutations in the precore region

Hepatitis B virus (HBV) transcribes two subsets of 3.5-kb RNAs: precore RNA for hepatitis B e antigen (HBeAg) expression, and pregenomic RNA for core and P protein translation as well as genome replication. HBeAg expression could be prevented by mutations in the precore region, while an upstream open reading frame (uORF) has been proposed as a negative regulator of core protein translation. We employed replication competent HBV DNA constructs and transient transfection experiments in Huh7 cells to verify the uORF effect and to explore the alternative function of precore RNA. Optimized Kozak sequence for the uORF or extra ATG codons as present in some HBV genotypes reduced core protein expression. G1896A nonsense mutation promoted more efficient core protein expression than mutated precore ATG, while a +1 frameshift mutation was ineffective. In conclusion, various HBeAg-negative precore mutations and mutations affecting uORF differentially regulate core protein expression and genome replication.

Molecular cloning and characterization of S-adenosylmethionine decarboxylase gene in rubber tree (Hevea brasiliensis).

S-Adenosylmethionine decarboxylase (SAMDC) is a key rate-limiting enzyme involved in polyamines biosynthesis, and it plays important roles in plant growth, development and stresses response. However, no SAMDC gene was reported in rubber tree. Here we report characteristics of an SAMDC gene (HbSAMDC1) in rubber tree. HbSAMDC1 contains a 1080bp open reading frame (ORF) encoding 359 amino acids. Quantitative real-time PCR analyses revealed that HbSAMDC1 exhibited distinct expression patterns in different tissues and was regulated by various stresses, including drought, cold, salt, wounding, and H2O2 treatments. HbSAMDC1 5' untranslated region (UTR) contains a highly conserved overlapping tiny and small upstream ORFs (uORFs), encoding 2 and 52 amino acid residues, respectively. No introns were located in the main ORF of HbSAMDC1, whereas two introns were found in the 5' UTR. In transgenic tobaccos, the highly conserved small uORF of HbSAMDC1 is found to be responsible for translational repression of downstream β-glucuronidase reporter. To our knowledge, this is the first report on molecular cloning, expression profiles, and 5' UTR characteristics of HbSAMDC1. These results lay solid foundation for further elucidating HbSAMDC1 function in rubber tree.

Arabidopsis Polyamine oxidase-2 uORF is required for downstream translational regulation

In eukaryotic mRNAs, small upstream open reading frames (uORFs) located in the 5′-untranslated region control the translation of the downstream main ORF. Polyamine oxidase (PAO) enzymes catalyze the oxidation of higher polyamines such as spermidine and spermine, and therefore contribute to the maintenance of intracellular polyamine content and to the regulation of physiological processes through their catabolic products. Recently, we reported that the Arabidopsis thaliana Polyamine Oxidase 2 (AtPAO2) is post-transcriptionally regulated by its 5′-UTR region through an uORF. In the present study, we analyzed whether the translation of the uORF is needed for the translational repression of the main ORF, and whether the inactivation of the uORF had an effect on the translational control mediated by polyamines. To this aim, we generated diverse single mutations in the uORF sequence; these mutant 5′-UTRs were fused to the GUS reporter gene, and tested in onion monolayer cells and A. thaliana transgenic seedlings. Removal of the start codon or introduction of a premature stop codon in the AtPAO2 uORF sequence abolished the negative regulation of the GUS expression exerted by the wild-type AtPAO2 uORF. An artificial uORF (32 amino acids in length) generated by the addition of a single nucleotide in AtPAO2 uORF proved to be less repressive than the wild-type uORF. Thus, our findings suggest that translation of the AtPAO2 uORF is necessary for the translational repression of the main ORF.

Diversity in boron toxicity tolerance of Australian barley (Hordeum vulgare L.) genotypes.

BackgroundBoron (B) is an important micronutrient for plant growth, but is toxic when levels are too high. This commonly occurs in environments with alkaline soils and relatively low rainfall, including many of the cereal growing regions of southern Australia. Four major genetic loci controlling tolerance to high soil B have been identified in the landrace barley, Sahara 3771. Genes underlying two of the loci encode the B transporters HvBot1 and HvNIP2;1.ResultsWe investigated sequence and expression level diversity in HvBot1 and HvNIP2;1 across barley germplasm, and identified five novel coding sequence alleles for HvBot1. Lines were identified containing either single or multiple copies of the Sahara HvBot1 allele. We established that only the tandemly duplicated Sahara allele conferred B tolerance, and this duplicated allele was found only in a set of nine lines accessioned in Australian collections as Sahara 3763–3771. HvNIP2;1 coding sequences were highly conserved across barley germplasm. We identified the likely causative SNP in the 5’UTR of Sahara HvNIP2;1, and propose that the creation of a small upstream open reading frame interferes with HvNIP2;1 translation in Sahara 3771. Similar to HvBot1, the tolerant HvNIP2;1 allele was unique to the Sahara barley accessions. We identified a new source of the 2H B tolerance allele controlling leaf symptom development, in the landrace Ethiopia 756.ConclusionsEthiopia 756, as well as the cultivar Sloop Vic which carries both the 2H and HvBot1 B tolerance alleles derived from Sahara 3771, may be valuable as alternative parents in breeding programs targeted to high soil B environments. There is significant diversity in B toxicity tolerance among contemporary Australian barley varieties but this is not related to variation at any of the four known B tolerance loci, indicating that novel, as yet undiscovered, sources of tolerance exist.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0607-1) contains supplementary material, which is available to authorized users.

Cellular interference in craniofrontonasal syndrome: males mosaic for mutations in the X-linked EFNB1 gene are more severely affected than true hemizygotes

Craniofrontonasal syndrome (CFNS), an X-linked disorder caused by loss-of-function mutations of EFNB1, exhibits a paradoxical sex reversal in phenotypic severity: females characteristically have frontonasal dysplasia, craniosynostosis and additional minor malformations, but males are usually more mildly affected with hypertelorism as the only feature. X-inactivation is proposed to explain the more severe outcome in heterozygous females, as this leads to functional mosaicism for cells with differing expression of EPHRIN-B1, generating abnormal tissue boundaries—a process that cannot occur in hemizygous males. Apparently challenging this model, males occasionally present with a more severe female-like CFNS phenotype. We hypothesized that such individuals might be mosaic for EFNB1 mutations and investigated this possibility in multiple tissue samples from six sporadically presenting males. Using denaturing high performance liquid chromatography, massively parallel sequencing and multiplex-ligation-dependent probe amplification (MLPA) to increase sensitivity above standard dideoxy sequencing, we identified mosaic mutations of EFNB1 in all cases, comprising three missense changes, two gene deletions and a novel point mutation within the 5′ untranslated region (UTR). Quantification by Pyrosequencing and MLPA demonstrated levels of mutant cells between 15 and 69%. The 5′ UTR variant mutates the stop codon of a small upstream open reading frame that, using a dual-luciferase reporter construct, was demonstrated to exacerbate interference with translation of the wild-type protein. These results demonstrate a more severe outcome in mosaic than in constitutionally deficient males in an X-linked dominant disorder and provide further support for the cellular interference mechanism, normally related to X-inactivation in females.

Cell-cycle and DNA damage regulation of the DNA mismatch repair protein Msh2 occurs at the transcriptional and post-transcriptional level

DNA mismatch repair during replication is a conserved process essential for maintaining genomic stability. Mismatch repair is also implicated in cell-cycle arrest and apoptosis after DNA damage. Because yeast and human mismatch repair systems are well conserved, we have employed the budding yeast Saccharomyces cerevisiae to understand the regulation and function of the mismatch repair gene MSH2. Using a luciferase-based transcriptional reporter, we defined a 218-bp region upstream of MSH2 that contains cell-cycle and DNA damage responsive elements. The 5′ end of the MSH2 transcript was mapped by primer extension and was found to encode a small upstream open reading frame (uORF). Mutagenesis of the uORF start codon or of the uORF stop codon, which creates a continuous reading frame with MSH2, increased Msh2 steady-state protein levels ∼2-fold. Furthermore, we found that the cell-cycle transcription factors Swi6, Swi4, and Mbp1—along with SCB/MCB cell-cycle binding sites upstream of MSH2—are all required for full basal expression of MSH2. Mutagenesis of the cell-cycle boxes resulted in a minor reduction in basal Msh2 levels and a 3-fold defect in mismatch repair. Disruption of the cell-cycle boxes also affected growth in a DNA polymerase-defective strain background where mismatch repair is essential, particularly in the presence of the DNA damaging agent methyl methane sulfonate (MMS). Promoter replacements conferring constitutive expression of MSH2 revealed that the transcriptional induction in response to MMS is required to maintain induced levels of Msh2. Turnover experiments confirmed an elevated rate of degradation in the presence of MMS. Taken together, the data show that the DNA damage regulation of Msh2 occurs at the transcriptional and post-transcriptional levels. The transcriptional and translational control elements identified are conserved in mammalian cells, underscoring the use of yeast as a model system to examine the regulation of MSH2.

An AUG codon upstream of rev and env open reading frames ensures optimal translation of the simian immunodeficiency virus Env protein

The mRNAs encoding the Rev and Env proteins of simian immunodeficiency virus (SIV) are unique because upstream translation start codons are present that may modulate the expression of these viral proteins. We previously reported the regulatory effect of a small upstream open reading frame (ORF) on Rev and Env translation. Here we study this mechanism in further detail by modulating the strength of the translation signals upstream of the open reading frames in subgenomic reporters. Furthermore, the effects of these mutations on SIV gene expression and viral replication are analyzed. An intricate regulatory mechanism is disclosed that allows the virus to express a balanced amount of these two proteins.

Regulation of tumor suppressor gene FUS1 expression by the untranslated regions of mRNA in human lung cancer cells

FUS1, also known as tumor suppressor candidate 2 (TUSC2), is a tumor suppressor gene located in the human chromosome 3p21.3 region. FUS1 mRNA transcripts could be detected on Northern blots in both normal lung and some lung cancer cell lines, but no endogenous FUS1 protein could be detected in a majority of lung cancer cell lines and small cell and non-small cell lung tumor tissues. However, mechanisms regulating FUS1 protein expression and its inactivation in primary lung cancer cells are largely unknown. In this study, we investigated the role of the 5′- and 3′-untranslated regions (UTRs) of the FUS1 gene transcript in the regulation of FUS1 protein expression. We identified RNA sequence elements in FUS1 UTRs that regulate FUS1 protein expression. We found that two small upstream open-reading frames in the 5′UTR of FUS1 mRNA could inhibit the translational initiation of FUS1 protein by interfering with the “scanning” of the ribosome initiation complexes. Several secondary RNA structural elements/motifs on the 3′UTR of FUS1 also exhibited a significant inhibitory effect on FUS1 protein expression. The 3′UTR-mediated regulatory effect on FUS1 protein expression was also differentially detected in normal lung epithelial and fibroblast cells compared with lung cancer cells. Our results provide new insight into the molecular mechanisms involved in the regulation of FUS1 expression.

S-Adenosylmethionine decarboxylase 유전자의 upstream open reading frame이 in vivo에서 translational inhibitor 로서의 작용 기작

SAMDC는 폴리아민 생합성 과정에서 주효소로 작용하며 항상성을 유지하기위해 정교하게 조절된다. 카네이션 SAMDC 유전자는 5'-leader sequence에 54개 아미노산으로 구성된 small uORF가 존재한다. Translation 과정을 조절하는 uORF의 작용기작을 연구하기 위하여 35S 프로모터에 SAMDC 유전자의 uORF 부위와 GUS 유전자를 재조합한 형질전환 담배 식물체를 이용하였다. 본 실험에서는 SAMDC uORF 염기서열 혹은 SAMDC uORF 단백질에 의해서 downstream GUS ORF의 translation이 억제되었다. 특히 translation 억제는 개시코돈이 point-mutation된 construct에서 효과적으로 이루어졌다. 따라서 이러한 결과는 ribosomal stalling이 translation 억제 과정에 관여한 것으로 사료된다. 개시 코돈과 종결코돈을 가진 SAMDC uORF의 아미노산 서열을 frame shift 시키면 GUS 활성이 증가하였는데 이는 translation inhibitor로서 작용할 때 아미노산 서열이 중요하다는 것을 의미하며, 결국은 SAMDC uORF의 단백질 구조가 중요하게 작용할 가능성을 제시한다. 또한 유식물과 담배 꽃 등의 in vivo 상에서도 GUS 발현을 조직화학적으로 분석했을 때 small uORF가 존재할 경우 GUS 염색이 크게 저하되었지만, 개시코돈이나 혹은 종결코돈이 제거되도록 point-mutation 시킨 construct가 도입된 형질전환식물체에서는 SAMDC uORF의 억제효과가 크게 완화 되었다. 또한 가장 중요한 관찰 결과로는 small uORF 염기서열로부터 in vitro 시스템에서 5.7 kDa의 단백질이 실제적으로 합성되었음을 관찰하였다. 폴리아민 처리 후 GUS 단백질이 억제된 결과는 uORF로부터 합성된 단백질이 폴리아민 뿐 만 아니라 translation 과정에 관여하는 다른 요소들과 상호작용을 이루어 조절될 수 있음을 암시한다. S-Adenosylmethionine decarboxylase (SAMDC; EC 4.1.4.50), a key enzyme for polyamines biosynthesis, was tightly regulated for homeostatic levels. Carnation SAMDC gene (CSDC9) has an small upstream open reading frame (uORF) of 54 amino acids in 5'-leader sequence. To explore the functional mechanism of uORFs in controlling translation, we used a GUS reporter gene driven with the 35S promoter and uORF region of SAMDC gene for making transgenic tobacco plants. In our experiment, there were a translational inhibition of its downstream GUS ORF by SAMDC uORF sequence or SAMDC uORF protein. Expecially, translational inhibition was most effective in point-mutated construct, in which the start codon was changed. Therefore, this results suggested the ribosomal stalling might be involved in this translational inhibitory process. The frame shift in amino acid sequence of SAMDC uORF with start codon and stop codon resulted in a moderate increasing in GUS activity, suggesting the native amino acid sequence was important for a function as a translational inhibitor. Also, we showed that the production of GUS protein was significantly inhibited in the presence of the small uORF using histochemical analysis of GUS expression in seedlings and tobacco flowers. Importantly, the small uORF sequence induced a real peptide of 5.7 kDa, which was provided the presence of SAMDC uORF peptide band using an in vitro transcription/translation system. The peptide product of uORF might interact with other components of translational machinery as well as polyamines, which was resulted from that polyamine treatment was inhibited GUS protein band in SDS-PAGE experiment.

Interesting twists on small RNA themes in Pseudomonas aeruginosa

While many of the initial studies of regulatory small RNA (sRNA) function focused on Escherichia coli and Salmonella, there is expanding characterization of sRNAs in other bacteria. These studies are revealing unique permutations of the themes found in E. coli and Salmonella. This point is nicely illustrated by the Pseudomonas aeruginosa Hfq-binding RNA, PhrS, which activates the synthesis of the key quorum sensing transcription regulator PqsR as described by Sonnleitner et al. in the current issue of Molecular Microbiology. The PhrS RNA combines features of several previously characterized sRNAs; its expression is induced by low oxygen, it encodes a small protein, and it modulates the expression of a quorum sensing regulator by preventing the formation of an inhibitory secondary structure. However, in contrast to other base pairing sRNAs that activate translation, PhrS does not act directly on pqsR but rather increases the translation of a small upstream open reading frame (uof) whose translation is coupled to pqsR.

Abstract 3083: Regulation of novel tumor suppressor gene FUS1 expression through 5’- and 3’-UTRs and in response to DNA damage in human lung cancer cells

Abstract FUS1 is a novel tumor suppressor gene located in the human chromosome 3p21.3 region. FUS1 mRNA transcripts could be detected on Northern blots in both normal lung and some lung cancer cell lines, but no endogenous FUS1 protein could be detected in a majority of lung cancer cell lines. Loss or reduction of FUS1 expression was observed in all small cell lung cancer and in more than 70 % of non-small cell lung carcinomas and was associated with significantly worse overall patient survival. However, mechanisms regulating FUS1 protein expression and its inactivation in primary lung cancer cells are largely unknown. In this study, we investigated the role of the 5’- and 3’-untranslated regions (UTRs) of the FUS1 gene in the regulation of FUS1 protein expression and determined changes in FUS1 gene and protein expression in response to oncogenic stress in normal lung and cancer cell lines. We constructed a series of expression plasmids containing the FUS1 coding region with or without the intrinsic UTRs and transfected them into normal lung bronchial epithelial and tumor cells to assess their roles in regulation of FUS1 protein expression. We identified some critical structure elements in FUS1 UTRs that could significantly regulate FUS1 protein expression. We found that two small upstream open-reading frames (uORFs) encoding an 8-amino-acid peptide and another 6-amino-acid peptide, respectively, in the 5’UTR of FUS1 could markedly inhibit the translational initiation of FUS1 protein by interfering with the “scanning” of the ribosome initiation complexes. Several secondary RNA structural elements/motifs on the 3’UTR of FUS1 also showed a significant inhibitory effect on FUS1 protein expression. The 3’UTR-mediated regulatory effect on FUS1 proteins expression was differentially detected in normal lung epithelial and fibroblast cells compared to lung cancer cells. We found that FUS1 expression could be induced or activated in the presence of DNA damaging agents, such as UV irradiation, etoposide, and cisplatin. UV irradiation could induce FUS1 protein and mRNA expression independent of the status of FUS1 UTRs. The FUS1 expression activated by DNA damaging agents such as etoposide or cisplatin facilitated FUS1-induced apoptosis, prevented cells from dividing and accelerated cell death. Our results provide new insight into the molecular mechanisms in the regulation of FUS1 expression in lung cancer cells. (Supported by NIH SPORE P50CA70907 and RO1CA116322 grants) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3083.

Cytochrome P450 1B1 mRNA untranslated regions interact to inhibit protein translation

CYP1B1 mRNA is expressed constitutively in all normal extrahepatic human tissues, though the protein is usually undetectable. In contrast, CYP1B1 protein is expressed at high levels in tumors. In this study CYP1B1 mRNA and protein expression was measured in a panel of cell lines indicating that CYP1B1 regulation is altered in tumor cell lines in vitro. Interrogation of ONCOMINE revealed that CYP1B1 mRNA is not significantly overexpressed in tumors compared to normal tissues, suggesting CYP1B1 is subject to posttranscriptional control. Analysis of the CYP1B1 mRNA revealed a complex 5' untranslated region (UTR) containing a small upstream open-reading frame (uORF). These features are present in mRNAs subject to translational control so the effect of the 5'UTR was tested using in vitro translation in CHO-K1 cells. The 5'UTR significantly inhibited luciferase reporter gene translation, and mutation of the uORF start codon abolished the inhibitory effect. The 5'UTR also interacted with the microRNA-27b recognition element in the CYP1B1 mRNA 3'UTR to almost completely inhibit translation. CYP1B1 is subject to a high degree of translational control, which may explain the absence of protein expression in normal cells. Alterations in translational control during malignant transformation may help to explain the tumor-specific expression of CYP1B1 protein.

An Upstream Open Reading Frame Controls Translation of var2csa, a Gene Implicated in Placental Malaria

Malaria, caused by the parasite Plasmodium falciparum, is responsible for substantial morbidity, mortality and economic losses in tropical regions of the world. Pregnant women are exceptionally vulnerable to severe consequences of the infection, due to the specific adhesion of parasite-infected erythrocytes in the placenta. This adhesion is mediated by a unique variant of PfEMP1, a parasite encoded, hyper-variable antigen placed on the surface of infected cells. This variant, called VAR2CSA, binds to chondroitin sulfate A on syncytiotrophoblasts in the intervillous space of placentas. VAR2CSA appears to only be expressed in the presence of a placenta, suggesting that its expression is actively repressed in men, children or non-pregnant women; however, the mechanism of repression is not understood. Using cultured parasite lines and reporter gene constructs, we show that the gene encoding VAR2CSA contains a small upstream open reading frame that acts to repress translation of the resulting mRNA, revealing a novel form of gene regulation in malaria parasites. The mechanism underlying this translational repression is reversible, allowing high levels of protein translation upon selection, thus potentially enabling parasites to upregulate expression of this variant antigen in the presence of the appropriate host tissue.